Application of phycocyanin in preparation of medicine for reducing expression level of pyroptosis marker

Abstract

The invention discloses application of phycocyanin in preparation of a medicine for reducing the expression level of a pyroptosis marker, and belongs to the technical field of biological medicine. And the pyroptosis marker is one or more of NLRP3, GSDMD, N-GSDMD, Caspase1 and Cleave-caspase1, and the pyroptosis marker is one or more of NLRP3, GSDMD, N-GSDMD, Caspase1 and Cleave-caspase1. The concentration of the phycocyanin is 2.5 to 10 mM. The application of the phycocyanin to preparation of the medicine for reducing the expression level of the pyroptosis marker has the beneficial effects that the phycocyanin can remarkably reduce the protein expression level of the pyroptosis key markers NLRP3, N-GSDMD and Cleved-caspase1, and has a good synergistic effect with NAC (N-acetyl-L-cysteine) (combined use of the phycocyanin and the NAC) for inhibiting pyroptosis, so that the application of the phycocyanin to the preparation of the medicine for reducing the expression level of the pyroptosis marker is realized, and the application of the phycocyanin to the preparation of the medicine for reducing the expression level of the pyroptosis marker is wide. The invention provides a novel research method for reducing pyroptosis and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to use of phycocyanin in preparing a drug for reducing the expression level of cell pyroptosis markers. Background Art

[0002] Pyroptosis, also known as inflammatory necrosis, is a type of programmed cell death, characterized by continuous cell swelling until the cell membrane ruptures, leading to the release of cell contents and activating a strong inflammatory response. Pyroptosis is an important natural immune response of the body and plays an important role in fighting infection and inflammation. Pyroptosis is a programmed cell necrosis mediated by gasdermin. Inhibiting pyroptosis can regulate the body's immune response, which is of great significance for the treatment and prevention of autoimmune diseases. In tumor treatment, inducing tumor cell pyroptosis can enhance anti-tumor immune response and promote tumor regression. Therefore, how to effectively regulate pyroptosis has become a current research hotspot.

[0003] Phycocyanin (C-PC) is a dark blue powder isolated from Spirulina. It is mainly found in cyanobacteria, red algae and cryptophytes. Phycocyanin is usually divided into C-phycocyanin and R-phycocyanin. The former is mainly found in cyanobacteria, while the latter is mainly found in red algae. Cryptophytes contain both. Its function is to absorb light (orange-yellow) energy and transmit light energy. It is not only a protein, but also an excellent natural food pigment and a good health food. However, there is no report on the effect of phycocyanin on the expression level of cell pyroptosis markers. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to provide the use of phycocyanin in influencing cell pyroptosis.

[0005] The present invention solves the above technical problems through the following technical means:

[0006] The first aspect of the present invention proposes the use of phycocyanin in the preparation of a drug for reducing the expression level of a cell pyroptosis marker, wherein the cell pyroptosis marker is one or more of NLRP3, GSDMD, and Caspase1.

[0007] Preferably, the concentration of the phycocyanin is 2.5-10 mM.

[0008] Preferably, the GSDMD comprises N-GSDMD.

[0009] Preferably, the Caspase 1 includes Cleaved-caspase1.

[0010] (The markers of cell pyroptosis are NLRP3, GSDMD and Caspase 1. As cell pyroptosis occurs, GSDMD and Caspase 1 are further broken down into N-GSDMD and Cleaved-Caspase 1. Therefore, the key markers of cell pyroptosis are: NLRP3, N-GSDMD and Cleaved-Caspase 1)

[0011] The second aspect of the present invention proposes the use of phycocyanin and NAC (N-acetyl-L-cysteine) (the two are used in combination) in the preparation of a pharmaceutical composition for inhibiting cell pyroptosis.

[0012] Preferably, the concentrations of phycocyanin and NAC are both 4-6 mM, and more preferably both are 5 mM.

[0013] The third aspect of the present invention proposes the use of hydrogen peroxide or ML385 (NRF2 inhibitor) in the preparation of a drug that promotes cell pyroptosis.

[0014] Preferably, the concentration of hydrogen peroxide is 180-220 μM, more preferably 200 μM.

[0015] Preferably, the concentration of ML385 is 8-12 mM, more preferably 10 mM.

[0016] A fourth aspect of the present invention provides a drug for inhibiting cell pyroptosis, wherein the active ingredients of the drug include phycocyanin and / or NAC (N-acetyl-L-cysteine).

[0017] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0018] Preferably, the pharmaceutically acceptable excipient is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

[0019] The fifth aspect of the present invention provides a drug for promoting cell pyroptosis, wherein the active ingredient of the drug includes hydrogen peroxide or ML385.

[0020] The beneficial effects of the present invention are:

[0021] 1. The present invention proposes the use of phycocyanin in the preparation of a drug for reducing the expression level of cell pyroptosis markers. Phycocyanin can significantly reduce the protein expression levels of cell pyroptosis markers NLRP3, GSDMD, and Caspase1, and has a good synergistic effect with NAC (N-acetyl-L-cysteine) (the two are used in combination) for inhibiting cell pyroptosis, providing a new research method for inhibiting cell pyroptosis and having good application prospects.

[0022] 2. The present invention also found that a certain concentration of hydrogen peroxide and ML385 (NRF2 inhibitor) can effectively promote cell pyroptosis, which is also of certain importance for effectively regulating cell pyroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The immunoblot images of oxidative stress and pyroptosis markers NRF2, NQO-1, NLRP3, GSDMD, N-GSDMD, Caspase 1 and Cleaved-caspase1 in ovarian tissues of all groups of mice in Example 1 of the present invention (left) and the Image J quantitative statistical graph of the corresponding proteins (right);

[0024] Figure 2 The immunofluorescence staining images of oxidative stress and pyroptosis markers NRF2, N-GSDMD and NLRP3 in ovarian tissues of all groups of mice in Example 1 of the present invention;

[0025] Figure 3 The immunoblot image of the oxidative stress marker NRF2 protein expression in human ovarian granulosa cells of all groups in Example 2 of the present invention (left figure) and the corresponding Image J quantitative statistical graph of NRF2 protein (right figure);

[0026] Figure 4 This is a statistical graph of the detection of cell viability of human ovarian granulosa cells in all groups of Example 2 of the present invention;

[0027] Figure 5 These are immunofluorescence staining images of the oxidative stress marker NRF2 (left), pyroptosis marker NLRP3 (middle) and N-GSDMD (right) proteins of human ovarian granulosa cells in all groups of Example 2 of the present invention;

[0028] Figure 6 The scanning electron micrographs of human ovarian granulosa cells of all groups in Example 2 of the present invention (left, used to observe cell pyroptosis, including increase in cell diameter) and the quantitative statistical graph of the corresponding cell diameter (right);

[0029] Figure 7 The protein expression diagram of oxidative stress and pyroptosis markers of all groups of human ovarian granulosa cells treated with phycocyanin, NAC, and the combination of the two (left figure) and the corresponding protein Image J quantitative statistical diagram (right figure);

[0030] Figure 8 The protein expression diagram of oxidative stress and pyroptosis markers of all groups of human ovarian granulosa cells treated with phycocyanin, hydrogen peroxide, and the combination of the two (left figure) and the corresponding protein Image J quantitative statistical diagram (right figure);

[0031] Fig. 9 The expression graph of oxidative stress and pyroptosis marker proteins of all groups of human ovarian granulosa cells treated with phycocyanin alone or in combination with ML385 (left) and the corresponding protein Image J quantitative statistical graph (right) are shown in Example 3 of the present invention.

[0032] In the figure, “*” represents p<0.05; “**” represents p<0.01; “***” represents p<0.001. “#” represents p<0.05; “##” represents p<0.01; “###” represents p<0.001; “&” represents p<0.05; “&&” represents p<0.01; “&&&” represents p<0.001; “+” represents presence, and “-” represents absence. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0035] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

[0036] Embodiment 1:

[0037] Constructing animal models to study the effects of the natural pigment phycocyanin (C-PC) on key markers of cell pyroptosis:

[0038] Construction of PCOS mouse model: 8-week-old ICR mice were used as research subjects (10 mice per group). After 1 week of adaptive feeding, ICR mice were induced to suffer from PCOS by subcutaneous injection of dehydroepiandrosterone (DHEA, 6 mg / 100 g body weight, 0.1 mL / mouse) for 21 consecutive days. Then, phycocyanin (20 mg / kg, 0.1 mL per mouse dissolved in saline) was injected intraperitoneally for 4 consecutive weeks. At the end of the experiment, the mice were killed for subsequent analysis;

[0039] At the same time, a control group was set up: PCOS control mice were subcutaneously injected with dehydroepiandrosterone (DHEA, 6mg / 100g body weight, 0.1mL / mouse) for 21 consecutive days to induce PCOS in ICR mice, and normal saline was injected intraperitoneally for 4 consecutive weeks (0.1ml per mouse). The normal control mice were given intraperitoneal injections of normal saline (0.1ml per mouse) for 4 consecutive weeks during the same period.

[0040] Western blotting was used to detect the application of phycocyanin in improving the oxidative stress (NRF2) and cell pyroptosis (NLRP3 / GSDMD / N-GSDMD / Caspase 1 / Cleaved-caspase 1) pathways in PCOS mice.

[0041] a. Extract protein from the ovarian tissues of normal mice, PCOS mice, and PCOS mice treated with phycocyanin. Add pre-cooled protein extraction reagent containing inhibitors (a mixture of protease inhibitors, PMSF, and phosphatase inhibitors) to the tissues of the three groups, and lyse on ice for 20 minutes. Centrifuge the lysate at 12,000 rpm in a pre-cooled centrifuge for 15 minutes. Discard the precipitate, take the supernatant (protein of each group) and immediately transfer it to a new centrifuge tube, add Loading Buffer at a ratio of supernatant: Loading Buffer = 4:1, mix thoroughly, boil at 100℃ for 10 minutes, and store at -80℃ for later use.

[0042] b. Western blotting was used to detect markers of oxidative stress and cell pyroptosis caused by the accumulation of cellular inflammatory factors. The protein samples of the three groups were separated on a 10%-15% SDS-polyacrylamide gel electrophoresis gel, then transferred to a PVDF membrane, and detected with antibodies to NRF2, NQO-1, NLRP3, GSDMD, N-GSDMD, Caspase 1, and cleaved-caspase1, which are related indicators in the oxidative stress and cell pyroptosis pathways.

[0043] The results are as follows Figure 1 and 2 As shown in the data, compared with the normal group mice, the expression levels of key markers of cell pyroptosis NLRP3, GSDMD, N-GSDMD, Caspase 1 and cleaved-caspase1 in the ovaries of PCOS group mice were significantly increased, while the expression levels of key markers of cell pyroptosis in the ovarian tissues of PCOS mice treated with phycocyanin (C-PC) were significantly decreased.

[0044] Embodiment 2:

[0045] Construct a cell model to study the effect of natural pigment phycocyanin (C-PC) on key markers of cell pyroptosis:

[0046] (1) Construction of human ovarian granulosa cell inflammatory injury model in vitro: Human ovarian granulosa cell line (KGN, ATCC, RRID: CVCL_0603) was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (4.5 g / L D-glucose, HyClone, USA) supplemented with 10% FBS (Gibco, USA), penicillin (100 IU / mL) and streptomycin sulfate (100 μg / mL). The cell culture medium was renewed every 2-3 days, and the culture was cultured in a fully humidified incubator at 37°C and 5% CO2. The cells were pretreated with 5 μM phycocyanin for 2 hours, then treated with 100 ng / mL LPS for 5 hours, and then treated with ATP (5 mM) for 1 hour. A control group was also set up: the human ovarian granulosa cells in the control group were treated with 100 ng / mL LPS for 5 hours, and then treated with ATP (5 mM) for 1 hour.

[0047] a. 1×10 4 Human ovarian granulosa cells KGN were inoculated in a 35mm confocal microscope cell culture dish. When the cells were completely attached to the wall, different concentrations of phycocyanin solution were added. The phycocyanin concentration gradient was set to 2.5mM, 5mM and 10mM. After pre-treatment with phycocyanin for 2 hours, the cells were then treated with 100ng / mL LPS for 5 hours, and then treated with ATP (5mM) for 1 hour. It included a normal group, an LPS / ATP-induced damage group, and an LPS / ATP group after phycocyanin treatment (phycocyanin experimental group). Proteins from each group were extracted separately (same method as above), and the NRF2 protein expression level of each group was checked in turn. Figure 3 As shown, with the increase of phycocyanin concentration, the expression level of NRF2 protein increased, but the expression level of 5mM phycocyanin NRF2 was significantly higher than that of 10mM phycocyanin NRF2, so 5mM phycocyanin was selected for subsequent experiments.

[0048] b. CCK-8 assay for cell viability: CCK-8 assay was used to assess cell viability. Human ovarian granulosa cells (1×10 4 ) were seeded into 96-well plates. The drugs were administered 24 hours before the addition of CCK-8 reagent (10 μL / well). One hour later, the absorbance was measured using a microplate reader and the values ​​of each group were counted. Figure 4 As shown, the cell viability was significantly decreased after LPS / ATP treatment, and the viability of human ovarian granulosa cells was significantly increased after 5mM phycocyanin treatment.

[0049] (2) After human ovarian granulosa cells were treated with phycocyanin, the examination indicators included: immunofluorescence examination of the expression of the oxidative stress indicator NRF2, immunofluorescence detection of the expression of cell pyroptosis markers NLRP3 and N-GSDMD, and protein immunoblotting detection of oxidative stress and cell pyroptosis markers.

[0050] Step 1: 1×10 4 Human ovarian granulosa cells KGN were inoculated in a 35mm confocal microscope cell culture dish. When the cells were completely attached to the wall, different concentrations of phycocyanin solution were added. The phycocyanin concentration gradient was set to 2.5mM, 5mM and 10mM. After phycocyanin pretreatment of cells for 2 hours, they were then treated with 100ng / mL LPS for 5 hours, followed by ATP (5mM) for 1 hour. The human ovarian granulosa cell group included a normal group, an LPS / ATP-induced injury group, and an LPS / ATP group treated with phycocyanin.

[0051] Step 2: After the cell treatment, wash the cells gently with 1×PBS to remove the residual culture medium and dead cells; fix with 4% formaldehyde at room temperature for 20 minutes. After fixation, wash with TBS-Tx to remove the residual fixative; add an appropriate amount of blocking solution to completely cover the coverslip, and block at room temperature for 20 minutes;

[0052] Step 3: Use tweezers to take out the coverslip with fixed cells and place it in a light-proof humidified box with the cell attached side facing up; aspirate the blocking solution, dilute the primary antibody with antibody diluent AbDil-Tx, take 50 μL and drop it on the coverslip, and incubate according to the instructions; after the primary antibody incubation is completed, aspirate and discard the primary antibody, and wash away the unbound primary antibody with TBS-Tx; according to the properties of the primary antibody, select the fluorescent secondary antibody, dilute the secondary antibody with AbDil-Tx at a ratio of 1:250, take 50 μL of the secondary antibody and drop it on the coverslip, incubate at room temperature for 1 hour, and wash away the unbound secondary antibody with TBS-Tx;

[0053] Step 4. After staining with the antibody to be labeled, take 50 μL of 1 μg / mL DAPI to stain the nucleus for 5 minutes. After staining, wash off the unbound DAPI with PBS; wipe the slide clean, add a drop of anti-fluorescence quenching mounting medium, turn the coverslip upside down on the mounting medium, so that the cell surface contacts the mounting medium; select the corresponding fluorescent channel, observe under the microscope, and take representative pictures.

[0054] The results are as follows Figure 5 As shown, compared with the normal group cells, the expression levels of NLRP3 and N-GSDMD, the key markers of pyroptosis, in the LPS / ATP-induced injury group cells were significantly increased, while the expression levels of the key markers of pyroptosis in the LPS / ATP group cells treated with phycocyanin (C-PC) were significantly decreased.

[0055] (3) Observation of cell pyroptosis using scanning electron microscopy

[0056] Step 1: First, remove the cells attached to the coverslip from the cell culture dish and gently wash them with PBS (phosphate buffered saline) for 3 times, 10 minutes each time. Then, fix them with 2.5% glutaraldehyde at 4°C overnight to maintain the cell morphology.

[0057] Step 2: Wash the fixed cells with PBS 3 times, 10 minutes each time, to remove the residual glutaraldehyde. Fix with 1% osmium acid at 4°C for 2 hours to enhance the morphological fixation of the cells. Then wash with PBS 3 times, 10 minutes each time.

[0058] Step 3: Use ethanol to dehydrate step by step, from 30% to 100% concentration, soak for 15 minutes at each concentration. Finally, soak in isoamyl acetate for more than 20 minutes. Use a critical point dryer to dry to avoid shrinkage and deformation of the sample during the drying process. Place the dried sample on an ion sputtering instrument for gold spraying, usually spraying a 50-300 angstrom thick metal film to improve the conductivity of the sample.

[0059] Step 4: Place the processed sample under a scanning electron microscope for observation and photography.

[0060] The results are as follows Figure 6 As shown, the degree of pyroptosis of human ovarian granulosa cells after LPS / ATP treatment was significantly increased, and the degree of pyroptosis of human ovarian granulosa cells after 5mM phycocyanin (C-PC) treatment was significantly reduced.

[0061] Embodiment 3:

[0062] Further research on the regulation of cell pyroptosis by the natural pigment phycocyanin (C-PC):

[0063] GCs were seeded in 6-well plates and waited for the cells to fully adhere to the wall. Human ovarian granulosa cells were pretreated with 5mM NAC (N-acetyl-L-cysteine), 200μM hydrogen peroxide (H2O2) and 10mM ML385 (NRF2 protein inhibitor) for 2 hours, and then pretreated with phycocyanin (5mM) for 2 hours, and then treated with 100ng / mL LPS for 5 hours, followed by treatment with ATP (5mM) for 1 hour, and protein immunoblotting was performed after 0.25% trypsin digestion.

[0064] (1) Research on inhibition of cell pyroptosis:

[0065] The combination of phycocyanin and NAC can significantly inhibit cell pyroptosis. Figure 7As shown in the figure, through western blot detection, we found that phycocyanin + LPS / ATP treatment can effectively reduce the protein expression levels of key markers of cell pyroptosis NLRP3, N-GSDMD and Cleaved-caspase 1. Furthermore, when phycocyanin was combined with NAC, the expression level of pyroptosis markers in human ovarian granulosa cells treated with LPS / ATP was significantly reduced, and the combined effect was better than the effect of phycocyanin and NAC alone. It can be seen that the combination of phycocyanin and NAC has a significant effect.

[0066] (2) Promote cell pyroptosis research:

[0067] a. Hydrogen peroxide (H2O2) promotes cell pyroptosis

[0068] The results are as follows Figure 8 As shown, through protein immunoblotting detection, we found that hydrogen peroxide + LPS / ATP treatment can effectively increase the protein expression levels of cell pyroptosis markers NLRP3, N-GSDMD and Cleaved-caspase 1, indicating that hydrogen peroxide has the effect of promoting cell pyroptosis.

[0069] b. ML385 (NRF2 protein inhibitor) promotes cell pyroptosis

[0070] The results are as follows Fig. 9 As shown in the figure, protein immunoblotting detected that ML385+LPS / ATP treatment could effectively increase the protein expression levels of cell pyroptosis markers NLRP3, N-GSDMD and Cleaved-caspase 1, indicating that ML385 has the effect of promoting cell pyroptosis.

[0071] In the above animal and cell experiments, the data are expressed as mean ± standard deviation. Data difference analysis was performed by GraphPadPrism V6.02 and t-test. Animal experiments: between PCOS group and normal group mice, between PCOS group mice treated with phycocyanin and PCOS mice. Cell experiments: between LPS / ATP induced group and normal control group, between LPS / ATP induced group and LPS / ATP induced group after phycocyanin pretreatment.

[0072] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of phycocyanin in the preparation of a drug for reducing the expression level of cell pyroptosis markers, characterized in that: The cell pyroptosis marker is one or more of NLRP3, GSDMD, and Caspase1.

2. The use according to claim 1, characterized in that: The concentration of the phycocyanin is 2.5-10 mM.

3. The use according to claim 1, characterized in that: The GSDMD includes N-GSDMD; and the Caspase1 includes Cleaved-caspase1.

4. Use of phycocyanin and NAC (N-acetyl-L-cysteine) in the preparation of a pharmaceutical composition for inhibiting cell pyroptosis.

5. The use according to claim 4, characterized in that: The concentrations of phycocyanin and NAC are both 4 to 6 mM, and more preferably both are 5 mM.

6. Use of hydrogen peroxide or ML385 in the preparation of a drug that promotes cell pyroptosis, characterized in that: The concentration of the hydrogen peroxide is 180-220 μM, more preferably 200 μM; the concentration of the ML385 is 8-12 mM, more preferably 10 mM.

7. A drug for inhibiting cell pyroptosis, characterized in that: Its active ingredients include phycocyanin and / or NAC (N-acetyl-L-cysteine).

8. The drug according to claim 7, characterized in that: The medicine also includes pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that: The pharmaceutically acceptable pharmaceutical excipient is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

10. A drug for promoting cell pyroptosis, characterized in that: Its active ingredients include hydrogen peroxide or ML385.

Images