Study On The Regulation And Its Mechanim Of Bauhinia Championâ…¡ Flavones On PI3K/Akt Signaling Pathway In Myocardial Ischemia Reperfusion Injury Rats

Objective:To investigate the effects and mechanisms of Bauhinia championii flavones (BCF) on myocardial ischemia/reperfusion injury (MI/RI) in rats by modulating the PI3K/Akt signaling pathway.Methods:Rats with normal ECG were randomly divided into six groups with 10 rats (half male and half female) per group:(1) sham-operated (sham) group, in which animals underwent a similar procedure without ligation of the coronary artery; (2) myocardial ischemia/reperfusion (MI/RI) group, in which the MI/RI model was established by ligating the left anterior descending (LAD) coronary artery for 30 min followed by loosening the ligature for 3 h; (3) BCF group, in which MI/RI rats pretreated with BCF (20 mg/kg); (4) WM group, in which MI/RI rats pretreated with wortmannin (the PI3K inhibitor, WM,24 μg/kg); (5) BCF+WM group, in which MI/RI rats pretreated with BCF (20 mg/kg) plus WM (24 μg/kg), and WM was given 10 min prior to BCF treatment; (6) BCF+Rap group, in which MI/RI rats pretreated with BCF (20 mg/kg) plus rapamycin (the mTOR inhibitor, Rap,1 mg/kg). BCF or WM was given 20 min before ischemia via sublingual intravenous injection. Rap was administered 1 h before ischemia via intraperitoneal injection. The rats in the sham group and MI/RI group received the same volume of saline respectively. After reperfusion, myocardial infarction area was measured by Evans blue-TTC staining. The creatine kinase (CK), CK-MB, lactate dehydrogenase (LDH) and nitric oxide (NO) contents were assessed by colorimetry. The serum levels of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and tumor necrosis factor-a (TNF-a) were determined by enzyme-linked immunosorbent assay (ELISA). The opening of mitochondrial permeability transition pore (MPTP) was assayed by spectrophotometry. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The ultrastructure of autophagosome was observed by transmission electronmicroscopy. PI3K, eNOS, caspase-3, Beclin1 and Atg5 mRNA expression was evaluated by fluorescence quantitative polymerase chain reaction (FQ-PCR). The protein levels of PI3K, Akt, phosphorylated (p-) Akt, eNOS, p-eNOS, mTOR, p-mTOR, P70S6K, p-P70S6K, Caspase-3, Beclinl and LC3-II/I were detected by western blotting.Results:1.BCF pretreatment alleviated MI/RI via the PI3K/Akt signaling pathway.The infarction area was expanded in the MI/RI group, and pretreatment with BCF significantly reduced the infarction area (P<0.05). No significant difference was observed in the risk area between groups (P>0.05). The serum CK, CK-MB, LDH, iNOS and TNF-a levels in the MI/RI group were significantly higher than those in the sham group (P<0.01), and were reduced remarkably by BCF pretreatment (P<0.05). In addition, the eNOS and NO levels were conspicuous increased after BCF administration (P<0.05). However, in the presence of WM, a specific PI3K inhibitor, the effects of BCF were almost completely abolished (P<0.05). Thus, no significant difference among the MI/RI, BCF+WM and WM groups with regard to these indexes were detected (P>0.05).2. BCF pretreatment inhibited apoptosis in MI/RI rats via the PI3K/Akt signaling pathway.Compared with the MI/RI group, BCF inhibited the openning of MPTP (P<0.05) and reduced the apoptosis rate (P<0.05). It also decreased the mRNA and protein expression of Caspase-3, and concomitantly increased PI3K, p-Akt and p-eNOS expression (P<0.05). These effects of BCF were reversed by the addition of WM (P<0.05), no significant difference was found among the groups of MI/RI, BCF+WM and WM (P>0.05).3. BCF pretreatment suppressed overautophagy in MI/RI rats via the PI3K/Akt signaling pathway.Compared with the MI/RI group, the BCF group had a reduction of autophagosome formation (P<0.05). BCF up-regulatedp-mTOR and p-P70S6K, while down-regulated the autophagy-related genes including Beclinl and Atg5, and reduced the ratio of LC3-Ⅱ/Ⅰ (P<0.05). The anti-autophagic effect of BCF was blocked by the co-administration of the PI3K inhibitor WM or the mTOR inhibitor Rap (P<0.05), no significant difference was found among the groups of MI/RI, BCF+WM and WM (P>0.05).Conclusion:This study indicated that BCF protected the heart against MI/RI via the inhibition of apoptosis and excessive autophagy, which protective effect is regulated by the activation of the PI3K/Akt signaling pathway.