“Er-xian” Regulates AMPK-PGC1α Pathway To Improve Myocardial Ischemia-reperfusion Injury

Objective:The aim of this study was to explore the potential protective effects of “ErXian”(Curculigo orchioides Gaertn and Herba Epimedii)against myocardial ischemia reperfusion injury(MIRI)in SD rats,which might exert its effect on ameliorating MIRI through the AMPK-PGC1α pathway.Methods:Sixty 160-180 g male SD rats were randomly divided into sham-operated(Sham)group,myocardial ischemia-reperfusion model(I/R)group,“Er-Xian” low-dose(Er-XianL)group,“Er-Xian” medium-dose(Er-Xian-M)group and “Er-Xian” high-dose(Er-XianH)group,12 rats in each group.Both Sham and I/R groups were given 10ml/kg of equal saline by continuous gavage for 2 weeks,once a day.Er-Xian low,medium and high dose groups were given 2.5g/kg,5g/kg and 10g/kg of the original dose of Er-Xian respectively by gavage with extract for 2 weeks,once a day.After the last gavage,the MIRI model was created by ligating of the anterior descending branch of the coronary artery,while the Sham group was only threaded but not ligated.After ligation of the anterior descending branch of the coronary artery for 30 min of ischemia followed by reperfusion for 2 h,the area of myocardial infarction was measured by TTC staining;the serum CK-MB level was measured by creatine kinase isoenzyme(CK-MB)ELISA kit;HE staining was performed to observe the myocardial histopathology;TUNEL staining was performed to detect the apoptosis rate of myocardial cells;Western Blot was performed to detect P AMPKα(T172),PGC1α protein expression levels.Data were statistically analyzed using Graphpad 9.4.1software.One-way analysis of variance(ANOVA)was used and data were expressed as mean ± standard deviation(Mean ± SD)and P < 0.05 was considered statistically significant.Results:1.The effects of Er-Xian on myocardial infarct area in MIRI rats: TTC staining results showed that myocardial infarct area was significantly increased in the I/R group compared to the Sham group,and myocardial infarct area was significantly decreased in all dose groups of Er-Xian compared to the I/R group.When comparing the Er-Xian dose groups two by two,there was a statistical difference between all Er-Xian dose groups.This indicated that Er-Xian pretreatment reduced the myocardial infarct area in MIRI rats in a dose-dependent manner.2.The effects of Er-Xian on serum CK-MB levels in MIRI rats: The CK-MB results showed that CK-MB levels were significantly higher in the I/R group compared to the Sham group,and CK-MB levels were significantly lower in all dose groups of Er-Xian compared to the I/R group.Comparing the Er-Xian dose groups two by two,CK-MB levels in the Er-Xian-M and Er-Xian-H groups were significantly different compared to the ErXian-L group,while CK-MB levels in the Er-Xian-M and Er-Xian-H groups were not statistically significant.This indicated that Er-Xian pretreatment dose-dependently attenuated the release of CK-MB from cardiomyocytes in MIRI rats and exerted myocardial protective effects.3.The effects of Er-Xian on the myocardial pathology of MIRI rats: HE staining showed that the overall structure of the heart tissue in the Sham group was basically normal,with neat and tight arrangement of myocardial cells and visible transverse lines,and no obvious inflammatory cell infiltration in the tissue.In the I/R group,the overall structure of the myocardial tissue was abnormal,with disturbed arrangement of myocardial cells,bifurcation and rupture of some myocardial fibres,heavy edema in the interstitial space and inflammatory cell infiltration visible in the tissue.The myocardial cells and interstitial edema were reduced to different degrees in the Er-Xian dose groups compared to the I/R group,and the inflammatory cell infiltration was significantly suppressed in a dose-dependent manner.This indicated that Er-Xian dose-dependently improved the morphological structure of myocardial tissue in MIRI rats.4.The effects of Er-Xian on the apoptotic rate of cardiomyocytes in MIRI rats: The TUNEL staining results showed that the apoptotic rate of cardiomyocytes in the I/R group was significantly higher than that in the Sham group,while the apoptotic rate of cardiomyocytes in all dose groups of Er-Xian decreased to different degrees compared with that in the I/R group.When comparing the Er-Xian dose groups two by two,the levels of apoptosis in cardiac myocytes were all significantly different.This indicated that Er-Xian pretreatment reduced the apoptosis rate in MIRI rats in a dose-dependent manner.5.The effects of Er-Xian on P-AMPKα(T172)and PGC1α protein expression in MIRI rats: Western blot results showed that the expression of P-AMPKα(T172)and PGC1α in Sham group and I/R group were statistically significant,and the expression of P-AMPKα(T172)and PGC1α in each dose group of Er-Xian was statistically significant compared with that in I/R group.When comparing the Er-Xian dose groups two by two,the expression of P-AMPKα(T172)and PGC1α were statistically significant between the groups.It was suggested that P-AMPKα(T172)and PGC1α protein expression were significantly increased in MIRI rats pretreated with Er-Xian,which dose-dependently upregulated the AMPK-PGC1α pathway.Conclusion:“Er-xian” exerted its effects on improving MIRI by reducing infarct size,lowering CK-MB level,improving myocardial tissue morphology and structure,and decreasing apoptosis rate of cardiomyocytes in a dose-dependent manner,and its mechanism of action was related to the upregulation of AMPK-PGC1α pathway.