ABSTRACT
ObjectiveTo explore the mechanism of Wutou Chishizhi Wan in regulating autophagy and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/glycogen synthase kinase-3β （GSK-3β） signaling pathway in rats with myocardial ischemia-reperfusion injury （MIRI）. MethodSixty male SD rats were randomly assigned into the normal group （normal saline）， model group （normal saline）， positive control （trimetazidine， 5.4 mg·kg-1） group， and low-， medium-， and high-dose （1.63， 4.9， 14.7 g·kg-1， respectively） Wutou Chishizhi Wan groups， with 10 rats in each group. The rats in other groups except the normal group underwent left anterior descending coronary artery ligation for modeling. Electrocardiogram was employed to detect the ST-segment elevation to evaluate the modeling. Hematoxylin-eosin （HE） staining was performed to reveal the damage of myocardial tissue. The levels of aspartate aminotransferase （AST） and creatine kinase （CK） were determined by colorimetry， and those of cardiac troponin T （cTnT） and myoglobin （MYO） by enzyme-linked immunosorbent assay （ELISA）. Western blot was carried out to determine the protein levels of microtubule-associated proteins 1 light chain 3 （LC3）， autophagy-related gene Beclin-1， PI3K， Akt， GSK-3β， p-GSK-3β， and p-Akt. ResultCompared with the normal group， the modeling elevated the serum levels of AST， CK， cTnT， and MYO （P<0.01）， destroyed the arrangement of myocardial cells abd nucle， twisted and broken myocardial fibers， up-regulated the protein levels of LC3Ⅱ/Ⅰ and Beclin-1 （P<0.01）， and down-regulated the protein levels of PI3K， p-Akt， and p-GSK-3β （P<0.01）. Compared with the model group， trimetazidine and Wutou Chishizhi Wan （all the doses） lowered the levels of AST， CK， cTnT， and MYO in serum （P<0.01）， restored the arrangement of myocardial cells and muscle fibers， reduced necrosis， down-regulated the protein level of Beclin-1 （P<0.01）， and up-regulated the protein levels of PI3K， p-Akt， and p-GSK-3β （P<0.01）. Additionally， Wutou Chishizhi Wan （all the doses） down-regulated the protein level of LC3Ⅱ/Ⅰ （P<0.05， P<0.01）. Compared with those in the trimetazidine group， the serum AST level rose in the low-dose Wutou Chishizhi Wan group （P<0.05） and declined in the high-dose group （P<0.01）， and the protein level of Beclin-1 was down-regulated in the medium-dose group （P<0.01）. Additionally， the trimetazidine group had higher protein level of LC3Ⅱ/Ⅰ than medium- and high-dose Wutou Chishizhi Wan groups （P<0.05， P<0.01）， higher protein level of PI3K than low-， medium-， and high-dose groups （P<0.01）， lower protein level of p-Akt than low- and medium-dose groups （P<0.01）， and higher p-GSK-3β protein level than the medium-dose group （P<0.01）. ConclusionDifferent doses of Wutou Chishizhi Wan can ameliorate MIRI， and the high dose has the best effect. Wutou Chishizhi Wan can reduce the activity of myocardial injury markers AST， CK， cTnT， and MYO， and alleviate the pathological damage of myocardial tissue. It can down-regulate the protein levels of beclin-1， LC3Ⅱ/Ⅰ， and up-regulate those of PI3K， p-Akt， and p-GSK-3β. In summary， Wutou Chishizhi Wan may inhibit excessive autophagy and regulate the PI3K/Akt/GSK-3β signaling pathway to exert protective effect on MIRI rats.
ABSTRACT
Objective:To investigate the protective effect of Wutou Chishizhi Wan on myocardial ischemia reperfusion injury （MIRI） in rats， and observe its effect on such mechanisms as coagulation function， vascular endothelial cells and oxidative stress in rats. Method:A total of 40 SD rats were randomly divided into normal group， model group， positive drug group （Urokinase group） and Wutou Chishizhi Wan group， with 10 rats in each group. Except for the normal group， rat myocardial ischemia-reperfusion injury models were established. The changes of heart rate （HR） at 10 min before ischemia， 30 min after ischemia and 30， 60， 120 min （T0，T1，T2，T3，T4）， and the change of electrocardiogram （ECG） J point after modeling in rats were observed. The pathological changes of rat myocardial tissue were observed by hematoxylin-eosin （HE） staining. The changes of four indexes of coagulation ［prothrombin time （PT）， activated partial thromboplastin time （APTT）， thrombin time （TT）， fibrinogen content decreased significantly （FIB）］ in rats were observed. The contents of endothelin-1 （ET-1）， thromboxane A2 （TXA2） and prostacyclin （PGI2） in serum and myocardium levels of superoxide dismutase （SOD）， malondialdehyde （MDA） and glutathione peroxidase （GSH-Px） of MIRI rats were observed. Western blot assay was used for the detection of oxidative stress protein Keap1 and transcription factor-E2-related factor （Nrf2） expression levels in rat myocardial tissue. Result:Compared with the normal group， the ECG of MIRI rats showed significant myocardial ischemic injury-like changes， ST segment was significantly elevated， J point was significantly increased， and the incidences of HR in T1， T2， T3 and T4 were significantly reduced （P<0.05， P<0.01）. Compared with the model group， Wutou Chishizhi Wan significantly reduced ECG J-point changes in MIRI rats， while increased the incidence of HR in T1， T2， T3 and T4 （P<0.05， P<0.01）. Compared with the normal group， PT， APTT and TT in the model group were significantly shortened （P<0.01）， FIB content was significantly increased （P<0.01）， and the serum PGI2 level decreased and TXA2 and ET-1 levels increased significantly in the model group （P<0.01）. SOD content and GSH-Px activities of myocardial tissue in the model group were significantly reduced （P<0.01）， whereas the MDA content was increased （P<0.01）. Compared with the model group， PT of the Wutou Chishizhi Wan group was prolonged （P<0.05） and APTT slightly prolonged， TT significantly prolonged （P<0.01）， FIB content decreased （P<0.05）， serum PGI2 increased （P<0.05）， TXA2 and ET-1 decreased significantly in the Wutou Chishizhi Wan group （P<0.01）， myocardial MDA content decreased， and SOD content and GSP-Px activity increased significantly （P<0.01）. Meanwhile， the Wutou Chishizhi Wan group was able to activate the Keap1/Nrf2 signaling pathway， which significantly increased Nrf2 expression and significantly decreased Keap1 expression （P<0.01）. Conclusion:Wutou Chishizhi Wan group can protect myocardial injury in MIRI rats. The specific mechanism is to protect MIRI by regulating vascular endothelial cell homeostasis and oxidative stress levels and activating Keap1/Nrf2 signaling pathway.