Application and method of lycium barbarum polysaccharide in preparation of medicine for inhibiting pyroptosis of cardiac muscle cells

Abstract

The invention discloses application and a method of lycium barbarum polysaccharide in preparation of drugs for inhibiting pyroptosis of cardiac muscle cells, and relates to the technical field of biological medicines.The method specifically comprises the following steps that S1, an H9C2 cardiac muscle cell hypoxia / reoxygenation model is established; s2, exploring the optimal action concentration of the lycium barbarum polysaccharide on the H9C2 myocardial-like cells; s3, detection results of the cell survival rate and LDH activity are obtained; s4, obtaining an apoptosis staining result of the Hoechst33342 / PI cell; s5, detection of inflammatory factors IL-1beta and IL-18; s6, mRNA detection of the pyroptosis markers ASC, NLRP3 and Caspase-1 is carried out; and S7, carrying out protein detection on the pyroptosis markers ASC, NLRP3 and Caspase-1. According to the application and the method of the lycium barbarum polysaccharide in the preparation of the medicine for inhibiting the pyroptosis of the cardiac muscle cells, the novel application of the lycium barbarum polysaccharide (LBP) in the preparation of the medicine for inhibiting the pyroptosis of the cardiac muscle cells is disclosed, experiments prove that 90 mu g / mL of LBP can obviously inhibit H / R induced H9C2 pyroptosis of the cardiac muscle cells, and the mechanism relates to activation of an Nrf2 / HO-1 signal channel and down-regulation of the activity of NLRP3 inflammasomes. And a new strategy is provided for development of myocardial protection drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to an application and method of Lycium barbarum polysaccharide in preparing a drug for inhibiting myocardial cell pyroptosis. Background Art

[0002] Myocardial ischemia-reperfusion (I / R) injury is a major issue in the treatment of cardiovascular diseases. Its mechanisms include oxidative stress, inflammatory response, and pyroptosis. Current clinically used antioxidant drugs and ischemic preconditioning methods have limited efficacy. Pyroptosis is a form of programmed cell death activated by the NLRP3 inflammasome and is closely associated with the release of inflammatory cytokines such as IL-1β and IL-18. The Nrf2 / HO-1 pathway is a key pathway in regulating resistance to oxidative stress, but its role in cardiomyocyte pyroptosis remains unclear.

[0003] Lycium barbarum polysaccharides (LBP) have been reported to have antioxidant and anti-inflammatory activities, but their application in cardiomyocyte pyroptosis has not been reported. Therefore, the development of new cardioprotective drugs based on LBP is of great clinical significance. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides an application and method of Lycium barbarum polysaccharide in the preparation of a drug for inhibiting myocardial cell pyroptosis, which solves the problems raised in the above-mentioned background technology.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: the use of Lycium barbarum polysaccharides in the preparation of drugs for inhibiting myocardial cell pyroptosis and the method thereof, comprising the following steps:

[0006] S1. Establishment of the hypoxia / reoxygenation model of H9C2 cardiomyocytes; S2. Exploration of the optimal concentration of Lycium barbarum polysaccharides on H9C2 cardiomyocytes; S3. Detection results of cell viability and LDH activity; S4. Hoechst 33342 / PI cell apoptosis staining results; S5. Detection of inflammatory factors IL-1β and IL-18; S6. Detection of mRNA levels of pyroptosis markers ASC, NLRP3, and Caspase-1; S7. Protein detection of pyroptosis markers ASC, NLRP3, and Caspase-1; S8. Detection of mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway; S9. Protein detection of cytoplasmic Nrf2, nuclear Nrf2, and HO-1, markers of the Nrf2 / HO-1 signaling pathway; S10. Selection of the best siRNA by RT-qPCR; S11: Western The optimal siRNA was selected by blotting experiment; S12: Cell viability and LDH activity detection results; S13: Hoechst 33342 / PI cell apoptosis staining results; S14: Detection of inflammatory factors IL-1β and IL-18; S15: mRNA detection of pyroptosis markers ASC, NLRP3, and Caspase-1; S16: mRNA detection of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway; S17: Protein detection of pyroptosis markers ASC, NLRP3, and Caspase-1.

[0007] Preferably, the establishment of the H9C2 cardiomyocyte hypoxia / reoxygenation model in S1 includes:

[0008] S1.1. Establish a method to place H9C2 cells in an incubator at 37°C containing 95% air and 5% CO2. After incubation for 3 hours, the cells were replaced with a simulated hypoxic solution saturated with high-purity N2 for 30 minutes, with an oxygen partial pressure below 7 kPa, and placed in a sealed container at 37°C containing 95% N2 and 5% CO2 for hypoxia for 0 hours, 3 hours, 6 hours, and 10 hours, respectively, followed by reoxygenation for 24 hours.

[0009] S1.2 Results and Analysis: Cell morphology of each group was photographed under white light. Cell morphology was damaged at 3, 6, and 10 hours of hypoxia. Cells died in aggregates at 10 hours of hypoxia, and morphological damage was most obvious. FITC Annexin V and PIApoptosis were used. The kit was used to detect the cell apoptosis rate of each group by flow cytometry. The cell apoptosis rate was 2.91% at 0 h of hypoxia, 10.27% at 3 h of hypoxia, 12.87% at 6 h of hypoxia, and 15.05% at 10 h of hypoxia. The cell supernatant was collected and the LDH activity was detected using a kit. Compared with 0 h of hypoxia, the LDH activity of 3 h of hypoxia increased (P < 0.05), the LDH activity of 6 h of hypoxia increased extremely significantly (P < 0.001), and the LDH activity of 10 h of hypoxia increased extremely significantly (P < 0.001). Based on the above results and reference data, the optimal conditions for establishing the H / R model of H9C2 cardiomyocytes are 6 h of hypoxia and 24 h of reoxygenation.

[0010] Preferably, the exploration of the optimal concentration of Lycium barbarum polysaccharide in S2 on H9C2 cardiomyocytes includes:

[0011] S2.1. Observe the effects of different concentrations of Lycium barbarum polysaccharides on H9C2 cardiomyocytes and divide them into 5 groups:

[0012] Control group (Control): H9C2 cardiomyocytes were cultured normally without special treatment; Lycium barbarum polysaccharide (30ug / mL): 30ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before the establishment of the H / R model; Lycium barbarum polysaccharide (60ug / mL): 60ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before the establishment of the H / R model; Lycium barbarum polysaccharide (90ug / mL): 90ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before the establishment of the H / R model; Lycium barbarum polysaccharide (120ug / mL): 120ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before the establishment of the H / R model;

[0013] S2.2 Results and analysis: In H9C2 cardiomyocytes, after pretreatment with 0ug / mL, 30ug / mL, 60ug / mL, 90ug / mL, and 120ug / mL of Lycium barbarum polysaccharides for 24h, the cell survival rates were as follows: Figure 4As shown in the figure, the effect on cells is concentration-dependent. In the concentration range of 30ug / mL to 120ug / mL, the cell survival rate is inversely proportional to the concentration of Lycium barbarum polysaccharides. At a concentration of 120ug / mL, there is a significant difference in cell survival rate compared with the blank group. Since 90ug / mL is used as the optimal concentration, Lycium barbarum polysaccharides will not have a toxic effect on cells due to excessively high concentrations.

[0014] Preferably, the detection results of cell viability and LDH activity in S3 include:

[0015] S3.1. CCK-8 assay for cell viability:

[0016] H9C2 cardiomyocytes were seeded in a 96-well plate and placed in a cell culture incubator. The culture medium of the 96-well plate was removed, the plates were washed twice with PBS, and 10 μl of CCK-8 and 100 μl of serum-free DMEM medium were added to each well. The 96-well plate was incubated in a dark incubator at 37°C for 2 h. The OD value was measured using a microplate reader at a wavelength of 450 nm. The cell survival rate was calculated according to the formula: cell survival rate (%) = (measured OD value - control OD value) / (standard OD value - blank OD value) × standard concentration × sample dilution factor × 1000 × 100%.

[0017] S3.2. Determination of lactate dehydrogenase (LDH) activity: After treatment of each cell group, the cell culture supernatant was collected and the assay was performed according to the kit instructions. The OD value was measured using a microplate reader at a wavelength of 450 nm. The LDH activity was calculated according to the formula: LDH activity (U / L) = (measured OD value - control OD value) / (standard OD value - blank OD value) × standard concentration × sample dilution factor × 1000.

[0018] S3.3 Results and Analysis

[0019] After treatment of each group of cells, the cell viability was detected by CCK-8. Compared with the Control group, the cell viability of the H / R group was extremely significantly decreased (P < 0.001), compared with the Control group, the cell viability of the H / R+ Lycium barbarum polysaccharide group was significantly decreased (P < 0.01), and compared with the H / R group, the cell viability of the H / R+ Lycium barbarum polysaccharide group was significantly increased (P < 0.01); LDH activity was detected using a kit. Compared with the Control group, the LDH activity of the H / R group was extremely significantly increased (P < 0.001), compared with the Control group, the LDH activity of the H / R+ Lycium barbarum polysaccharide group was extremely significantly increased (P < 0.001), and compared with the H / R group, the LDH activity of the H / R+ Lycium barbarum polysaccharide group was extremely significantly decreased (P < 0.001). This indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can increase the cell viability of H9C2 cardiomyocytes after H / R and reduce the LDH activity of H9C2 cardiomyocytes after H / R.

[0020] Preferably, the Hoechst 33342 / PI apoptosis staining results in S4 include:

[0021] S4.1. Cell plating: Take the normally cultured cells, remove the original culture medium, add PBS and then trypsin to digest for 1-3 minutes to terminate the digestion, use a pipette to blow into single cells, centrifuge the cell suspension at 1000 rpm, 4℃ for 5 minutes, and resuspend in culture medium.

[0022] Take 20 μL of cell suspension and add 20 μL of trypan blue for counting. Add 2*104 cells / well (24-well plate) to a 24-well plate covered with a slide and incubate overnight at 37°C with 5% CO2. For Hoechst-PI apoptosis detection, add 5 μL of Hoechst 33342 staining solution and 5 μL of PI staining solution, mix gently, and incubate on ice or at 4°C for 20-30 minutes before subsequent fluorescence detection.

[0023] S4.2. Results and analysis. After treatment of each group of cells, cell apoptosis was detected by Hoechst 33342 / PI double staining. Normal cells showed weak red fluorescence + weak blue fluorescence; apoptotic cells showed weak red fluorescence + strong blue fluorescence; necrotic cells showed strong red fluorescence + weak blue fluorescence. Compared with the Control group, the apoptosis rate of cells in the H / R group was extremely significantly increased (P < 0.001). Compared with the Control group, the apoptosis rate of cells in the H / R+lycium barbarum polysaccharide group was increased (P < 0.05). Compared with the H / R group, the apoptosis rate of cells in the H / R+lycium barbarum polysaccharide group was decreased (P < 0.05). This indicates that pretreatment with 90ug / mL of Lycium barbarum polysaccharide can reduce the apoptosis rate of H9C2 cardiomyocytes after H / R.

[0024] Preferably, the detection of inflammatory factors IL-1β and IL-18 in S5 includes:

[0025] S5.1 Collect cell supernatants according to experimental groups and freeze at -20°C for subsequent ELISA testing. Before starting the experiment, all reagents should be equilibrated to room temperature. When preparing reagents or samples, mix thoroughly and avoid foaming as much as possible.

[0026] 1. Washing: Add 300 μL of wash buffer to each well, let stand for 40 seconds, spin dry, wash the plate three times, and pat the liquid in the wells dry on absorbent paper. 2. Adding samples: Set up blank wells, standard wells, and test sample wells. Add 100 μL of sample diluent to the blank wells and 100 μL of standard or test sample to the remaining wells. Cover the plate with film and incubate at 37°C for 120 minutes.

[0027] 3. Discard the liquid, spin dry, wash the plate 3 times, soak for 1 minute each time, add 300 μL wash buffer to each well, spin dry and pat the liquid in the well on absorbent paper; 4. Add 100 μL diluted Biotin-Conjugate Antibody to each well, cover with film, and incubate at 37°C for 1 hour; 5. Discard the liquid in the well, spin dry, wash the plate 3 times, and repeat the same steps as step 3; 6. Add 100 μL diluted 1× Streptavidin-HRP to each well and incubate at 37°C for 30 minutes; 7. Discard the liquid in the well, spin dry, wash the plate 3 times, and repeat the same steps as step 3; 8. Add 90 μL TMB Substrate to each well and incubate at 37°C for 15 minutes to develop color; 9. Add 50 μL Stop Solution to each well to terminate the reaction, mix well, and immediately detect OD450;

[0028] S5.2. Results and analysis. After treatment of each group of cells, the inflammatory factors IL-1β and IL-18 in the cell supernatant were detected by ELISA experiments. Compared with the Control group, the inflammatory factors IL-1β and IL-18 in the H / R group were extremely significantly increased (P < 0.001); compared with the Control group, the inflammatory factors IL-1β and IL-18 in the H / R+ Lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001); compared with the H / R group, the inflammatory factors IL-1β and IL-18 in the H / R+ Lycium barbarum polysaccharide group were extremely significantly decreased (P < 0.001); this shows that pretreatment with 90ug / mL Lycium barbarum polysaccharide can reduce the inflammatory factors IL-1β and IL-18 after H / R of H9C2 cardiomyocytes.

[0029] Preferably, the mRNA detection of pyroptosis markers ASC, NLRP3, and Caspase-1 in S6 includes:

[0030] S6.1. Extraction of total RNA using Trizol method:

[0031] 1. Pretreatment of cell samples: Add 1 mL of Trizol reagent, mix thoroughly by pipetting, transfer to an RNase-free 1.5 mL EP tube, and lyse for 10 minutes. 2. Add 200 μL of chloroform, mix vigorously by inversion several times, and let stand at room temperature for 5 minutes. 3. Centrifuge at 4°C, 12,000 rpm, for 15 minutes. Separation of the three phases, upper (RNA), middle (protein), and lower (DNA), will be visible. 4. Transfer the upper aqueous phase (approximately 400 μL) to another fresh 1.5 mL EP tube, add 400 μL of isopropanol, mix thoroughly, and let stand at room temperature for 10 minutes. 5. Centrifuge at 4°C, 12,000 rpm, for 10 minutes. A white RNA precipitate will be visible at the bottom of the tube. 6. Discard the supernatant, add 1 mL of RNase-free 75% ethanol, vortex to mix thoroughly, and centrifuge at 4°C, 10,000 rpm, for 5 minutes. 7. Repeat step 6 once. 8. Discard the supernatant, air-dry the RNA precipitate for 5-10 minutes, and dissolve the precipitate in 20 μL of IX. Take 2 μL of the dissolved RNA and measure OD260, OD280, and OD260 / OD280 using a micro-spectrophotometer to calculate RNA purity and concentration. Estimate RNA quality based on the OD260 / OD280 ratio; a ratio between 1.8 and 2.0 satisfies experimental requirements. Calculate sample RNA concentration based on the absorbance readings using the following formula: Total RNA concentration (μg / μL) = OD260 × 40 × 10⁻³. X. Store the total RNA in a -80°C freezer until ready for use.

[0032] S6.2. Reverse transcription into cDNA:

[0033] 1. For genomic DNA removal, thaw the RNA template, RNAase-free water, and 10×g DNA Remover Buffer on ice. Prepare the reaction system (10 μL) in a nuclease-free microcentrifuge tube. Mix the system by pipetting and centrifuge briefly. Incubate the system in a PCR instrument using the following protocol: incubate at 42°C for 2 minutes and then at 60°C for 5 minutes.

[0034] 4. Reverse transcription reaction: After the previous step, quickly place the system on ice to cool, centrifuge briefly, and add reverse transcription reaction

[0035] 5. Then, use a pipette to mix the system and centrifuge briefly. Place the reaction on a PCR instrument. The program is as follows: incubate at 25°C for 10 minutes, incubate at 50°C for 15 minutes, and then incubate at 85°C for 5 minutes. Then, place the reverse transcription product on ice or refrigerate until use.

[0036] S6.3. Real-time fluorescence quantitative PCR detection reaction system:

[0037] Use a pipette to mix the system and centrifuge briefly. Place the system on a fluorescent quantitative PCR instrument for amplification detection to obtain running data. The final data is analyzed using the 2-△△Ct method and a data analysis report is compiled.

[0038] Q-PCR algorithm (relative quantification, 2-ΔΔCt method)

[0039] algorithm:

[0040] 1. Target gene Ct value - reference gene Ct value,

[0041] 2. Ct value of the test sample - Ct value of the control sample,

[0042] 3. Take the logarithm of the negative value of the difference;

[0043] 4. Target gene Ct value - reference gene Ct value, e.g. 4.662 = 20.473 - 15.811;

[0044] 5. Calculate the Ct value of the test sample minus the Ct value of the reference sample. For example, if the control sample is sample 1, then calculate the Ct value of the target gene minus the Ct value of the reference gene, minus 4.662.

[0045] 6. The negative value of the Ct value of the test sample minus the Ct value of the control sample was taken as the logarithm with base 2, and the Excel function was "=power(2,-0.000)"; s6.4. Results and analysis. After treatment of each group of cells, the mRNA levels of ASC, NLRP3, and Caspase-1 were detected by RT-qPCR. Compared with the Control group, the mRNA levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R group were extremely significantly increased (P < 0.001); compared with the Control group, the mRNA levels of pyroptosis markers ASC and NLRP3 in the H / R+lycium barbarum polysaccharide group were significantly decreased (P < 0.001). The mRNA level of ASC, a pyroptosis marker, was extremely significantly increased (P < 0.001), and the mRNA level of Caspase-1, a pyroptosis marker, was increased (P < 0.05), but there was no significant difference; compared with the H / R group, the mRNA level of ASC, a pyroptosis marker, in the H / R+Lycium barbarum polysaccharide group was significantly increased (P < 0.01), and the mRNA levels of NLRP3 and Caspase-1 were extremely significantly increased (P < 0.001); this indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can reduce the mRNA levels of ASC, NLRP3, and Caspase-1, pyroptosis-specific markers, in H9C2 cardiomyocytes after H / R.

[0046] Preferably, the protein detection of pyroptosis markers ASC, NLRP3, and Caspase-1 in S7 includes:

[0047] S7.1. Detection Methods

[0048] 1. Extraction of total cell protein

[0049] Pour out the culture medium and turn the bottle upside down on absorbent paper to absorb the culture medium dry; add 3 mL of 4°C pre-cooled PBS (0.01M pH 7.2-7.3) to each bottle of cells; place it flat and gently shake for 1 minute to wash the cells, then discard the washing solution; repeat the above operation twice, wash the cells three times in total to wash away the culture medium; discard the PBS and place the culture bottle on ice; add 10 μL PMSF (100mM) to 1 mL of lysis buffer, shake well and place on ice; add 400 μL of lysis buffer containing PMSF to each bottle of cells and lyse on ice for 30 minutes. To ensure sufficient cell lysis, shake the culture bottle back and forth frequently; after lysis, scrape the cells on the side of the culture bottle with a clean scraper, and then use a gun to transfer the cell debris and lysis buffer to a 1.5 mL centrifuge tube; centrifuge at 12000 rpm for 5 minutes at 4°C; transfer the supernatant after centrifugation into 0.5 mL centrifuge tubes and store at -20°C;

[0050] 2. Extraction of nuclear and cytoplasmic proteins; 3. Determination of protein concentration; 4. Protein denaturation; 5. Electrophoresis; 6. Electrophoretic separation; 7. Western blot analysis;

[0051] s7.2 Results and Methods

[0052] After treatment of each group of cells, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 were detected by Western blotting experiments. Compared with the Control group, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R group were extremely significantly increased (P < 0.001); compared with the Control group, the protein levels of pyroptosis markers ASC and Caspase-1 in the H / R+Lycium barbarum polysaccharide group were significantly increased (P < 0.01), and the protein level of pyroptosis marker NLRP3 was increased, but there was no significant difference; compared with the H / R group, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R+Lycium barbarum polysaccharide group were extremely significantly decreased (P < 0.001); this indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can reduce the protein levels of pyroptosis-specific markers ASC, NLRP3, and Caspase-1 in H9C2 cardiomyocytes after H / R.

[0053] Preferably, the detection of the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway in s8, comprises:

[0054] s8.1. Detection methods

[0055] 1. Experimental Grouping and Treatment

[0056] Control group: H9C2 cardiomyocytes cultured in normal serum-containing medium; H / R group: H9C2 cardiomyocyte H / R model was established according to Conclusion 1; H / R+Lycium barbarum polysaccharide group: H9C2 cardiomyocyte H / R model was established by pretreatment with 90 μg / mL Lycium barbarum polysaccharide for 24 h;

[0057] 2. Real-time fluorescence quantitative PCR detection of mRNA

[0058] Total RNA was extracted using Trizol as described in Section 2.9.1 of the first experimental method. Reverse transcription to cDNA was performed as described in Section 2.9.2 of the first experimental method. Real-time fluorescence quantitative PCR was performed as described in Section 2.9.3 of the first experimental method.

[0059] s8.2. Results and analysis. After treatment of each group of cells, the mRNA levels of Nrf2 and HO-1 were detected by RT-qPCR experiments. Compared with the Control group, the mRNA level of HO-1, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R group was significantly increased (P < 0.01), and the mRNA level of Nrf2 was increased, but there was no significant difference; compared with the Control group, the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in the H / R+lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001); compared with the H / R group, the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in the H / R+lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001); this indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can increase the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in H9C2 cardiomyocytes after H / R.

[0060] Preferably, the protein detection of the Nrf2 / HO-1 signaling pathway markers cytoplasmic Nrf2, nuclear Nrf2, and HO-1 in S9 includes:

[0061] S9.1, Experimental method, same as S7;

[0062] S9.2. Results and analysis. After treatment, the protein levels of cytoplasmic Nrf2, nuclear Nrf2, and HO-1, markers of the Nrf2 / HO-1 signaling pathway, were detected by Western blotting. Compared with the Control group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R group was significantly decreased (P < 0.001), while the protein levels of nuclear Nrf2 and HO-1 were significantly increased (P < 0.001). Compared with the Control group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R+lycium barbarum polysaccharide group was significantly decreased. (P < 0.001), and the protein levels of nuclear Nrf2 and HO-1 were extremely significantly increased (P < 0.001). Compared with the H / R group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, was extremely significantly increased in the H / R+lycium barbarum polysaccharide group (P < 0.001), and the nuclear Nrf2 and HO-1 protein levels were extremely significantly increased (P < 0.001). This indicates that pretreatment with 90 μg / mL of Lycium barbarum polysaccharide can enable Nrf2 in the cytoplasm to enter the cell nucleus, thereby activating HO-1 and increasing the HO-1 protein level.

[0063] The selection of the best siRNA in the RT-qPCR experiment in S10 includes:

[0064] S10.1. RT-qPCR selection of optimal siRNA

[0065] For real-time fluorescence quantitative PCR, extract total RNA using Trizol as described in Section 1, Experimental Methods, 2.9.1; reverse transcribe to cDNA as described in Section 1, Experimental Methods, 2.9.2; and perform real-time fluorescence quantitative PCR as described in Section 1, Experimental Methods, 2.9.3.

[0066] Results and Analysis After treatment of each group of cells, the mRNA level of Nrf2 was detected by RT-qPCR. Compared with the blank group, the mRNA level of Nrf2 in the NC siRNA group was not significantly different; compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA1 group was decreased (P < 0.05); compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA3 group was extremely significantly decreased (P < 0.001); compared with the Nrf2 siRNA2 group, the mRNA level of Nrf2 in the Nrf2 siRNA1 group was extremely significantly increased (P < 0.001); compared with the Nrf2 siRNA2 group, the mRNA level of Nrf2 in the Nrf2 siRNA3 group was increased (P < 0.05); This showed that Nrf2 The mRNA level of Nrf2 was the lowest in the siRNA2 group, and Nrf2 siRNA2 was selected as the optimal siRNA;

[0067] The detection results of cell viability and LDH activity in the S12 include:

[0068] S12.1: Detection Methods

[0069] Experimental groups:

[0070] Control group (Control) + NC siRNA; HR model group + NC siRNA; HR model group + Lycium barbarum polysaccharide group + NC siRNA; Control group (Control) + Nrf2 siRNA2; HR model group + Nrf2 siRNA2; HR model group + Lycium barbarum polysaccharide group + Nrf2 siRNA2; Lycium barbarum polysaccharide experimental group was pretreated with Lycium barbarum polysaccharide for 24 hours, then hypoxic for 6 hours and reoxygenated at 37°C overnight for 24 hours, with three replicates per group;

[0071] Cell plating:

[0072] Adherent cells: Take normally cultured cells, remove the original culture medium, add PBS to wash, and then add trypsin to digest for 3 minutes to terminate the digestion. Use a pipette to blow into single cells, centrifuge the cell suspension at 1000 rpm for 5 minutes, resuspend in culture medium, take 20 μL of cell suspension and add 20 μL of trypan blue for counting, and plate 10,000 cells / well (96-well plate), 100 μL per well. At the same time, set up a blank group, add 100 μL of sterile PBS to the wells around the cell wells, 5% CO2, and culture overnight at 37°C;

[0073] Transfection (6-well plate, 2 mL culture medium):

[0074] siRNA transfection: 200 μL jetPRIME buffer was placed in EP tubes, siRNA was added to a final concentration of 15 nM, and vortexed to mix; 4 μL jetPRIME reagent was added, vortexed to mix, and incubated at room temperature for 10 min; 200 μL of the above mixture was added to the cell culture medium, and fresh medium was replaced after 4 h, incubated at 37°C with 5% CO2;

[0075] CCK-8 assay: Add 10 μL of CCK-8 solution to each well and incubate in a 5% CO2, 37°C incubator for 3 h. Measure the absorbance (OD 450) of each well with a microplate reader. Analyze the data.

[0076] S12.2: Results and Analysis

[0077] After each group of cells were treated, the cell viability was detected by CCK-8 and the LDH activity was detected using a kit.

[0078] Compared with the control group + NC siRNA group, the cell survival rate in the H / R model group + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + NC siRNA group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); compared with the control group + Nrf2 siRNA2 group, the cell survival rate in the H / R model group + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + Nrf2siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); Compared with the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Lycium barbarum polysaccharide + NC siRNA group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); this showed that pretreatment with 90ug / mL Lycium barbarum polysaccharide and Nrf2 siRNA2 could reduce the cell survival rate of H9C2 cardiomyocytes after H / R; compared with the control group + NC siRNA group, the LDH activity in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the LDH activity in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the control group + Nrf2 Compared with the H / R model group + Nrf2 siRNA2 group, the LDH activity in the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the LDH activity in the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + NC siRNA group, the LDH activity in the H / R model group + LBP + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the LDH activity in the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + LBP + NC siRNA group, the LDH activity in the H / R model group + LBP + Nrf2 The LDH activity in the siRNA2 group was extremely significantly increased (P < 0.001); this indicated that pretreatment with 90ug / mL Lycium barbarum polysaccharide and Nrf2 siRNA2 could increase the LDH activity of H9C2 cardiomyocytes after H / R.

[0079] The present invention provides the use and method of Lycium barbarum polysaccharide in preparing a drug for inhibiting myocardial cell pyroptosis, which has the following beneficial effects:

[0080] 1. Application and method of wolfberry polysaccharide in preparing drugs for inhibiting myocardial cell pyroptosis. Disclosed is a new use of wolfberry polysaccharide (LBP) in preparing drugs for inhibiting myocardial cell pyroptosis. Experiments have confirmed that 90 μg / mL of LBP can significantly inhibit H / R-induced H9C2 cardiomyocyte pyroptosis. Its mechanism involves activating the Nrf2 / HO-1 signaling pathway and downregulating the activity of the NLRP3 inflammasome, providing a new strategy for the development of myocardial protective drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The cell morphology of each group was photographed under white light for the present invention.

[0082] Figure 2 The flow cytometry apoptosis cytometry was used to detect the apoptosis rate of cells in each group.

[0083] Figure 3 The lactate dehydrogenase (LDH) activity of cells in each group was detected for the present invention (*, compared with 0 h hypoxia and reoxygenation, P < 0.05; ***, compared with 0 h hypoxia and reoxygenation, P < 0.001).

[0084] Figure 4 The results show that the different concentrations of Lycium barbarum polysaccharides (0ug / mL, 30ug / mL, 60ug / mL, 90ug / mL, 120ug / mL) of the present invention affect the survival rate of H9C2 cardiomyocytes (**, P<0.01 compared with the blank group).

[0085] Figure 5 The cell survival rate test results of the present invention are shown (**, compared with the Control group, P < 0.01; ***, compared with the Control group, P < 0.001).

[0086] Figure 6 This is the LDH activity detection result of the present invention (***, compared with the Control group, P < 0.001).

[0087] Figure 7 The results of Hoechst 33342 / PI cell apoptosis staining of the present invention are as follows: A is the fluorescence microscope photography result; B is the statistical analysis of the apoptosis ratio of each group (*, compared with the Control group, P<0.05; ***, compared with the Control group, P<0.001).

[0088] Figure 8 These are the detection results of the inflammatory factors IL-1β and IL-18 of the present invention (***, P<0.001 compared with the Control group).

[0089] Figure 9 These are the mRNA detection results of the pyroptosis markers ASC, NLRP3, and Caspase-1 in the present invention (***, P < 0.001 compared with the Control group).

[0090] Figure 10 The present invention shows the protein detection results of pyroptosis markers ASC, NLRP3, and Caspase-1 (**, compared with the Control group, P < 0.01; ***, compared with the Control group, P < 0.001).

[0091] Figure 11 These are the mRNA detection results of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway of the present invention (**, P<0.01 compared with the Control group; ***, P<0.001 compared with the Control group).

[0092] Figure 12 Protein detection of cytoplasmic Nrf2, nuclear Nrf2, and HO-1, markers of the Nrf2 / HO-1 signaling pathway of the present invention (***, P < 0.001 compared with the Control group).

[0093] Figure 13 The optimal siRNA detection results were selected for RT-qPCR of the present invention (*, P < 0.05 compared with the blank group; ***, P < 0.001 compared with the blank group).

[0094] Figure 14 The best siRNA detection results were selected for the Western blotting experiment of the present invention (**, P < 0.01 compared with the blank group; ***, P < 0.001 compared with the blank group).

[0095] Figure 15 The results of cell survival rate and LDH activity detection of the present invention are shown in Table 1 (***, compared with the control group + NC siRNA group, P < 0.001).

[0096] Figure 16 Figure 2 is the Hoechst 33342 / PI apoptosis staining result of the present invention: A is the fluorescence microscope photography result; B is the statistical analysis of the apoptosis ratio of each group (*, compared with the control + NC siRNA group, P < 0.05; ***, compared with the control + NC siRNA group, P < 0.001).

[0097] Figure 17These are the detection results of the inflammatory factors IL-1β and IL-18 of the present invention (**, compared with the control group + NC siRNA group, P<0.01; ***, compared with the control group + NC siRNA group, P<0.001).

[0098] Figure 18 These are the mRNA detection results of the pyroptosis markers ASC, NLRP3, and Caspase-1 of the present invention (*, compared with the control group + NC siRNA group, P < 0.05; **, compared with the control group + NC siRNA group, P < 0.01; ***, compared with the control group + NC siRNA group, P < 0.001).

[0099] Figure 19 These are the mRNA detection results of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway of the present invention (**, compared with the control group + NC siRNA group, P < 0.01; ***, compared with the control group + NC siRNA group, P < 0.001).

[0100] Figure 20 These are the protein detection results of the pyroptosis markers ASC, NLRP3, and Caspase-1 of the present invention (**, compared with the control group + NC siRNA group, P < 0.01; ***, compared with the control group + NC siRNA group, P < 0.001).

[0101] Figure 21 These are the protein detection results of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway of the present invention (*, compared with the control group + NC siRNA group, P < 0.05; **, compared with the control group + NC siRNA group, P < 0.01; ***, compared with the control group + NC siRNA group, P < 0.001). DETAILED DESCRIPTION

[0102] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0103] See also Figures 1 to 21 The present invention provides a technical solution: the application and method of wolfberry polysaccharide in the preparation of drugs for inhibiting myocardial cell pyroptosis; the effective concentration: the concentration of LBP is 90 μg / mL, which can inhibit H / R-induced H9C2 cardiomyocyte pyroptosis by regulating the Nrf2 / HO-1 signaling pathway.

[0104] Mechanism of action: LBP promotes the translocation of Nrf2 protein from the cytoplasm to the nucleus, activates HO-1 protein expression, and then inhibits the expression of NLRP3 inflammasome-related markers (ASC, Caspase-1) and inflammatory factors (IL-1β, IL-18).

[0105] Scope of application: Suitable for the prevention or treatment of cardiovascular diseases such as myocardial ischemia-reperfusion injury and heart failure.

[0106] 2 Technical Effects

[0107] Significantly reduced H / R-induced cell pyroptosis (reduced by ≥30%) and increased cell survival (increased by ≥25%).

[0108] Inhibits lactate dehydrogenase (LDH) activity (reduction ≥40%) and the release of inflammatory factors IL-1β and IL-18 (reduction ≥35%).

[0109] Silencing the Nrf2 gene by siRNA reversed the protective effect of LBP, verifying its pathway dependence.

[0110] Example 1: Establishment of an H9C2 cardiomyocyte hypoxia / reoxygenation model

[0111] 1. Method

[0112] H9C2 cells were placed in an incubator at 37°C containing 95% air and 5% CO2 for 3 h. The cells were then replaced with a simulated hypoxic solution saturated with high-purity N2 for 30 min and with an oxygen partial pressure of less than 7 kPa. The cells were then placed in a sealed container at 37°C containing 95% N2 and 5% CO2 for hypoxia for 0 h, 3 h, 6 h, and 10 h, respectively, and then reoxygenated for 24 h.

[0113] 2. Results and Analysis

[0114] The cell morphology of each group was photographed under white light. The cell morphology was damaged at 3h, 6h, and 10h of hypoxia. Cells died in aggregates at 10h of hypoxia, and the morphological damage was most obvious. Figure 1 The cell apoptosis rate of each group was detected by flow cytometry using FITC Annexin V and PIApoptosis Kit. The apoptosis rate of cells in each group was 2.91% at 0 h of hypoxia, 10.27% at 3 h of hypoxia, 12.87% at 6 h of hypoxia, and 15.05% at 10 h of hypoxia. Figure 2 The cell supernatant was collected and the LDH activity was detected using a kit. Compared with 0h of hypoxia, the LDH activity of 3h of hypoxia increased (P < 0.05), the LDH activity of 6h of hypoxia increased significantly (P < 0.001), and the LDH activity of 10h of hypoxia increased significantly (P < 0.001). The results are shown in Figure 3 Based on the above results and reference data, the optimal conditions for establishing the H9C2 cardiomyocyte H / R model are 6 hours of hypoxia and 24 hours of reoxygenation.

[0115] Example 2: Exploration of the optimal concentration of Lycium barbarum polysaccharides on H9C2 cardiomyocytes

[0116] 1. Method

[0117] The effects of different concentrations of Lycium barbarum polysaccharides on H9C2 cardiomyocytes were observed and divided into 5 groups:

[0118] Control group (Control): H9C2 cardiomyocytes were cultured normally without special treatment.

[0119] Lycium barbarum polysaccharide (30ug / mL): 24h before establishing the H / R model, 30ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes.

[0120] Lycium barbarum polysaccharide (60ug / mL): 24h before establishing the H / R model, 60ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes.

[0121] Lycium barbarum polysaccharide (90ug / mL): 24h before establishing the H / R model, 90ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes.

[0122] Lycium barbarum polysaccharide (120ug / mL): 120ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before establishing the H / R model.

[0123] 2. Results and Analysis

[0124] In H9C2 cardiomyocytes, after 24h pretreatment with 0ug / mL, 30ug / mL, 60ug / mL, 90ug / mL, and 120ug / mL Lycium barbarum polysaccharides, the cell survival rate was as follows: Figure 4 As shown in the figure, the effect on cells is concentration-dependent. Within the concentration range of 30ug / mL to 120ug / mL, the cell survival rate is inversely proportional to the concentration of Lycium barbarum polysaccharides. At a concentration of 120ug / mL, the cell survival rate is significantly different from that of the blank group. Therefore, in this experiment, 90ug / mL was used as the optimal concentration to prevent Lycium barbarum polysaccharide from causing toxic effects on cells due to excessively high concentrations.

[0125] Example 3: Detection results of cell viability and LDH activity

[0126] 1. Method

[0127] CCK-8 assay for cell viability:

[0128] 1) H9C2 cardiomyocytes were seeded into 96-well plates and placed in a cell culture incubator.

[0129] 2) Remove the culture medium from the 96-well plate, wash twice with PBS, and add 10 μl of CCK-8 and 100 μl of serum-free DMEM medium to each well.

[0130] 3) Place the 96-well plate in a 37°C incubator in the dark for 2 h, and measure the OD value using a microplate reader at a wavelength of 450 nm.

[0131] 4) Calculate the cell survival rate according to the formula: Cell survival rate (%) = (measured OD value - control OD value) / (standard OD value - blank OD value) × standard concentration × sample dilution factor × 1000 × 100%.

[0132] Determination of lactate dehydrogenase (LDH) activity:

[0133] 1) After each group of cells was treated, the cell culture supernatant was collected and the procedure was performed according to the kit instructions.

[0134] 2) Measure the OD value using a microplate reader at a wavelength of 450 nm.

[0135] 3) Calculate the LDH activity according to the formula: LDH activity (U / L) = (measured OD value - control OD value) / (standard OD value - blank OD value) × standard concentration × sample dilution factor × 1000.

[0136] 2. Results and Analysis

[0137] After each group of cells were treated, the cell survival rate was detected by CCK-8. The test results were as follows: Figure 5 As shown in the figure, compared with the Control group, the cell survival rate in the H / R group was extremely significantly decreased (P < 0.001), compared with the Control group, the cell survival rate in the H / R + Lycium barbarum polysaccharide group was significantly decreased (P < 0.01), and compared with the H / R group, the cell survival rate in the H / R + Lycium barbarum polysaccharide group was significantly increased (P < 0.01); the LDH activity was detected using a kit, and the test results are shown in the figure. Figure 6 As shown in the results, compared with the Control group, the LDH activity of the H / R group was extremely significantly increased (P < 0.001), compared with the Control group, the LDH activity of the H / R + Lycium barbarum polysaccharide group was extremely significantly increased (P < 0.001), and compared with the H / R group, the LDH activity of the H / R + Lycium barbarum polysaccharide group was extremely significantly decreased (P < 0.001); thus, it was shown that pretreatment with 90 μg / mL Lycium barbarum polysaccharide could increase the cell survival rate of H9C2 cardiomyocytes after H / R and decrease the LDH activity of H9C2 cardiomyocytes after H / R.

[0138] Example 4: Hoechst 33342 / PI cell apoptosis staining results

[0139] method

[0140] 1) Cell Plating: Take normally cultured cells, aspirate the original culture medium, add PBS and then trypsin to digest for 1-3 minutes to terminate digestion, pipette to separate into single cells, centrifuge the cell suspension at 1000 rpm and 4°C for 5 minutes, resuspend in culture medium, take 20 μL of cell suspension and add 20 μL of trypan blue for counting, add 2 x 104 cells / well (24-well plate) to a 24-well plate with a slide, and culture overnight at 37°C with 5% CO2.

[0141] 2) Hoechst-PI apoptosis detection: Add 5 μL of Hoechst 33342 staining solution and 5 μL of PI staining solution, mix gently, incubate on ice or at 4°C for 20-30 min, and then perform subsequent fluorescence detection.

[0142] 2. Results and Analysis

[0143] After treatment of each group of cells, cell apoptosis was detected by Hoechst 33342 / PI double staining. The staining results are shown in Figure 2. Figure 7 As shown, normal cells show weak red fluorescence + weak blue fluorescence; apoptotic cells show weak red fluorescence + strong blue fluorescence; and necrotic cells show strong red fluorescence + weak blue fluorescence. Compared with the Control group, the apoptosis rate of cells in the H / R group was extremely significantly increased (P < 0.001). Compared with the Control group, the apoptosis rate of cells in the H / R + Lycium barbarum polysaccharide group was increased (P < 0.05). Compared with the H / R group, the apoptosis rate of cells in the H / R + Lycium barbarum polysaccharide group was decreased (P < 0.05). This indicates that pretreatment with 90 μg / mL Lycium barbarum polysaccharide can reduce the apoptosis rate of H9C2 cardiomyocytes after H / R.

[0144] Example 5: Detection of inflammatory factors IL-1β and IL-18

[0145] 1. Method

[0146] Collect cell supernatants according to experimental groups and freeze at -20°C for subsequent ELISA testing. Before starting the experiment, all reagents should be equilibrated to room temperature; when preparing reagents or samples, mix thoroughly and avoid foaming as much as possible.

[0147] 1) Washing: Add 300 μL of wash buffer to each well, let stand for 40 seconds, spin dry, wash the plate three times, and pat dry on absorbent paper.

[0148] 2) Sample addition: Set up blank wells, standard wells, and wells for the sample to be tested. Add 100 μL of sample diluent to the blank well and 100 μL of the standard or sample to be tested to the remaining wells. Cover the plate with film and incubate at 37°C for 120 minutes.

[0149] 3) Discard the liquid, spin dry, and wash the plate three times, soaking for 1 minute each time. Add 300 μL of wash buffer to each well, spin dry, and tap the wells dry with absorbent paper.

[0150] 4) Add 100 μL of diluted Biotin-Conjugate antibody to each well, cover with film, and incubate at 37°C for 1 hour;

[0151] 5) Discard the liquid in the wells, spin dry, and wash the plate three times as in step 3;

[0152] 6) Add 100 μL of 1× diluted Streptavidin-HRP to each well and incubate at 37°C for 30 min;

[0153] 7) Discard the liquid in the wells, spin dry, and wash the plate three times as in step 3;

[0154] 8) Add 90 μL of TMB Substrate to each well and incubate at 37°C for 15 min to develop color;

[0155] 9) Add 50 μL Stop Solution to each well to terminate the reaction, mix well, and immediately measure OD450.

[0156] 2. Results and Analysis

[0157] After each group of cells were treated, the inflammatory factors IL-1β and IL-18 in the cell supernatant were detected by ELISA. Figure 8 As shown, compared with the Control group, the inflammatory factors IL-1β and IL-18 in the H / R group were significantly increased (P < 0.001); compared with the Control group, the inflammatory factors IL-1β and IL-18 in the H / R + Lycium barbarum polysaccharide group were significantly increased (P < 0.001); compared with the H / R group, the inflammatory factors IL-1β and IL-18 in the H / R + Lycium barbarum polysaccharide group were significantly decreased (P < 0.001). This shows that pretreatment with 90ug / mL Lycium barbarum polysaccharide can reduce the inflammatory factors IL-1β and IL-18 in H9C2 cardiomyocytes after H / R.

[0158] Example 6: Detection of mRNA of Pyroptosis Markers ASC, NLRP3, and Caspase-1

[0159] 1. Method

[0160] Trizol method for total RNA extraction:

[0161] 1) Pretreatment of cell samples: Add 1 mL of Trizol reagent, mix thoroughly by pipetting, transfer to a 1.5 mL EP tube without RNase, and lyse for 10 min.

[0162] 2) Add 200 μL of chloroform, mix vigorously by inverting several times, and let it stand at room temperature for 5 minutes.

[0163] 3) Centrifuge at 4°C, 12,000 rpm for 15 min. The solution will separate into three phases: upper (RNA), middle (protein), and lower (DNA).

[0164] 4) Transfer the upper aqueous phase (approximately 400 μL) to another new 1.5 mL EP tube, add 400 μL of isopropanol, mix well, and let stand at room temperature for 10 minutes.

[0165] 5) Centrifuge at 4°C, 12,000 rpm for 10 min. A white RNA precipitate will be visible at the bottom of the tube.

[0166] 6) Discard the supernatant, add 1 mL of RNase-free 75% ethanol, vortex to mix, and centrifuge at 10,000 rpm at 4°C for 5 minutes.

[0167] 7) Repeat step 6 once.

[0168] 8) Discard the supernatant, air-dry the RNA pellet for 5-10 minutes, and dissolve the pellet in 20 μL of DEPC water.

[0169] 9) Take 2 μL of dissolved RNA and measure OD260, OD280, and OD260 / OD280 using a micro-spectrophotometer to calculate RNA purity and concentration. Estimate RNA quality based on the OD260 / OD280 ratio; a ratio between 1.8 and 2.0 satisfies experimental requirements. Calculate sample RNA concentration based on absorbance using the following formula: Total RNA concentration (μg / μL) = OD260 × 40 × 10⁻³.

[0170] 10) Store the total RNA in a -80°C refrigerator for future use.

[0171] 2.9.2 Reverse transcription into cDNA:

[0172] 1) Genomic DNA removal reaction

[0173] Thaw the RNA template, RNAase-free water, and 10×g DNA Remover Buffer on ice. In a nuclease-free microcentrifuge tube, prepare the reaction mixture (10 μL) on ice according to the table below. The reaction mixture is as follows:

[0174] Table 2.1 Genomic DNA removal response system

[0175]

[0176] Note: When the template is total RNA, the dosage is 10ng-2μg.

[0177] Subsequently, the system was mixed by pipetting and briefly centrifuged, and then placed on a PCR instrument for reaction. The procedure was as follows: incubate at 42°C for 2 minutes and incubate at 60°C for 5 minutes.

[0178] 2) Reverse transcription reaction

[0179] After the previous step of the reaction is completed, quickly place the system on ice to cool, centrifuge briefly, and add the reverse transcription reaction components as follows:

[0180] Table 2.2 mRNA reverse transcriptional response system

[0181]

[0182]

[0183] Subsequently, use a pipette to mix the system and centrifuge briefly, then place it on a PCR instrument for reaction. The procedure is as follows: incubate at 25°C for 10 minutes, incubate at 50°C for 15 minutes, and then incubate at 85°C for 5 minutes. Then, place the reverse transcription product on ice or refrigerate it for later use.

[0184] Real-time fluorescence quantitative PCR detection reaction system:

[0185] Table 2.3 RT-qPCR reaction system

[0186]

[0187] Subsequently, the system was mixed by pipetting and centrifuged briefly, and then placed on a fluorescent quantitative PCR instrument for amplification detection. The procedure is as follows:

[0188] Table 2.4 RT-qPCR reaction procedures

[0189]

[0190] The running data were obtained, and the final data were analyzed using the 2-△△Ct method and a data analysis report was compiled.

[0191] Q-PCR algorithm (relative quantification, 2-ΔΔCt method)

[0192] algorithm:

[0193] 1) Target gene Ct value - reference gene Ct value,

[0194] 2) Ct value of the test sample - Ct value of the control sample,

[0195] 3) Take the logarithm of the negative value of the difference.

[0196]

[0197]

[0198] 1) Target gene Ct value minus reference gene Ct value, e.g., 4.662 = 20.473 - 15.811;

[0199] 2) Ct value of the test sample minus Ct value of the control sample. If the control sample is sample 1, then subtract 4.662 from all the results in (1);

[0200] 3) Take the base-2 logarithm of the negative value of (2). The Excel function is "=power(2,-0.000)".

[0201] Note: This experiment was performed with three replicate wells, and the final differential expression was obtained by first calculating separately and then averaging.

[0202] Primer sequences

[0203] Table 2.5 Primer pairs and primer sequences required for the experiment

[0204]

[0205] 2. Results and Analysis

[0206] After treatment of each group of cells, the mRNA levels of ASC, NLRP3, and Caspase-1 were detected by RT-qPCR experiments. The results are shown in Figure 2. Figure 9As shown in the results, compared with the Control group, the mRNA levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R group were extremely significantly increased (P < 0.001); compared with the Control group, the mRNA levels of pyroptosis markers ASC and NLRP3 in the H / R+Lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001), and the mRNA level of pyroptosis marker Caspase-1 was increased (P < 0.05), but there was no significant difference; compared with the H / R group, the mRNA level of pyroptosis marker ASC in the H / R+Lycium barbarum polysaccharide group was significantly increased (P < 0.01), and the mRNA levels of NLRP3 and Caspase-1 were extremely significantly increased (P < 0.001). This indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can reduce the mRNA levels of pyroptosis-specific markers ASC, NLRP3, and Caspase-1 in H9C2 cardiomyocytes after H / R.

[0207] Example 7: Protein Detection of Pyroptosis Markers ASC, NLRP3, and Caspase-1

[0208] 1. Method

[0209] Extraction of total cell protein

[0210] 1) Pour off the culture medium and place the bottle upside down on absorbent paper to absorb the culture medium.

[0211] 2) Add 3 mL of 4°C pre-chilled PBS (0.01 M, pH 7.2-7.3) to each flask of cells. Gently shake the flask for 1 minute to wash the cells, then discard the wash solution. Repeat this process two more times, washing the cells three times to remove the culture medium. Discard the PBS and place the flask on ice.

[0212] 3) Add 10 μL PMSF (100 mM) to 1 mL of lysis buffer, shake well, and place on ice.

[0213] 4) Add 400 μL of lysis buffer containing PMSF to each bottle of cells and lyse on ice for 30 minutes. To ensure complete cell lysis, shake the culture bottle frequently.

[0214] 5) After lysis, scrape the cells to the side of the culture flask with a clean scraper, and then use a pipette to transfer the cell fragments and lysate to a 1.5 mL centrifuge tube.

[0215] 6) Centrifuge at 12000 rpm at 4°C for 5 min.

[0216] 7) Transfer the supernatant after centrifugation into 0.5 mL centrifuge tubes and store at -20°C.

[0217] Nuclear and cytoplasmic protein extraction

[0218] 1) Prepare the solutions: Thaw all three reagents in the kit at room temperature. Immediately place on ice and mix thoroughly. Prepare an appropriate amount of Cytoplasmic Protein Extraction Reagent A and add PMSF within a few minutes before use to a final concentration of 1 mM. Prepare an appropriate amount of Nuclear Protein Extraction Reagent and add PMSF within a few minutes before use to a final concentration of 1 mM.

[0219] 2) For adherent cells: Wash once with PBS and scrape off the cells with a cell scraper, or treat the cells with EDTA solution to loosen their adhesion and then pipette off the cells. Collect the cells by centrifugation and aspirate the supernatant as much as possible, reserving the cell pellet for later use. Try to avoid digesting the cells with trypsin to prevent the enzyme from degrading the target protein to be extracted.

[0220] 3) Add 200 μL of Cytoplasmic Protein Extraction Reagent A supplemented with PMSF to every 20 μL of cell pellet. Vortex vigorously at maximum speed for 5 seconds to completely suspend and disperse the cell pellet.

[0221] 4) Incubate on ice for 15 minutes, then add 10 μL of Cytoplasmic Protein Extraction Reagent B. Vortex vigorously at maximum speed for 5 seconds, then incubate on ice for 1 minute.

[0222] 5) Vortex vigorously at maximum speed for 5 seconds, then centrifuge at 12,000 rpm for 5 minutes at 4°C. Immediately transfer the supernatant to a pre-chilled plastic tube. This is the extracted cytoplasmic protein. It can be used immediately or frozen.

[0223] 6) Completely aspirate the remaining supernatant and add 50 μL of nuclear protein extraction reagent supplemented with PMSF. Vortex vigorously at top speed for 30 seconds to completely suspend and disperse the cell pellet. Return the tube to the ice bath and vortex vigorously at top speed for 30 seconds every 2 minutes for a total of 30 minutes.

[0224] 7) Centrifuge at 12,000 rpm for 10 minutes at 4°C. Immediately transfer the supernatant to a pre-chilled plastic tube. This is the extracted nuclear protein. Use immediately or freeze at -70°C. For every 2 million cells lysed with 50 μL of the nuclear protein extraction reagent in this product, the supernatant obtained will have a nuclear protein concentration of approximately 1.2-3.0 mg / mL.

[0225] Determination of protein concentration

[0226] 1) Dilute the protein sample appropriately (take 1 μL of each sample and mix with 19 μL of PBS for dilution, that is, the test sample is diluted 20 times.

[0227] 2) Dilute the BSA standard to prepare standard proteins with protein concentrations of 1, 0.8, 0.6, 0.4, and 0.2.

[0228] 3) Add PBS-diluted protein samples and diluted standard proteins to a 96-well plate, with 20 μL added to each well. The two wells containing PBS serve as blank controls.

[0229] 4) Mix Solution A and Solution B in the BCA kit at a ratio of 50:1. Add 200 μL of the mixture to each well of a 96-well plate. Be careful not to create bubbles to prevent them from affecting the reaction.

[0230] 5) Incubate at 37°C in the dark for 30 min.

[0231] 6) Measure OD570 using a microplate reader.

[0232] 7) Calculate the linear regression equation based on the standard protein concentration and the corresponding OD value, and calculate the sample protein concentration using the regression equation based on the protein sample OD value.

[0233] Protein denaturation

[0234] Add the extracted protein supernatant to 5× protein loading buffer and place in a metal bath for 5 minutes. Check if the sample is translucent and then pipette to see if it is viscous. If it is not translucent or viscous, extend the boiling time. After denaturation, cool to room temperature and store at -20°C.

[0235] electrophoresis

[0236] 1) Preparation of electrophoresis gel

[0237] After cleaning and drying the glass plate, secure it to the gel dispenser and prepare the separating gel. Pour the separating gel into the gaps between the glass plates to the appropriate height. Cover the separating gel with anhydrous ethanol until the gel is completely polymerized. Pour out the anhydrous ethanol, gently rinse with double-distilled water, and blot dry with filter paper. Then, add concentrated gel to the appropriate height and insert the comb. Remove the comb after the concentrated gel is completely polymerized.

[0238] Table 2.6 Separation glue configuration system

[0239]

[0240] Table 2.7 Concentrated glue configuration system

[0241]

[0242]

[0243] 2) Electrophoretic separation

[0244] Mount the prepared gel on the electrophoresis tank and pour electrophoresis solution into the reservoir. Use a micropipette to add the prepared protein sample and MAKER to the sample well. After loading, run the gel at a constant voltage of 80V until the bromophenol blue indicator forms a line at the junction of the stacking gel and the separating gel. Then, switch to a constant voltage of 120V until the bromophenol blue reaches the bottom of the gel. This process takes approximately 1.5 hours.

[0245] Western blot detection

[0246] 1) Transfer: Remove the gel and cut the target band according to the marker. Rinse with distilled water. Cut a PVDF membrane and filter paper of the same size as the PAGE gel. Soak the PVDF membrane in methanol for a few seconds, then soak it and the filter paper in electrotransfer buffer. Arrange the membrane in the order of black plate, fiber pad, filter paper, gel, PVDF membrane, filter paper, fiber pad, and white plate. Clamp the plates and place them in the transfer apparatus, with the black plate facing the black negative electrode. Fill the transfer tank with electrotransfer buffer and begin transfer.

[0247] Transfer conditions:

[0248]

[0249] 2) Blocking: Take out the electroporated PVDF membrane, wash it once with TBST, and block it with 5% milk / TBST on a shaker at room temperature for 1 hour.

[0250] 3) Washing: Rinse with TBST for 2 minutes.

[0251] 4) Binding of primary antibody to target protein: dilute the primary antibody with 1% BSA / PBST (1:1000), seal the membrane with a hybridization bag, and store in a refrigerator at 4°C overnight.

[0252] 5) Washing: Wash three times with PBST, 10 min each time.

[0253] 6) Incubation of secondary and primary antibodies: Place the PVDF membrane in horseradish peroxidase-labeled secondary antibody (1:5000) diluted in 5% milk / PBST and incubate on a shaker at room temperature for 1 h.

[0254] 7) Washing: Wash three times with PBST, 10 min each time.

[0255] 8) Color development: Take equal amounts of Enhanced Luminol Reagent and Oxidizing Reagent, dilute with an appropriate amount of ddH2O, mix thoroughly, and dropwise onto the sealing film. Place the PVDF membrane face down on the luminescent reagents and develop color for 2.0 minutes. Flip the PVDF membrane over and visualize the results using a gel imaging system.

[0256] Appendix 1: The gel used in this experiment is as follows:

[0257]

[0258]

[0259] Appendix 2: The dilution ratio of the primary antibody used in this experiment is as follows:

[0260]

[0261] Appendix 3: The dilution ratio of the secondary antibody used in this experiment is as follows:

[0262]

[0263] 2. Results and Methods

[0264] After treatment of each group of cells, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 were detected by Western blotting. Figure 10 As shown in the results, compared with the Control group, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R group were significantly increased (P < 0.001); compared with the Control group, the protein levels of pyroptosis markers ASC and Caspase-1 in the H / R+Lycium barbarum polysaccharide group were significantly increased (P < 0.01), and the protein level of pyroptosis marker NLRP3 was increased, but there was no significant difference; compared with the H / R group, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R+Lycium barbarum polysaccharide group were significantly decreased (P < 0.001). This indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can reduce the protein levels of pyroptosis-specific markers ASC, NLRP3, and Caspase-1 in H9C2 cardiomyocytes after H / R.

[0265] Example 8: Detection of mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway

[0266] 1. Method

[0267] Experimental groups and treatments

[0268] Control group: H9C2 cardiomyocytes cultured in normal serum-containing medium.

[0269] H / R group: Based on Conclusion 1, the H9C2 cardiomyocyte H / R model was established.

[0270] H / R+ Lycium barbarum polysaccharide group: 90ug / mL Lycium barbarum polysaccharide was pretreated for 24h to establish the H / R model of H9C2 cardiomyocytes.

[0271] Real-time fluorescence quantitative PCR detection of mRNA

[0272] 1) Extract total RNA using Trizol method: Follow the procedure described in Section 2.9.1 of the experimental method in Part 1.

[0273] 2) Reverse transcription into cDNA: Follow the procedures described in Section 2.9.2 of the experimental method in Part I.

[0274] 3) Real-time fluorescence quantitative PCR detection: Follow the procedures described in Section 2.9.3 of the experimental method in Part I.

[0275] Primer sequences

[0276] Table 2.3 Primer pairs and primer sequences required for the experiment

[0277]

[0278] 2. Results and Analysis

[0279] After treatment of each group of cells, the mRNA levels of Nrf2 and HO-1 were detected by RT-qPCR experiments. The results are as follows Figure 11 As shown, compared with the Control group, the mRNA level of HO-1, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R group was significantly increased (P < 0.01), while the mRNA level of Nrf2 was increased, but there was no significant difference. Compared with the Control group, the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in the H / R+Lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001). Compared with the H / R group, the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in the H / R+Lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001). This indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can increase the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in H9C2 cardiomyocytes after H / R.

[0280] Example 9: Protein detection of Nrf2 / HO-1 signaling pathway markers: cytoplasmic Nrf2, nuclear Nrf2, and HO-1

[0281] 1. Method

[0282] The same method as in Case 7 was used.

[0283] 2. Results and Analysis

[0284] After treatment of each group of cells, the protein levels of Nrf2 / HO-1 signaling pathway markers, cytoplasmic Nrf2, nuclear Nrf2, and HO-1, were detected by Western blotting. Figure 12 As shown, compared with the Control group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R group was extremely significantly decreased (P < 0.001), and the protein levels of nuclear Nrf2 and HO-1 were extremely significantly increased (P < 0.001); compared with the Control group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R+lycium barbarum polysaccharide group was extremely significantly decreased (P < 0.001), and the protein levels of nuclear Nrf2 and HO-1 were extremely significantly increased (P < 0.001); compared with the H / R group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R+lycium barbarum polysaccharide group was extremely significantly increased (P < 0.001), and the protein levels of nuclear Nrf2 and HO-1 were extremely significantly increased (P < 0.001). This indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can enable Nrf2 in the cytoplasm to enter the cell nucleus, thereby activating HO-1 and increasing the level of HO-1 protein.

[0285] Example 10: RT-qPCR experiment to select the best siRNA

[0286] 1. Method

[0287] RT-qPCR selection of optimal siRNA

[0288] Real-time fluorescence quantitative PCR detection

[0289] 1) Extract total RNA using Trizol method: Follow the procedure described in 2.9.1 of the first experimental method.

[0290] 2) Reverse transcription into cDNA: Follow the procedure described in Section 2.9.2 of the experimental method in Part I.

[0291] 3) Real-time fluorescence quantitative PCR detection: Follow the procedures described in Section 2.9.3 of the experimental method in Part 1.

[0292] Primer sequences

[0293] Table 2.4 Primer pairs and primer sequences required for the experiment

[0294]

[0295] Table 2.5 siRNA primer pair and primer sequence table required for experiment

[0296]

[0297] 2. Results and Analysis

[0298] After treatment of each group of cells, the mRNA level of Nrf2 was detected by RT-qPCR experiment. The results are as follows Figure 13 As shown, compared with the blank group, the mRNA level of Nrf2 in the NC siRNA group had no significant difference; compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA1 group was decreased (P < 0.05); compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA3 group was extremely significantly decreased (P < 0.001); compared with the Nrf2 siRNA2 group, the mRNA level of Nrf2 in the Nrf2 siRNA1 group was extremely significantly increased (P < 0.001); compared with the Nrf2 siRNA2 group, the mRNA level of Nrf2 in the Nrf2 siRNA3 group was increased (P < 0.05). This showed that the Nrf2 mRNA level in the Nrf2 siRNA2 group was the lowest, and Nrf2 siRNA2 was the best siRNA.

[0299] Example 11: Western blotting experiment to select the best siRNA

[0300] 1. Method

[0301] The same method as in Case 7 was used.

[0302] 2. Results and Analysis

[0303] After treatment of each group of cells, the protein level of Nrf2 was detected by Western blotting experiment. The results are shown in Figure 2. Figure 14As shown in the figure, compared with the blank group, there was no significant difference in the protein level of Nrf2 in the NC siRNA group; compared with the blank group, the protein level of Nrf2 in the Nrf2 siRNA1 group was significantly decreased (P < 0.01); compared with the blank group, the protein level of Nrf2 in the Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the blank group, the protein level of Nrf2 in the Nrf2 siRNA3 group was extremely significantly decreased (P < 0.001); compared with the Nrf2 siRNA2 group, the protein level of Nrf2 in the Nrf2 siRNA1 group was extremely significantly increased (P < 0.001); compared with the Nrf2 siRNA2 group, the protein level of Nrf2 in the Nrf2 siRNA3 group was increased (P < 0.05). This showed that the protein level of Nrf2 in the Nrf2 siRNA2 group was the lowest, which was consistent with the mRNA detection results, and Nrf2 siRNA2 was selected as the optimal siRNA.

[0304] Example 12: Test results of cell viability and LDH activity

[0305] 1. Method

[0306] Experimental groups

[0307] Control group (Control) + NC siRNA

[0308] HR model group + NC siRNA

[0309] HR model group + Lycium barbarum polysaccharide group + NC siRNA

[0310] Control group (Control) + Nrf2 siRNA2

[0311] HR model group + Nrf2 siRNA2

[0312] HR model group+lycium barbarum polysaccharide group+Nrf2 siRNA2

[0313] The Lycium barbarum polysaccharide experimental group was pretreated with Lycium barbarum polysaccharide for 24 hours, then hypoxic for 6 hours and reoxygenated at 37°C overnight for 24 hours, with three replicates per group.

[0314] Cell plating

[0315] Adherent cells: Take normally cultured cells, aspirate the original culture medium, add PBS to wash, and then add trypsin to digest for 3 minutes to terminate the digestion. Use a pipette to blow into single cells, centrifuge the cell suspension at 1000 rpm for 5 minutes, resuspend in culture medium, take 20 μL of cell suspension and add 20 μL of trypan blue for counting, and plate 10,000 cells / well (96-well plate), 100 μL per well. At the same time, set up a blank group, add 100 μL of sterile PBS to the wells around the cell wells, 5% CO2, and culture overnight at 37°C.

[0316] Transfection (6-well plate, 2 mL culture medium)

[0317] 1) siRNA transfection: Take 200 μL jetPRIME buffer and place it into EP tubes, add siRNA at a final concentration of 15 nM, and vortex to mix;

[0318] 2) Add 4 μL jetPRIME reagent, vortex to mix, and incubate at room temperature for 10 min;

[0319] 3) Take 200 μL of the above mixture and add it to the cell culture medium respectively. After 4 hours, replace with fresh culture medium and culture at 37°C with 5% CO2.

[0320] CCK-8 assay

[0321] 1) Add 10 μL of CCK-8 solution to each well and incubate in a 5% CO2, 37°C incubator for 3 h;

[0322] 2) Measure the absorbance OD 450 of each well using a microplate reader;

[0323] 3) Data analysis.

[0324] 2. Results and Analysis

[0325] After each group of cells were treated, the cell viability was detected by CCK-8 and the LDH activity was detected using a kit. The test results were as follows: Figure 15 As shown,

[0326] 1. Compared with the control group + NC siRNA group, the cell survival rate in the H / R model group + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + NC siRNA group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001).

[0327] Compared with the control group + Nrf2 siRNA2 group, the cell survival rate in the H / R model group + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001).

[0328] Compared with the H / R model group + NC siRNA group, the cell survival rate of the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the cell survival rate of the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the cell survival rate of the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); this indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can reduce the cell survival rate of H9C2 cardiomyocytes after H / R.

[0329] 2. Compared with the control group + NC siRNA group, the LDH activity in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the LDH activity in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001).

[0330] Compared with the control group + Nrf2 siRNA2 group, the LDH activity in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the LDH activity in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001).

[0331] Compared with the H / R model group + NC siRNA group, the LDH activity of the H / R model group + LBP + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the LDH activity of the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + LBP + NC siRNA group, the LDH activity of the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); this indicates that pretreatment with 90ug / mL LBP and Nrf2 siRNA2 can increase the LDH activity of H9C2 cardiomyocytes after H / R.

[0332] Example 13: Hoechst 33342 / PI cell apoptosis staining results

[0333] 1. Method

[0334] Same as implementation case 4.

[0335] 2. Results and Analysis

[0336] After treatment of each group of cells, cell apoptosis was detected by Hoechst 33342 / PI double staining. The staining results are shown in Figure 2. Figure 16 As shown, normal cells show weak red fluorescence + weak blue fluorescence; apoptotic cells show weak red fluorescence + strong blue fluorescence; and necrotic cells show strong red fluorescence + weak blue fluorescence.

[0337] Compared with the control group + NC siRNA group, the apoptosis rate in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the apoptosis rate in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001).

[0338] Compared with the control group + Nrf2 siRNA2 group, the apoptosis rate in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the apoptosis rate in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001).

[0339] Compared with the H / R model group + NC siRNA group, the apoptosis rate of the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly reduced (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the apoptosis rate of the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly reduced (P < 0.001); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the apoptosis rate of the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was increased, with no significant difference; this indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2siRNA2 can increase the apoptosis rate of H9C2 cardiomyocytes after H / R.

[0340] Example 14: Detection of inflammatory factors IL-1β and IL-18

[0341] 1. Method

[0342] Same as implementation case 3

[0343] 2. Results and Analysis

[0344] After each group of cells were treated, the inflammatory factors IL-1β and IL-18 in the cell supernatant were detected by ELISA. Figure 17 As shown:

[0345] 1. Compared with the control group + NC siRNA group, the IL-1β level in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the control group + NC siRNA group, the IL-1β level in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was significantly increased (P < 0.01); compared with the H / R model group + NC siRNA group, the IL-1β level in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001).

[0346] Compared with the control group + Nrf2 siRNA2 group, the IL-1β level in the H / R model group + Nrf2 siRNA2 group had no significant difference; compared with the H / R model group + Nrf2 siRNA2 group, the IL-1β level in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001).

[0347] Compared with the H / R model group + NC siRNA group, the IL-1β level in the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the IL-1β level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the IL-1β level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA group was significantly increased (P < 0.01); this indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can increase the level of inflammatory factor IL-1β in H9C2 cardiomyocytes after H / R.

[0348] 2. Compared with the control group + NC siRNA group, the IL-18 level in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the IL-18 level in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001).

[0349] Compared with the control group + Nrf2 siRNA2 group, the IL-18 level in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the IL-18 level in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001).

[0350] Compared with the H / R model group + NC siRNA group, the IL-18 level in the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the IL-18 level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the IL-18 level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was increased (P < 0.05); this indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can increase the level of inflammatory factor IL-1β in H9C2 cardiomyocytes after H / R.

[0351] Example 15: Detection of mRNA of Pyroptosis Markers ASC, NLRP3, and Caspase-1

[0352] 1. Method

[0353] Same as implementation case 6.

[0354] 2. Results and Analysis

[0355] After treatment of each group of cells, the mRNA levels of ASC, NLRP3, and Caspase-1 were detected by RT-qPCR experiments. The results are shown in Figure 2. Figure 18 As shown:

[0356] 1. Compared with the control group + NC siRNA group, the mRNA level of ASC in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the mRNA level of ASC in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was decreased (P < 0.05).

[0357] Compared with the control group + Nrf2 siRNA2 group, the mRNA level of ASC in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the mRNA level of ASC in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001).

[0358] Compared with the H / R model group + NC siRNA group, the mRNA level of ASC in the H / R model group + L. barbarum polysaccharides + NC siRNA group was decreased (P < 0.05); compared with the H / R model group + Nrf2 siRNA2 group, the mRNA level of ASC in the H / R model group + L. barbarum polysaccharides + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + L. barbarum polysaccharides + NC siRNA group, the mRNA level of ASC in the H / R model group + L. barbarum polysaccharides + Nrf2 siRNA2 group was increased, with no significant difference; this indicates that pretreatment with 90ug / mL L. barbarum polysaccharides and Nrf2 siRNA2 can increase the mRNA level of ACS in H9C2 cardiomyocytes after H / R.

[0359] 2. Compared with the control group + NC siRNA group, the mRNA level of NLRP3 in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the mRNA level of NLRP3 in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was decreased, with no significant difference.

[0360] Compared with the control group + Nrf2 siRNA2 group, the mRNA level of NLRP3 in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the mRNA level of NLRP3 in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001).

[0361] Compared with the H / R model group + NC siRNA group, the mRNA level of NLRP3 in the H / R model group + LBP + NC siRNA group was decreased, with no significant difference; compared with the H / R model group + Nrf2 siRNA2 group, the mRNA level of NLRP3 in the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + LBP + NC siRNA group, the mRNA level of NLRP3 in the H / R model group + LBP + Nrf2 siRNA2 group was increased, with no significant difference; this indicates that pretreatment with 90ug / mL LBP and Nrf2 siRNA2 can increase the mRNA level of NLRP3 in H9C2 cardiomyocytes after H / R.

[0362] 3. Compared with the control group + NC siRNA group, the mRNA level of Caspase-1 in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the mRNA level of Caspase-1 in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001).

[0363] Compared with the control group + Nrf2 siRNA2 group, the mRNA level of Caspase-1 in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the mRNA level of Caspase-1 in the H / R model group + Lycium barbarum polysaccharide + Nrf2siRNA2 group was extremely significantly decreased (P < 0.001).

[0364] Compared with the H / R model group + NC siRNA group, the mRNA level of Caspase-1 in the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the mRNA level of Caspase-1 in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the mRNA level of Caspase-1 in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was increased, with no significant difference; this indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can increase the mRNA level of Caspase-1 in H9C2 cardiomyocytes after H / R.

[0365] Example 16: Detection of Nrf2 and HO-1 mRNA markers of the Nrf2 / HO-1 signaling pathway

[0366] 1. Method

[0367] Same as implementation case 6.

[0368] 2. Results and Analysis

[0369] After treatment of each group of cells, the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, were detected by RT-qPCR. Figure 19 As shown:

[0370] Compared with the H / R model group + NC siRNA group, the Nrf2 mRNA level in the H / R model group + LBP + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the Nrf2 mRNA level in the H / R model group + LBP + Nrf2 siRNA2 group was increased, with no significant difference; compared with the H / R model group + LBP + NC siRNA group, the Nrf2 mRNA level in the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001). This indicates that pretreatment with 90ug / mL LBP and Nrf2 siRNA2 can reduce the Nrf2 mRNA level in H9C2 cardiomyocytes after H / R.

[0371] 2. Compared with the H / R model group + NC siRNA group, the HO-1 mRNA level in the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the HO-1 mRNA level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the HO-1 mRNA level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001). This indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can reduce the HO-1 mRNA level in H9C2 cardiomyocytes after H / R.

[0372] Example 17: Protein Detection of Pyroptosis Markers ASC, NLRP3, and Caspase-1

[0373] 1. Method

[0374] Same as implementation case 7.

[0375] 2. Results and Analysis

[0376] After treatment of each group of cells, the protein levels of ASC, NLRP3, and Caspase-1 were detected by Western blotting experiments. The results are shown in Figure 2. Figure 20 As shown:

[0377] 1. Compared with the control group + NC siRNA group, the ASC protein level in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the ASC protein level in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001).

[0378] Compared with the control group + Nrf2 siRNA2 group, the ASC protein level in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the ASC protein level in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001).

[0379] Compared with the H / R model group + NC siRNA group, the ASC protein level in the H / R model group + LBP + NC siRNA group increased less (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the ASC protein level in the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + LBP + NC siRNA group, the ASC protein level in the H / R model group + LBP + Nrf2 siRNA2 group was significantly increased (P < 0.01); this indicates that pretreatment with 90ug / mL LBP and Nrf2 siRNA2 can increase the ACS protein level in H9C2 cardiomyocytes after H / R.

[0380] 2. Compared with the control group + NC siRNA group, the protein level of NLRP3 in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the protein level of NLRP3 in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001).

[0381] Compared with the control group + Nrf2 siRNA2 group, the NLRP3 protein level in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the NLRP3 protein level in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001).

[0382] Compared with the H / R model group + NC siRNA group, the protein level of NLRP3 in the H / R model group + LBP + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the protein level of NLRP3 in the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + LBP + NC siRNA group, the protein level of NLRP3 in the H / R model group + LBP + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); this indicates that pretreatment with 90ug / mL LBP and Nrf2 siRNA2 can increase the NLRP3 protein level in H9C2 cardiomyocytes after H / R.

[0383] 3. Compared with the control group + NC siRNA group, the protein level of Caspase-1 in the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the protein level of Caspase-1 in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.01).

[0384] Compared with the control group + Nrf2 siRNA2 group, the protein level of Caspase-1 in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the protein level of Caspase-1 in the H / R model group + Lycium barbarum polysaccharide + Nrf2siRNA2 group was extremely significantly decreased (P < 0.001).

[0385] Compared with the H / R model group + NC siRNA group, the protein level of Caspase-1 in the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the protein level of Caspase-1 in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.01); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the protein level of Caspase-1 in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); this indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can increase the Caspase-1 protein level in H9C2 cardiomyocytes after H / R.

[0386] Example 18: Protein detection of Nrf2 / HO-1 signaling pathway markers: cytoplasmic Nrf2, nuclear Nrf2, and HO-1

[0387] 1. Method

[0388] Same as implementation case 9.

[0389] 2. Results and Analysis

[0390] After treatment of each group of cells, the protein levels of cytoplasmic Nrf2, nuclear Nrf2, and HO-1 were detected by Western blotting. Figure 21 As shown:

[0391] 1. Compared with the H / R model group + NC siRNA group, the cytoplasmic Nrf2 protein level in the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the cytoplasmic Nrf2 protein level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was increased, with no significant difference; compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the cytoplasmic Nrf2 protein level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001). This indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can reduce the cytoplasmic Nrf2 protein level in H9C2 cardiomyocytes after H / R.

[0392] 2. Compared with the H / R model group + NC siRNA group, the nuclear Nrf2 protein level in the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the nuclear Nrf2 protein level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was increased (P < 0.001); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the nuclear Nrf2 protein level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001). This indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can reduce the nuclear Nrf2 protein level in H9C2 cardiomyocytes after H / R.

[0393] 3. Compared with the H / R model group + NC siRNA group, the HO-1 protein level in the H / R model group + L. barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the HO-1 protein level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was increased (P < 0.001); compared with the H / R model group + L. barbarum polysaccharide + NC siRNA group, the HO-1 protein level in the H / R model group + L. barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001). This indicates that pretreatment with 90ug / mL L. barbarum polysaccharide and Nrf2 siRNA2 can reduce the HO-1 protein level in H9C2 cardiomyocytes after H / R.

[0394] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. Application of Lycium barbarum polysaccharide in the preparation of a drug for inhibiting myocardial cell pyroptosis and a method thereof, characterized in that: The following steps are involved: S1. Establishment of the H9C2 cardiomyocyte hypoxia / reoxygenation model; S2: Exploration of the optimal concentration of Lycium barbarum polysaccharide on H9C2 cardiomyocytes; S3: Cell viability and LDH activity assay results; S4: Hoechst 33342 / PI cell apoptosis staining results; S5: Detection of inflammatory factors IL-1β and IL-18; S6: mRNA detection of pyroptosis markers ASC, NLRP3, and Caspase-1; S7: Protein detection of pyroptosis markers ASC, NLRP3, and Caspase-1; S8: mRNA level detection of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway; S9: Protein detection of cytoplasmic Nrf2, nuclear Nrf2, and HO-1, markers of the Nrf2 / HO-1 signaling pathway; S10: Optimal siRNA selection by RT-qPCR; S11: Optimal siRNA selection by Western blotting; S12: Cell viability and LDH activity detection results; S13: Hoechst 33342 / PI cell apoptosis staining results; S14: Detection of inflammatory factors IL-1β and IL-18; S15: mRNA detection of pyroptosis markers ASC, NLRP3, and Caspase-1; S16: mRNA detection of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway; S17: Protein detection of pyroptosis markers ASC, NLRP3, and Caspase-1.

2. The use and method of Lycium barbarum polysaccharide in preparing a drug for inhibiting myocardial cell pyroptosis according to claim 1, characterized in that: The H9C2 cardiomyocyte hypoxia / reoxygenation model established in S1 includes: S1.

1. Establish a method to place H9C2 cells in an incubator at 37°C containing 95% air and 5% CO2. After incubation for 3 hours, the cells were replaced with a simulated hypoxic solution saturated with high-purity N2 for 30 minutes, with an oxygen partial pressure below 7 kPa, and placed in a sealed container at 37°C containing 95% N2 and 5% CO2 for hypoxia for 0 hours, 3 hours, 6 hours, and 10 hours, respectively, followed by reoxygenation for 24 hours. S1.2 Results and Analysis: Cell morphology of each group was photographed under white light. Cell morphology was damaged at 3, 6, and 10 hours of hypoxia. Cells died in aggregates at 10 hours of hypoxia, and morphological damage was most obvious. FITC Annexin V and PIApoptosis were used. The kit was used to detect the cell apoptosis rate of each group by flow cytometry. The cell apoptosis rate was 2.91% at 0 h of hypoxia, 10.27% at 3 h of hypoxia, 12.87% at 6 h of hypoxia, and 15.05% at 10 h of hypoxia. The cell supernatant was collected and the LDH activity was detected using a kit. Compared with 0 h of hypoxia, the LDH activity of 3 h of hypoxia increased (P < 0.05), the LDH activity of 6 h of hypoxia increased extremely significantly (P < 0.001), and the LDH activity of 10 h of hypoxia increased extremely significantly (P < 0.001). Based on the above results and reference data, the optimal conditions for establishing the H / R model of H9C2 cardiomyocytes are 6 h of hypoxia and 24 h of reoxygenation.

3. The use and method of Lycium barbarum polysaccharide in preparing a drug for inhibiting myocardial cell pyroptosis according to claim 1, characterized in that: The exploration of the optimal concentration of Lycium barbarum polysaccharide in S2 on H9C2 cardiomyocytes includes: S2.

1. Observe the effects of different concentrations of Lycium barbarum polysaccharides on H9C2 cardiomyocytes and divide them into 5 groups: Control group (Control): H9C2 cardiomyocytes were cultured normally without special treatment; Lycium barbarum polysaccharide (30ug / mL): 30ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before the establishment of the H / R model; Lycium barbarum polysaccharide (60ug / mL): 60ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before the establishment of the H / R model; Lycium barbarum polysaccharide (90ug / mL): 90ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before the establishment of the H / R model; Lycium barbarum polysaccharide (120ug / mL): 120ug / mL of Lycium barbarum polysaccharide was added to the culture medium of H9C2 cardiomyocytes 24h before the establishment of the H / R model; S2.

2. Results and analysis: In H9C2 cardiomyocytes, after 24h pretreatment with 0ug / mL, 30ug / mL, 60ug / mL, 90ug / mL, and 120ug / mL of Lycium barbarum polysaccharides, the cell survival rates are shown in Figure 4. The effect on cells was concentration-dependent. Within the concentration range of 30ug / mL to 120ug / mL, the cell survival rate was inversely proportional to the Lycium barbarum polysaccharide concentration. At a concentration of 120ug / mL, there was a significant difference in cell survival rate compared with the blank group. Since 90ug / mL was used as the optimal concentration, the Lycium barbarum polysaccharide would not have a toxic effect on the cells due to excessively high concentrations.

4. The use and method of Lycium barbarum polysaccharide in preparing a drug for inhibiting myocardial cell pyroptosis according to claim 1, characterized in that: The detection results of cell viability and LDH activity in the S3 include: S3.

1. CCK-8 assay for cell viability: H9C2 cardiomyocytes were seeded in a 96-well plate and placed in a cell culture incubator. The culture medium of the 96-well plate was removed, the plates were washed twice with PBS, and 10 μl of CCK-8 and 100 μl of serum-free DMEM medium were added to each well. The 96-well plate was incubated in a dark incubator at 37°C for 2 h. The OD value was measured using a microplate reader at a wavelength of 450 nm. The cell survival rate was calculated according to the formula: cell survival rate (%) = (measured OD value - control OD value) / (standard OD value - blank OD value) × standard concentration × sample dilution factor × 1000 × 100%. S3.

2. Determination of lactate dehydrogenase (LDH) activity: After treatment of each cell group, the cell culture supernatant was collected and the assay was performed according to the kit instructions. The OD value was measured using a microplate reader at a wavelength of 450 nm. The LDH activity was calculated according to the formula: LDH activity (U / L) = (measured OD value - control OD value) / (standard OD value - blank OD value) × standard concentration × sample dilution factor × 1000. S3.3 Results and Analysis After treatment of each group of cells, the cell viability was detected by CCK-8. Compared with the Control group, the cell viability of the H / R group was extremely significantly decreased (P < 0.001), compared with the Control group, the cell viability of the H / R+ Lycium barbarum polysaccharide group was significantly decreased (P < 0.01), and compared with the H / R group, the cell viability of the H / R+ Lycium barbarum polysaccharide group was significantly increased (P < 0.01); LDH activity was detected using a kit. Compared with the Control group, the LDH activity of the H / R group was extremely significantly increased (P < 0.001), compared with the Control group, the LDH activity of the H / R+ Lycium barbarum polysaccharide group was extremely significantly increased (P < 0.001), and compared with the H / R group, the LDH activity of the H / R+ Lycium barbarum polysaccharide group was extremely significantly decreased (P < 0.001). This indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can increase the cell viability of H9C2 cardiomyocytes after H / R and reduce the LDH activity of H9C2 cardiomyocytes after H / R.

5. The use of Lycium barbarum polysaccharide in the preparation of a drug for inhibiting myocardial cell pyroptosis and the method according to claim 1, characterized in that: The Hoechst 33342 / PI apoptosis staining results in S4 include: S4.

1. Cell plating: Take the normally cultured cells, remove the original culture medium, add PBS and then trypsin to digest for 1-3 minutes to terminate the digestion, use a pipette to blow into single cells, centrifuge the cell suspension at 1000 rpm, 4℃ for 5 minutes, and resuspend in culture medium. Take 20 μL of cell suspension and add 20 μL of trypan blue for counting. Add 2*104 cells / well (24-well plate) to a 24-well plate covered with a slide and incubate overnight at 37°C with 5% CO2. For Hoechst-PI apoptosis detection, add 5 μL of Hoechst 33342 staining solution and 5 μL of PI staining solution, mix gently, incubate on ice or at 4°C for 20-30 minutes, and perform subsequent fluorescence detection. S4.

2. Results and analysis. After treatment of each group of cells, cell apoptosis was detected by Hoechst 33342 / PI double staining. Normal cells showed weak red fluorescence + weak blue fluorescence; apoptotic cells showed weak red fluorescence + strong blue fluorescence; necrotic cells showed strong red fluorescence + weak blue fluorescence. Compared with the Control group, the apoptosis rate of cells in the H / R group was extremely significantly increased (P < 0.001). Compared with the Control group, the apoptosis rate of cells in the H / R+lycium barbarum polysaccharide group was increased (P < 0.05). Compared with the H / R group, the apoptosis rate of cells in the H / R+lycium barbarum polysaccharide group was decreased (P < 0.05). This indicates that pretreatment with 90ug / mL of Lycium barbarum polysaccharide can reduce the apoptosis rate of H9C2 cardiomyocytes after H / R.

6. The use and method of Lycium barbarum polysaccharide in preparing a drug for inhibiting myocardial cell pyroptosis according to claim 1, characterized in that: The detection of inflammatory factors IL-1β and IL-18 in S5 includes: S5.1 Collect cell supernatants according to experimental groups and freeze at -20°C for subsequent ELISA testing. Before starting the experiment, all reagents should be equilibrated to room temperature. When preparing reagents or samples, mix thoroughly and avoid foaming as much as possible.

1. Washing: Add 300 μL of wash buffer to each well, let stand for 40 seconds, spin dry, wash the plate three times, and pat the liquid in the wells dry on absorbent paper.

2. Adding samples: Set up blank wells, standard wells, and test sample wells. Add 100 μL of sample diluent to the blank wells and 100 μL of standard or test sample to the remaining wells. Cover the plate with film and incubate at 37°C for 120 minutes.

3. Discard the liquid, spin dry, and wash the plate three times, soaking for 1 minute each time. Add 300 μL of wash buffer to each well, spin dry, and pat dry on absorbent paper.

4. Add 100 μL of diluted Biotin-Conjugate antibody to each well, cover with film, and incubate at 37°C for 1 hour.

5. Discard the liquid in the wells, spin dry, and wash the plate three times, repeating step 3.

6. Add 100 μL of 1× diluted Streptavidin-HRP to each well and incubate at 37°C for 30 min.

7. Discard the liquid in the wells, spin dry, and wash the plate three times as in step 3.

8. Add 90 μL of TMB Substrate to each well and incubate at 37°C for 15 minutes to develop color.

9. Add 50 μL of Stop Solution to each well to terminate the reaction, mix well, and immediately measure OD450. S5.2 Results and Analysis: After treatment, the inflammatory factors IL-1β and IL-18 in the cell supernatant were detected by ELISA. Compared with the control group, the inflammatory factors IL-1β and IL-18 in the H / R group were significantly increased (P < 0.001). Compared with the Control group, the inflammatory factors IL-1β and IL-18 in the H / R+ Lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001); compared with the H / R group, the inflammatory factors IL-1β and IL-18 in the H / R+ Lycium barbarum polysaccharide group were extremely significantly decreased (P < 0.001); this indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can reduce the inflammatory factors IL-1β and IL-18 after H / R in H9C2 cardiomyocytes.

7. The use of Lycium barbarum polysaccharide in the preparation of a drug for inhibiting myocardial cell pyroptosis and the method according to claim 1, characterized in that: The mRNA detection of pyroptosis markers ASC, NLRP3, and Caspase-1 in S6 includes: S6.

1. Extraction of total RNA using Trizol method:

1. Pretreatment of cell samples: Add 1 mL of Trizol reagent, mix thoroughly by pipetting, transfer to an RNase-free 1.5 mL EP tube, and lyse for 10 minutes.

2. Add 200 μL of chloroform, mix vigorously by inversion several times, and let stand at room temperature for 5 minutes.

3. Centrifuge at 4°C, 12,000 rpm, for 15 minutes. Separation of the three phases, upper (RNA), middle (protein), and lower (DNA), will be visible.

4. Transfer the upper aqueous phase (approximately 400 μL) to another fresh 1.5 mL EP tube, add 400 μL of isopropanol, mix thoroughly, and let stand at room temperature for 10 minutes.

5. Centrifuge at 4°C, 12,000 rpm, for 10 minutes. A white RNA precipitate will be visible at the bottom of the tube.

6. Discard the supernatant, add 1 mL of RNase-free 75% ethanol, vortex to mix thoroughly, and centrifuge at 4°C, 10,000 rpm, for 5 minutes.

7. Repeat step 6 once.

8. Discard the supernatant, air-dry the RNA precipitate for 5-10 minutes, and dissolve the precipitate in 20 μL of IX. Take 2 μL of the dissolved RNA and measure OD260, OD280, and OD260 / OD280 using a micro-spectrophotometer to calculate RNA purity and concentration. Estimate RNA quality based on the OD260 / OD280 ratio; a ratio between 1.8 and 2.0 satisfies experimental requirements. Calculate sample RNA concentration based on the absorbance readings using the following formula: Total RNA concentration (μg / μL) = OD260 × 40 × 10⁻³. X. Store the total RNA in a -80°C freezer until ready for use. S6.

2. Reverse transcription into cDNA:

1. To remove genomic DNA, thaw the RNA template, RNAase-free water, and 10×gDNA Remover Buffer on ice. Prepare the reaction system (10 μL) in a nuclease-free microcentrifuge tube. Mix the system by pipetting and centrifuge briefly. Incubate the reaction in a PCR instrument using the following protocol: incubate at 42°C for 2 min and then at 60°C for 5 min.

2. Reverse transcription reaction: After the previous step, quickly place the system on ice to cool, centrifuge briefly, and add reverse transcription reaction 3. Then, use a pipette to mix the system and centrifuge briefly. Place the reaction on a PCR instrument. The procedure is as follows: incubate at 25°C for 10 minutes, incubate at 50°C for 15 minutes, and then incubate at 85°C for 5 minutes. Then, place the reverse transcription product on ice or refrigerate until use. S6.

3. Real-time fluorescence quantitative PCR detection reaction system: Use a pipette to mix the system and centrifuge briefly. Place the system on a fluorescent quantitative PCR instrument for amplification detection to obtain running data. The final data is analyzed using the 2-△△Ct method and a data analysis report is compiled. Q-PCR algorithm (relative quantification, 2-ΔΔCt method) algorithm:

1. Target gene Ct value - reference gene Ct value, 2. Ct value of the test sample - Ct value of the control sample, 3. Take the logarithm of the negative value of the difference; 4. Target gene Ct value - reference gene Ct value, e.g. 4.662 = 20.473 - 15.811; 5. Calculate the Ct value of the test sample minus the Ct value of the reference sample. For example, if the control sample is sample 1, then calculate the Ct value of the target gene minus the Ct value of the reference gene, minus 4.

662. VI. The negative of the Ct value of the test sample minus the Ct value of the control sample was logarithmic to base 2. The Excel function is "=power(2, -0.000)". s6.

4. Results and Analysis. After treatment, the mRNA levels of ASC, NLRP3, and Caspase-1 were detected by RT-qPCR. Compared with the control group, the mRNA levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R group were significantly increased (P < 0.001). Compared with the Control group, the mRNA levels of the pyroptosis markers ASC and NLRP3 in the H / R+Lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001), and the mRNA level of the pyroptosis marker Caspase-1 was increased (P < 0.05), but there was no significant difference; compared with the H / R group, the mRNA level of the pyroptosis marker ASC in the H / R+Lycium barbarum polysaccharide group was significantly increased (P < 0.01), and the mRNA levels of NLRP3 and Caspase-1 were extremely significantly increased (P < 0.001); These results indicate that pretreatment with 90 μg / mL Lycium barbarum polysaccharide can reduce the mRNA levels of pyroptosis-specific markers ASC, NLRP3, and Caspase-1 in H9C2 cardiomyocytes after H / R.

8. The use of Lycium barbarum polysaccharide in the preparation of a drug for inhibiting myocardial cell pyroptosis and the method according to claim 1, characterized in that: The protein detection of pyroptosis markers ASC, NLRP3, and Caspase-1 in S7 includes: S7.

1. Detection Methods 1. Extraction of total cell protein Pour out the culture medium and turn the bottle upside down on absorbent paper to absorb the culture medium dry; add 3 mL of 4°C pre-cooled PBS (0.01M pH 7.2-7.3) to each bottle of cells; place it flat and shake gently for 1 minute to wash the cells, and then discard the washing solution; repeat the above operation twice, wash the cells three times in total to wash away the culture medium; discard the PBS and place the culture bottle on ice; add 10 μL PMSF (100mM) to 1 mL of lysis buffer, shake well and place on ice; add 400 μL of lysis buffer containing PMSF to each bottle of cells and lyse on ice for 30 minutes. To ensure sufficient cell lysis, shake the culture bottle back and forth frequently; after lysis, scrape the cells on one side of the culture bottle with a clean scraper, and then use a gun to transfer the cell fragments and lysis buffer to a 1.5 mL centrifuge tube; centrifuge at 12000 rpm for 5 minutes at 4°C; transfer the supernatant after centrifugation into 0.5 mL centrifuge tubes and store at -20°C; 2. Extraction of nuclear and cytoplasmic proteins; 3. Determination of protein concentration; 4. Protein denaturation; 5. Electrophoresis; 6. Electrophoretic separation; 7. Western blot analysis; s7.2 Results and Methods After treatment of each group of cells, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 were detected by Western blotting experiments. Compared with the Control group, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R group were extremely significantly increased (P < 0.001); compared with the Control group, the protein levels of pyroptosis markers ASC and Caspase-1 in the H / R+Lycium barbarum polysaccharide group were significantly increased (P < 0.01), and the protein level of pyroptosis marker NLRP3 was increased, but there was no significant difference; compared with the H / R group, the protein levels of pyroptosis markers ASC, NLRP3, and Caspase-1 in the H / R+Lycium barbarum polysaccharide group were extremely significantly decreased (P < 0.001); this indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can reduce the protein levels of pyroptosis-specific markers ASC, NLRP3, and Caspase-1 in H9C2 cardiomyocytes after H / R.

9. The use of Lycium barbarum polysaccharide in the preparation of a drug for inhibiting myocardial cell pyroptosis and the method according to claim 1, characterized in that: The detection of mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway in s8 includes: s8.

1. Detection methods 1. Experimental Grouping and Treatment Control group: H9C2 cardiomyocytes cultured in normal serum-containing medium; H / R group: H9C2 cardiomyocyte H / R model was established according to Conclusion 1; H / R+Lycium barbarum polysaccharide group: H9C2 cardiomyocyte H / R model was established by pretreatment with 90 μg / mL Lycium barbarum polysaccharide for 24 h; 2. Real-time fluorescence quantitative PCR detection of mRNA Total RNA was extracted using Trizol as described in Section 2.9.1 of the first experimental method. Reverse transcription to cDNA was performed as described in Section 2.9.2 of the first experimental method. Real-time fluorescence quantitative PCR was performed as described in Section 2.9.3 of the first experimental method. s8.

2. Results and analysis. After treatment of each group of cells, the mRNA levels of Nrf2 and HO-1 were detected by RT-qPCR experiments. Compared with the Control group, the mRNA level of HO-1, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R group was significantly increased (P < 0.01), and the mRNA level of Nrf2 was increased, but there was no significant difference; compared with the Control group, the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in the H / R+lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001); compared with the H / R group, the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in the H / R+lycium barbarum polysaccharide group were extremely significantly increased (P < 0.001); this indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide can increase the mRNA levels of Nrf2 and HO-1, markers of the Nrf2 / HO-1 signaling pathway, in H9C2 cardiomyocytes after H / R.

10. The use of Lycium barbarum polysaccharide in preparing a drug for inhibiting myocardial cell pyroptosis according to claim 8, wherein: Protein detection of Nrf2 / HO-1 signaling pathway markers cytoplasmic Nrf2, nuclear Nrf2, and HO-1 in S9 includes: S9.1, Experimental method, same as S7; S9.

2. Results and analysis. After treatment, the protein levels of cytoplasmic Nrf2, nuclear Nrf2, and HO-1, markers of the Nrf2 / HO-1 signaling pathway, were detected by Western blotting. Compared with the Control group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R group was significantly decreased (P < 0.001), while the protein levels of nuclear Nrf2 and HO-1 were significantly increased (P < 0.001). Compared with the Control group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, in the H / R+lycium barbarum polysaccharide group was significantly decreased. (P < 0.001), and the protein levels of nuclear Nrf2 and HO-1 were extremely significantly increased (P < 0.001). Compared with the H / R group, the cytoplasmic Nrf2, a marker of the Nrf2 / HO-1 signaling pathway, was extremely significantly increased in the H / R+lycium barbarum polysaccharide group (P < 0.001), and the nuclear Nrf2 and HO-1 protein levels were extremely significantly increased (P < 0.001). This indicates that pretreatment with 90 μg / mL of Lycium barbarum polysaccharide can enable Nrf2 in the cytoplasm to enter the cell nucleus, thereby activating HO-1 and increasing the HO-1 protein level. The selection of the best siRNA in the RT-qPCR experiment in S10 includes: S10.

1. RT-qPCR selection of optimal siRNA For real-time fluorescence quantitative PCR, extract total RNA using Trizol as described in Section 1, Experimental Methods, 2.9.1; reverse transcribe to cDNA as described in Section 1, Experimental Methods, 2.9.2; and perform real-time fluorescence quantitative PCR as described in Section 1, Experimental Methods, 2.9.

3. Results and Analysis After treatment of each group of cells, the mRNA level of Nrf2 was detected by RT-qPCR. Compared with the blank group, the mRNA level of Nrf2 in the NC siRNA group was not significantly different; compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA1 group was decreased (P < 0.05); compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the blank group, the mRNA level of Nrf2 in the Nrf2 siRNA3 group was extremely significantly decreased (P < 0.001); compared with the Nrf2 siRNA2 group, the mRNA level of Nrf2 in the Nrf2 siRNA1 group was extremely significantly increased (P < 0.001); compared with the Nrf2 siRNA2 group, the mRNA level of Nrf2 in the Nrf2siRNA3 group was increased (P < 0.05); This showed that Nrf2 The mRNA level of Nrf2 was the lowest in the siRNA2 group, and Nrf2 siRNA2 was selected as the optimal siRNA; The detection results of cell viability and LDH activity in the S12 include: S12.1: Detection Methods Experimental groups: Control group (Control) + NC siRNA; HR model group + NC siRNA; HR model group + Lycium barbarum polysaccharide group + NC siRNA; Control group (Control) + Nrf2 siRNA2; HR model group + Nrf2 siRNA2; HR model group + Lycium barbarum polysaccharide group + Nrf2 siRNA2; Lycium barbarum polysaccharide experimental group was pretreated with Lycium barbarum polysaccharide for 24 hours, then hypoxic for 6 hours and reoxygenated at 37°C overnight for 24 hours, with three replicates per group; Cell plating: Adherent cells: Take normally cultured cells, remove the original culture medium, add PBS to wash, and then add trypsin to digest for 3 minutes to terminate the digestion. Use a pipette to blow into single cells, centrifuge the cell suspension at 1000 rpm for 5 minutes, resuspend in culture medium, take 20 μL of cell suspension and add 20 μL of trypan blue for counting, and plate 10,000 cells / well (96-well plate), 100 μL per well. At the same time, set up a blank group, add 100 μL of sterile PBS to the wells around the cell wells, 5% CO2, and culture overnight at 37°C; Transfection (6-well plate, 2 mL culture medium): siRNA transfection: 200 μL jetPRIME buffer was placed in EP tubes, siRNA was added to a final concentration of 15 nM, and vortexed to mix; 4 μL jetPRIME reagent was added, vortexed to mix, and incubated at room temperature for 10 min; 200 μL of the above mixture was added to the cell culture medium, and fresh medium was replaced after 4 h, incubated at 37°C with 5% CO2; CCK-8 assay: Add 10 μL of CCK-8 solution to each well and incubate in a 5% CO2, 37°C incubator for 3 h. Measure the absorbance (OD 450) of each well with a microplate reader. Analyze the data. S12.2: Results and Analysis After each group of cells were treated, the cell viability was detected by CCK-8 and the LDH activity was detected using a kit. Compared with the control group + NC siRNA group, the cell survival rate in the H / R model group + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + NC siRNA group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); compared with the control group + Nrf2 siRNA2 group, the cell survival rate in the H / R model group + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + Nrf2siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the cell survival rate in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly increased (P < 0.001); Compared with the control group + NC siRNA2 group, the cell survival rate of the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Lycium barbarum polysaccharide + NC siRNA group, the cell survival rate of the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); this showed that pretreatment with 90ug / mL Lycium barbarum polysaccharide and Nrf2 siRNA2 could reduce the cell survival rate of H9C2 cardiomyocytes after H / R; compared with the control group + NC siRNA group, the LDH activity of the H / R model group + NC siRNA group was extremely significantly increased (P < 0.001); compared with the H / R model group + NC siRNA group, the H / R model group + Lycium barbarum polysaccharide + NC The LDH activity in the siRNA group was extremely significantly decreased (P < 0.001); compared with the control group + Nrf2 siRNA2 group, the LDH activity in the H / R model group + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the LDH activity in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); compared with the H / R model group + NC siRNA group, the LDH activity in the H / R model group + Lycium barbarum polysaccharide + NC siRNA group was extremely significantly decreased (P < 0.001); compared with the H / R model group + Nrf2 siRNA2 group, the LDH activity in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly decreased (P < 0.001); Compared with the siRNA group, the LDH activity in the H / R model group + Lycium barbarum polysaccharide + Nrf2 siRNA2 group was extremely significantly increased (P < 0.001); this indicates that pretreatment with 90ug / mL Lycium barbarum polysaccharide and Nrf2 siRNA2 can increase the LDH activity of H9C2 cardiomyocytes after H / R.

Images