| Worldwide,the morbidity and mortality of cardiovascular disease remain high,among which myocardial ischemia and acute myocardial infarction are high risk factors for death.In clinical practice,reperfusion is often used to treat patients.During the reperfusion process,the myocardial tissue undergoes stimulation such as hypoxia/reoxygenation and PH value changes,which leads to further damage to the myocardial tissue,aggravating the patient’s condition,and appearing myocardial abscess,arrhythmia.This pathological phenomenon is clinically called myocardial ischemia/reperfusion（MI/R）injury.The exact mechanism of myocardial ischemiareperfusion injury has not yet been clarified,so it is of great significance to find drugs for preventing and treating myocardial ischemia-reperfusion injury.Inonotus obliquus（IO）,also known as Chaga mushroom,is a sub-genus of Betula that grows on birch trees.Its main bioactive components are polysaccharides,polyphenols,total saponins,and total flavonoids.Current studies have shown that IO has pharmacological effects such as antioxidant,hypolipidemic,anti-tumor,and immune system enhancement,but it is unclear whether it has a protective effect on MI/R.Object:The main components of the Inonotus obliquus extract extracted in different concentrations of ethanol were measured by ultraviolet spectrophotometry,and70 %ethanol Inonotus obliquus extract was selected for subsequent studies.The protective effect of Inonotus obliquus alcohol extract on MI/R injury in rats was observed,and the mechanism of action was initially explored.Method:Use water and 20 %,30 %,40 %,50 %,70 %,95 %concentration ethanol to extract Inonotus obliquus respectively and measure the content of polysaccharides,polyphenols,total flavonoids and total saponins in each sample.Choose the Inonotus obliquus alcohol extract（IOE）obtained by 70 %ethanol to be used as a follow-up experimental drug.150 female Wistar rats were randomly divided into five groups according to body weight,namely Sham group,MI/R group,and low,medium and high dose groups of Inonotus obliquus alcohol extract（MI/R-IOE 150,300,600mg/kg）.Rats in Sham group and MI/R group were administered intragastrically（ig）0.5 %CMC-Na 10 m L/kg,while rats in MI/R-IOE 150,300,and 600 mg/kg groups were given corresponding drugs 10 m L/kg by ig respectively The rats were administrated with 0.5 %CMC-Na and IOE for 7 days,once a day.One hour after the last administration,the left anterior descending coronary artery（LAD）of the rat was ligated for 2 hours and then reperfused for 2 hours to establish a rat MI/R injury model.After reperfusion,the LVIDd,LVIDs,LVEF and LVFS of the rats were determined by Doppler ultrasound;the myocardial infarction area of ??the rats was determined by NBT staining;the degree of myocardial damage and pathological changes were observed by HE staining;CK-MB,AST,and LDH activity in rat serum were measured using ELISA kits;the content of MDA and the activities of SOD,CAT and GSH-Px in rat myocardial tissue were determined by biochemical kits;the apoptosis of rat myocardial cells was detected by TUNEL staining;the protein expression of SIRT1,the expression of endoplasmic reticulum stress-related proteins GRP78,Caspase-12,endoplasmic reticulum stress-related pathway PERK/e IF2α/CHOP protein,apoptosis-related proteins Bcl-2 and Bax,and Caspase family precursor proteins Procaspase-3 and Procaspase-9 in rat myocardial tissue were detected by Western Blot;Caspase-12 m RNA level was detected by QPCR.Result:1.The results of component analysis of IOE extracted with different concentrations of ethanol showed that the content of polysaccharide in IOE extracted with aqueous solution was the highest.The contents of polyphenols and total flavonoids in IOE extracted by 70 %ethanol solution were the highest.The content of total saponins in IOE extracted by 95 %ethanol was the highest..2.The results of Doppler ultrasound showed that compared with the Sham group,the LVIDd and LVIDs of the MI/R group were increased,while the LVEF and LVFS were significantly decreased（P<0.01）.Compared with MI/R group,MI/R-IOE 150,300 and 600 mg/kg could reduce LVIDd and LVIDs（P<0.05 or P<0.01）,while increased LVEF and LVFS（P<0.05 or P<0.01）,which proved that IOE could improve the cardiac function in MI/R rats.3.The results of NBT staining showed that compared with the Sham group,the MI/R group had obvious myocardial infarction（P<0.01）;compared with the MI/R group,MI/R-IOE 150,300 and 600 mg/kg could make the infarct size decreased（P<0.01）;HE results showed that,compared with the Sham group,the myocardial tissue of the MI/R group had severe pathological damage;MI/R-IOE 150,300 and600 mg/kg could improve myocardial tissue pathological damage in rats caused by MI/R to varying degrees;at the same time,compared with the Sham group,the activities of CK-MB,AST and LDH in the serum of the MI/R group were significantly increased（P<0.01）.);compared with MI/R group,MI/R-IOE 150,300,600 mg/kg could reduced the activities of CK-MB,AST and LDH in rat serum（P<0.05 or P<0.01）,the above results showed that IOE has protective effect on MI/R rat myocardium.4.The results of oxidative stress-related indicators showed that compared with the Sham group,the content of MDA in myocardial tissue of the MI/R group increased（P<0.01）,while the activities of SOD,CAT and GSH-Px decreased（P<0.01）;compared with the MI/R group,MI/R-IOE 150,300,and 600 mg/kg could reduced the increase of MDA content caused by MI/R injury（P<0.01）,and at the same time increased the activities of SOD,CAT and GSH-Px（P<0.01）,indicating that IOE could resist MI/R injury by anti-oxidative stress.5.The results of TUNEL staining showed that compared with the Sham group,myocardial cells in the MI/R group had obvious apoptosis（P<0.01）;compared with MI/R group,MI/R-IOE 150,300 and 600 mg/kg significantly reduced the apoptosis rate of myocardial cells（P<0.05 or P<0.01）,indicating that IOE could inhibit cardiomyocyte apoptosis in MI/R-injured rats.6.Western Blot results showed that compared with the Sham group,the expression of SIRT1 in the MI/R group was significantly decreased;compared with the MI/R group,MI/R-IOE 150,300,and 600 mg/kg could increase the SIRT1 protein expression in myocardial tissue（P<0.01）.Endoplasmic reticulum stress-related proteins and PERK/e IF2α/CHOP pathway proteins were measured at the same time.Compared with Sham group,GRP78,p-PERK,p-eIF2α,CHOP and Caspase-12 in myocardial tissue of MI/R group rats were significantly increased（P<0.01）.Compared with MI/R group,MI/R-IOE 150,300,600 mg/kg could reduce GRP78,pPERK,p-e IF2α,CHOP and Caspase-12（P<0.05 or P<0.01）,and the results of QPCR for Caspase-12 m RNA level were consistent with the Western Blot results.The above results indicated that IOE can inhibit PERK/e IF2α/CHOP by activating SIRT1 pathway and play a protective effect on myocardial ischemia-reperfusion injury in rats.7.Western Blot detection of apoptosis-related proteins showed that compared with the Sham group,the expression of Bax protein in myocardial tissue of MI/R group increased（P<0.01）,while Bcl-2,Procaspase-3 and Procaspase-9 proteins Compared with MI/R group,MI/R-IOE 150,300,600 mg/kg could reduce the protein expression of Bax（P<0.01）,and at the same time increase Bcl-2,Procaspase-3 and the protein expression levels of Procaspase-9（P<0.01）,indicating that IOE could inhibit myocardial cell apoptosis induced by inhibiting endoplasmic reticulum stress in MI/R rats.Conclusion:In this study,the Inonotus obliquus extracted by different conditions was analyzed,and 70 %Inonotus obliquus alcohol extract was selected for subsequent study.It has been found that Inonotus obliquus alcohol extract could protect against rat myocardial ischemia reperfusion injury,and the mechanism may involve improving the body’s antioxidative strength,activating SIRT1 by inhibiting PERK/e IF2α/CHOP pathway,relieving cardiomyocyte apoptosis induced by ER stress. |
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