Asiaticoside alleviates myocardial ischemia-reperfusion injury in rats by inhibiting NLRP3 inflammasome-mediated pyroptosis

doi:10.12122/j.issn.1673-4254.2025.05.10

Abstract

Abstract:

Objective To study the mechanism mediating the protective effect of asiaticoside (AS) against myocardial ischemia-reperfusion injury (MIRI) in rats. Methods Fifty SD rats were randomized into sham-operated group, MIRI model group and AS treatment group. AS treatment was administered at low, moderate and high doses by daily gavage for 2 weeks before MIRI modeling (n=10). Serum levels of lactate dehydrogenase (LDH), creatine kinase isoenzyme (CK-MB), interleukin-18 (IL-18) and IL-1β, the volume of myocardial infarction and ischemia, and myocardial pathologies of the rats were determined or observed. The protein expression levels of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 in the myocardial tissues were detected using Western blotting. The changes in the expression levels of these proteins were also detected in H9C2 cells with AS pretreatment prior to hypoxia-reoxygenation (H/R) injury. Results The rats models of MIRI exhibited significant myocardial infarction and ischemia with increased serum levels of LDH and CK-MB and myocardial expressions of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18. AS pretreatment effectively reduced myocardial infarction volume in the rat models and significantly reduced serum LDH and CK-MB levels and the protein levels in the myocardial tissue in a dose-dependent manner. In the H9C2 cell model of H/R injury, AS pretreatment significantly suppressed the elevation of the protein expressions of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18. Molecular docking studies showed that AS had a strong binding affinity with NLRP3. Conclusion Asiaticoside can alleviate MIRI in rats possibly by inhibiting NLRP3 inflammasome-mediated pyroptosis.

Key words: asiaticoside, myocardial ischemia-reperfusion injury, NLRP3, pyroptosis

Fenlan BIAN, Shiyao NI, Peng ZHAO, Maonanxing QI, Bi TANG, Hongju WANG, Pinfang KANG, Jinjun LIU. Asiaticoside alleviates myocardial ischemia-reperfusion injury in rats by inhibiting NLRP3 inflammasome-mediated pyroptosis[J]. Journal of Southern Medical University, 2025, 45(5): 977-985.

Figures/Tables 8

Fig.1 Molecular docking of AS with NLRP3.

Fig.2 TTC-Evans blue staining of rat cardiac tissues (A) and comparison of the extent of myocardial infarction (B) and ischemia (C) among the 5 groups. ***P<0.001 vs Sham group; ###P<0.001 vs I/R group (Mean±SD, n=3).

Fig.3 Effect of AS pretreatment on serum levels of LDH (A), CK-MB (B), IL-1β (C) and IL-18 (D) in MIRI rats. ***P<0.001 vs Sham group; ##P<0.01, ###P<0.001 vs I/R group (Mean±SD, n=6).

Fig.4 HE staining of myocardial tissue of the rats in each group.

Fig.5 Western blotting for detecting protein expressions in the myocardial tissue of the rats in each group. A: Western blots of NLRP3, caspase-1, ASC, GSDMD, GSDMD-N, IL-1β and IL-18 in the myocardial tissues of the rats. B-H: Quantitative analysis of pyroptosis-related proteins in each group. ***P<0.001 vs Sham group; #P<0.05, ##P<0.01, ###P<0.001 vs I/R group (Mean±SD, n=4).

Fig.6 Effects of different concentrations of AS on viability of H9C2 cells. A: Effect of AS on viability of H9C2 cells. B: Effect of AS pretreatment on viability of H9C2 cells with hypoxia-reoxygenation injury. ***P<0.001 vs Control group; #P<0.05,###P<0.001 vs H/R group (Mean±SD, n=3).

Fig.7 Immunofluorescence staining showing NLRP3 and caspase-1 protein expressions in H9C2 cells in each group. A: Immunofluorescence staining of NLRP3 in H9C2 cells in each group (Original magnification: ×20). B: Immunofluorescence staining of caspase-1 in H9C2 cells in each group (×20). C: Average fluorescence intensity of NLRP3 in each group. D: Average fluorescence intensity of caspase-1 in each group. ***P<0.001 vs Control group; ###P<0.001 vs H/R group (Mean±SD, n=3).

Fig.8 Western blotting for detecting protein expressions in H9C2 cells in each group. A: Western blots of NLRP3, caspase-1, ASC, GSDMD, GSDMD-N, IL-1β and IL-18 in H9C2 cells in each group. B-H: Quantitative analysis of expressions of the pyroptosis-related proteins in H9C2 cells in each group. **P<0.01,***P<0.001 vs Control group; #P<0.05, ##P<0.01, ###P<0.001 vs H/R group (Mean±SD, n=4).

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