To Study The Effect And Mechanism Of Bauhinia Championii Flavones On Anti-ischemia/ Reperfusion Injury In Myocardial Cells Via Inhibiting Necroptosis

Objective: The preliminary experimental results proved that Bauhinia Championii flavones(BCF) has the effect of antiautophagy and antiapoptosis. The purpose of this experiment is to study the effects of BCF on regulating myocardial cell necroptosis.Methods: 1. This study was divided into two parts: in vitro and in vivo. The cadiocytes were cultured in vitro for 3~4 D, with good growth and pulsation, and established the model of hypoxia / reoxygenation injury(H/R, hypoxia 2h. / reoxygenation 12 h. and 24 h.).The myocardial ischemia/reperfusion injury model in vivo were made. Sprague-Dawley(SD) rats of either sex were randomized into 5 groups, Sham、Model、BCF(20 mg?kg-1)、BCF+Nec-1、Nec-1(0.6 mg?kg-1)(n=8). By ligation of the left coronary artery anterior descending branch of the methods for preparing rat myocardial ischemia / reperfusion injury model(ischemia for 30 min and then reperfused for 12 h. and 24 h.).2. In vitro experiments, The contents of total antioxidative capacity(T-AOC) and TNF-α in serum of each group were determined according to the ultraviolet spectrophotometry, while necroptosis was detected by flow cytometry. The protein expression of RIPK3 was observed by western-blot.3. In vivo experiments,the changes of ST-segment on theⅡlead ECG were observed. The contents of creatine kinase-MB(CK-MB), inducible nitric oxide synthase(iNOS) and total antioxidative capacity(T-AOC) in serum of each group were determined according to the ultraviolet spectrophotometry. The protein expression of LC3- Ⅱ, Beclin-1 and RIPK3 was observed by western-blot.Results: 1. Effects of BCF on biochemical indexes in serum and the protein expression of RIPK3 and the necroptosis rate in the of hypoxia / re oxygenation injury on cadiocytes.Compared with the model group, preconditioning by BCF increased the activity of T-AOC and decreased the release of TNF-α(P<0.05). The flow cytometric analysis showed that BCF decreased the necroptosis rate(P<0.05). Besides, BCF down-regulated the protein expression of RIPK3, as measured by western-blot(P<0.05). Compared with the model group, the expression of RIPK3 decreased and the expression of RIPK3 were decreased significantly in BCF+Nec-1 treated group(P<0.05).2. Effects of BCF on biochemical indexes in serum and the protein expression of LC3-Ⅱ, Beclin-1 and RIPK3 in ischemia reperfusion injury ratsCompared with the model group, BCF does-dependently improved T-AOC, decreased CK-MB and iNOS(P<0.05). Meanwhile, BCF down-regulated the protein expression of LC3-Ⅱand Beclin-1, as well as RIPK3 protein expression(P<0.05). On the other hand, compared with Necrostatin-1 group, BCF reduce the contents of CK-MB, iNOS, and LC3-Ⅱ, Beclin-1, RIPK3 protein expression and increased the level of T-AOC at the weak degree; But it have stronger effect when combined BCF with Nec-1(P<0.05).Conclusions: BCF pretreatment could reduce myocardial ischemia/ reperfusion injury. Its protective mechanisms likely because of its effects on anti-myocardial oxidative damages, anti-necroptosis, and protecting damaged myocard.