Use of reb b in the preparation of a medicament for treating lung ischemia reperfusion injury

Abstract

The application discloses application of Reb B in preparation of a medicine for treating lung ischemia-reperfusion injury. The application screens a natural compound Reb B by establishing an alveolar type II cell A549 ischemia-reperfusion model and combining a high-throughput drug screening mode. The Reb B can significantly enhance the OGD / R tolerance of the A549 cell by inhibiting a mitochondrial apoptosis pathway, and provides a theoretical basis for clinical application of the Reb B in ischemia-reperfusion related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to the application of Reb B in the preparation of drugs for treating pulmonary ischemia-reperfusion injury. Background Technology

[0002] Lung ischemia-reperfusion injury (LIRI) has become a well-established cause of postoperative complications in clinical lung surgery. Exploring targeted prevention and treatment measures to mitigate or avoid LIRI has been a key focus of research both domestically and internationally. From the perspective of Traditional Chinese Medicine (TCM), LIRI manifests as qi deficiency and blood stasis. Due to ischemia, local qi and blood supply is insufficient, impairing the lung's function of governing qi. During reperfusion, the lung's propulsive force is weak, leading to poor qi and blood circulation, accumulation of stasis, and prolonged stasis resulting in toxin buildup, causing apoptosis and even necrosis of alveolar epithelial and vascular endothelial cells. High-throughput drug screening is currently the main method for new drug development, and screening sample libraries are an important component of drug screening.

[0003] Compared to combinatorial chemistry synthesis libraries and drug derivative libraries, natural compound libraries have significant advantages in terms of the number and diversity of compounds. Statistics show that more than half of currently marketed drugs are derived from natural products. Between 1981 and 2002, more than a quarter of the new drugs approved by the U.S. Food and Drug Administration were derivatives of natural products. With societal development, people are increasingly concerned about the negative impacts of chemical drugs on human health and the environment. Returning to nature and protecting the environment has become a trend in addressing the relationship between humanity and the environment. The development of natural medicines is an inevitable trend in new drug research and development and is currently an important source of compounds in the field. Summary of the Invention

[0004] In view of this, the present invention addresses the problems existing in the prior art by providing an application of Reb B in the preparation of drugs for treating pulmonary ischemia-reperfusion injury. Rebaudioside B (Reb B) can significantly enhance the tolerance of A549 cells to OGD / R by inhibiting the mitochondrial apoptosis pathway, providing a theoretical basis for the clinical application of Reb B in ischemia-reperfusion-related diseases.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides the use of Reb B in the preparation of drugs for treating pulmonary ischemia-reperfusion injury.

[0007] Furthermore, the molecular formula of Reb B is C 38 H 60 O18 Its chemical structure is:

[0008]

[0009] Furthermore, Reb B can reduce ischemia-reperfusion injury in alveolar type II cells A549.

[0010] Furthermore, Reb B exerted a protective effect in the A549 cell OGD / R model by inhibiting the mitochondrial apoptosis pathway.

[0011] The second invention provides a drug for preparing a treatment for pulmonary ischemia-reperfusion injury, comprising Reb B.

[0012] Furthermore, the drug is in the form of powder, capsule, injection, or tablet.

[0013] Compared with the prior art, the present invention can achieve the following technical effects:

[0014] This invention establishes an ischemia-reperfusion model of type II alveolar cells (A549) and, combined with high-throughput drug screening, identifies a natural compound, Reb B, which significantly enhances the ischemia-reperfusion tolerance of A549 cells. Therefore, this invention can provide a candidate drug with clinical application value for the treatment of pulmonary ischemia-reperfusion. Attached Figure Description

[0015] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0016] Figure 1 This is a flowchart of high-throughput natural compound screening;

[0017] Figure 2 The percentage of compounds at different cell viability levels in the A549 cell OGD12h / R12h model is shown.

[0018] Figure 3 This is a graph showing the results of Caspase 3 activity detection by ELISA.

[0019] Figure 4 The Hoechst 33258 fluorescence staining method was used to detect the effect of Reb B on apoptosis in A549 cells;

[0020] Figure 5 shows the results of Western blot analysis of apoptosis-related protein expression. Detailed Implementation

[0021] The following will describe the implementation of the present invention in detail with reference to the embodiments, so that the process of how the present invention uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0022] In recent years, the use of high-throughput synthetic small molecule libraries for drug screening has led to the discovery of a large number of new bioactive molecules. Based on the initial screening, secondary screening indicators are added to ultimately identify lead compounds with significant therapeutic effects, which can be used for drug development, with a focus on validating molecular mechanisms and studying their biological processes.

[0023] The purpose of this invention is to discover a natural compound that can enhance lung ischemia-reperfusion tolerance through high-throughput drug screening. Experiments have shown that Rebaudioside B can effectively reduce ischemia-reperfusion injury in alveolar type II cells (A549).

[0024] This invention provides a natural compound that can inhibit apoptosis in the A549 ischemia-reperfusion model, named Rebaudioside B (Reb B), with the molecular formula C. 38 H 60 O 18 Rebaudioside B is a natural compound extracted from the leaves of stevia rebaudianabertoni. It has poor water solubility, with a water solubility of only 0.01-0.02% at room temperature. This invention is the first to apply Rebaudioside B to an A549 ischemia-reperfusion model. Through a series of molecular biology techniques, it was found that Rebaudioside B can significantly inhibit apoptosis in A549 cells during ischemia-reperfusion.

[0025] This invention utilizes A549 alveolar epithelial cells to establish an oxygen-glucose deprivation / reperfusion (OGD / R) model to simulate pulmonary ischemia-reperfusion injury during clinical cardiopulmonary bypass. A preliminary screening of highly active drugs was conducted from a library of 2661 novel small molecule compounds using the CCK8 assay. Subsequent screening was performed by measuring Caspase3 activity to determine the optimal compound, rebaudioside B (Reb B). A concentration gradient for Rebaudioside B (Reb B) was established, and the appropriate drug concentration was determined using CCK8 assay and Hoechst 33258 staining. Western blotting and RT-qPCR were used to detect the mRNA transcription and protein expression levels of Bcl2, Bax, Caspase3, and Cleaved Caspase3. The results showed that the mRNA and protein expression levels of the pro-apoptotic genes Bax and Caspase3 in the OGD / R group were higher than those in the OGD / R+Reb B group. Furthermore, the mRNA and protein expression levels of the anti-apoptotic gene Bcl2 were significantly lower in the OGD / R+Reb B group. This invention has found that Rebaudioside B (Reb B) can significantly enhance the OGD / R tolerance of A549 cells by inhibiting the mitochondrial apoptosis pathway, providing a theoretical basis for the clinical application of Reb B in ischemia-reperfusion-related diseases.

[0026] Experimental methods

[0027] (I) Establishment and grouping of the A549 alveolar epithelial cell oxygen-glucose deprivation / reperfusion (OGD / R) model

[0028] A549 cells in logarithmic growth phase were replaced with glucose-free and serum-free DMEM medium and incubated in a tri-gas incubator (1% O2, 5% CO2) for 12 h. Subsequently, the glucose-free and serum-free DMEM medium was replaced again with DMEM complete medium, and the cells were reoxygenated in a normoxic incubator (21% O2, 5% CO2) for 24 h. The cells were then divided into a control group (con), an OGD / R group, and an OGD / D+Reb B group.

[0029] (II) Screening of high-throughput natural compound libraries

[0030] 2661 natural compounds were purchased from Selleck (USA). A549 cells were seeded in 96-well plates (5 × 10⁶ cells / well). 3 In A549 cells, an oxygen-glucose deprivation / reperfusion (OGD / R) model was established when the cells were in the logarithmic growth phase. 2661 compounds were added to each well at a concentration of 10 μM. After the experiment, the CCK8 absorbance value of each well was measured.

[0031] (III) CCK-8 assay for cell viability

[0032] After the A549 cell oxygen-glucose deprivation / reperfusion (OGD / R) model was established, the 96-well plate was removed from the incubator, the old culture medium was discarded, and 100 μl of CCK8 mixture was added to each well of the 96-well plate. The plate was then incubated at 37°C for 30 min in the dark, and the absorbance was measured at 450 nm using a microplate reader.

[0033] (iv) Measurement of caspase 3 activity in A549 cells

[0034] After establishing the A549 cell oxygen-glucose deprivation / reperfusion (OGD / R) model, cells were collected in 1.5 mL EP tubes, washed twice with pre-chilled PBS, and the supernatant was aspirated. 100 μL of cell lysis buffer was added to each EP tube, and the cells were resuspended and lysed on ice for 30 min. The cells were then centrifuged at 12000 rpm at 4°C for 15 min. Caspase 3 levels were detected using an ELISA kit, and the relative proportions of Caspase 3 activity in each group were compared with the control group.

[0035] (V) Hoechst 33258 staining to detect morphological changes in A549 cells

[0036] After the A549 cell oxygen-glucose deprivation / reperfusion (OGD / R) model was established, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde solution for 15 min. The fixative was then discarded, and the cells were washed three times with PBS solution for 3 min each time. Finally, 500 μl of Hoechst 33258 staining solution was added and stained for 8 min. The staining of A549 cell nuclei was observed under a fluorescence inverted microscope.

[0037] (VI) Western blot analysis of the expression of target proteins in A549 cells

[0038] After establishing the A549 cell oxygen-glucose deprivation / reperfusion (OGD / R) model, total protein was extracted from A549 cells using cell lysis buffer. The protein samples were then heated to boiling and centrifuged. Polyacrylamide gel electrophoresis (SDS-PAGE) was performed. After electrophoresis, the membrane was transferred at a constant current of 200 mA for 90 min, blocked with 10% skim milk at room temperature for 1 h, and incubated overnight at 4°C after adding primary antibody. The next day, secondary antibody was added and incubated at room temperature for 1 h. ECL luminescent agent was added for development, and images were acquired using an automated gel imaging system.

[0039] Experimental results

[0040] 1. High-throughput screening of natural compounds

[0041] The initial screening involved determining the cell viability of each CCK8 well to identify compounds with high activity. The secondary screening involved determining the caspase 3 activity of each well to identify the target compounds. See the flowchart for details. Figure 1 .

[0042] 2. CCK8 assay was used to detect the proliferation of A549 cells in each group.

[0043] CCK8 assay results showed that the viability of A549 cells in the OGD / R group was below 90% after intervention with 98.8% of the compounds, and only the viability of cells significantly improved after intervention with 1.2% of the compounds. Figure 2 .

[0044] 3. Detection of Caspase 3 activity using ELISA method

[0045] Thirty-two molecular compounds that could increase the viability of A549 cells in the OGD / R model to over 90% were selected from the initial screening for secondary screening. The expression level of Caspase 3 in the A549 cell OGD / R model treated with these 32 molecular compounds was detected using a Caspase 3 ELISA kit. The results revealed the presence of a compound, Rebaudioside B (Reb B), in stevia leaves, with the molecular formula C... 38 H 60 O 18 It is one of the steviol glycosides (SGs) found in stevia leaves, and its chemical structure is as follows:

[0046]

[0047] The Caspase 3 level in the A549 cell OGD / R model treated with Reb B was significantly lower than that in the A549 cell OGD / R model. Figure 3 P < 0.001 (OGD / R + Reb B group vs. OGD / R). Therefore, Rebaudioside B was chosen as the research subject.

[0048] 4. Hoechst 33258 staining was used to detect morphological changes in A549 cells.

[0049] In the control group, A549 cells exhibited uniform chromatin distribution in their nuclei and a uniform pale blue fluorescence. Compared to the control group, A549 cells in the OGD / R group showed chromatin condensation in their nuclei and exhibited varying degrees of strong blue fluorescence. The apoptosis rate in the OGD / R group was significantly higher than that in the control group. Figure 4 ###P<0.001; OGD / R group vs. control group). Compared with the OGD / R group, the apoptosis rate of the OGD / R+RebB group was significantly reduced (P<0.001). Figure 4 , ###P<0.001; OGD / R+Reb group B vs. OGD / R).

[0050] 5. Western blot detection of apoptosis-related protein expression

[0051] To investigate whether Reb B exerts an anti-apoptotic effect in the A549 cell OGD / R model, protein expression in the mitochondrial pathway was detected by Western blot. Figure 5A Western blot results showed that the relative expression levels of Caspase3 protein in the control group, OGD / R group, and OGD / R+Reb B group were 0.27±0.03, 1.30±0.08, and 0.88±0.04, respectively. The relative expression levels of Cleaved Caspase3 protein were 0.23±0.01, 1.43±0.08, and 0.50±0.07, respectively. The relative expression levels of Bax / Bcl2 protein were 0.66±0.06, 1.26±0.11, and 0.74±0.02, respectively. Compared with the control group, the expression rates of Caspase3, Cleaved Caspase3, and Bax / Bcl2 were significantly increased in the OGD / R group. Figure 5B , 5C 5D; ***P<0.001, ***P<0.001, **P<0.01; OGD / R group vs. control group). After Reb B intervention, the expression of Caspase 3 and Cleaved Caspase 3 was significantly lower than that in the OGD / R group ( Figure 5B and 5C ; ###P<0.001, ##P<0.01; OGD / R+RebB group vs. OGD / R), Bax / Bcl2 showed no significant difference ( Figure 5D ;P>0.05; OGD / R+Reb group B vs. OGD / R).

[0052] in conclusion

[0053] In summary, this invention demonstrates that Reb B exerts a protective effect in the A549 cell OGD / R model by inhibiting the mitochondrial apoptosis pathway. This result lays a theoretical foundation for the treatment and clinical application of Reb B in acute lung injury.

[0054] The foregoing description illustrates and describes several preferred embodiments of the invention. However, as previously stated, it should be understood that the invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the invention should be within the protection scope of the appended claims.

Claims

1. Use of Reb B in the preparation of a drug for treating lung ischemia-reperfusion injury; the Reb B can reduce the ischemia-reperfusion injury of alveolar type II cells A549; The Reb B plays a protective role in an A549 cell OGD / R model by inhibiting a mitochondrial apoptosis pathway; the molecular formula of the Reb B is C 38 H 60 O 18 , and a chemical structure thereof is:

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