The Study Of Mongolian Medicine Zhenbao Pill Inhibits Oxidative Stress Injury By Regulating Autophagy Of Human Umbilical Vein Endothelial Cells And Its Mechanism

Background Mongolian medicine is an important part of traditional Chinese medicine.It is the wisdom crystallization accumulated and summarized by the Mongolian people in their long-term production and life.Clinical studies have confirmed the therapeutic effect of Zhenbao Pill（ZBP）on neuroprotection and functional recovery after acute cerebral ischemia.However,the protective effect of ZBP on vascular endothelial cells is still unclear.Autophagy is a self-clearance mechanism existing in normal cells.Under physiological conditions,autophagosomes are responsible for including aging,misfolded proteins and damaged organelles,etc.They combine with lysosomes to degrade and reuse the included substances in acidic environment,which plays an important role in maintaining the homeostasis and survival of the intracellular environment.With the in-depth studies on ischemia reperfusion and cardiovascular and cerebrovascular ischemia and hypoxia diseases,scholars at home and abroad have found that autophagy may be a new and effective protective mechanism in the process of ischemia reperfusion injury,but how autophagy participates in the pathophysiological changes of cerebral ischemia reperfusion injury is controversial.Therefore,it is very important to study the relationship between the protective mechanism and autophagy of ZBP for exploring the drug target of ZBP.Objective Whether ZBP has protective effect on vascular endothelial cells is unclear.In this study,H₂O₂was used to establish an in vitro model of oxidative stress in human umbilical vein endothelial cells（HUVEC）,and to explore the relationship and mechanism between ZBP and autophagy.Methods The first part:SD rats were randomly given low-dose（0.25 g/kg）group,medium-dose（0.5 g/kg）group,high-dose（1.0 g/kg）group and blank（1%CMC-NA）group.ZBP was gavaged continuously for 7 days.After the last gavage for 1h,blood was collected to prepare drug-containing serum and blank serum（rat serum without drug）.1.The effect of different concentrations of H₂O₂（100,200,400,800μM）on cell activity was detected by CCK-8 method.2.CCK-8 method was used to screen the protective effects of different concentrations（0.25 g/kg,0.5 g/kg,1.0 g/kg）of drug-containing serum on HUVEC.3.The cells were divided into normal control group,H₂O₂group,H₂O₂+CMC-Na group and H₂O₂+ZBP group according to the H₂O₂induced concentration and the drug-containing serum concentration of ZBP,and the protective effect of ZBP on oxidative stress injury of HUVEC was investigated.Cell apoptosis level in each group was detected by flow cytometry using Annexin V-FITC/PI kit;The level of reactive oxygen species（ROS）production in each group was detected by fluorescent probe DCFH-DA.Lactate dehydrogenase（LDH）kit was used to detect the level of LDH in each group.Mitochondrial transmembrane potential（MMP）level of each group was detected by Rhodamine-123.The second part:on the basis of the first part group,the group of H₂O₂+ZBP+3-MA（autophagy inhibitor）was added.1.GFP-m Cherry LC3 adenovirus was used to infect cells,and the fluorescence intensity,i.e.the level of autophagy,was observed under confocal microscopy.2.The number of autophagosomes in each group was observed under electron microscope.3.Cell apoptosis in each group was determined by flow cytometry using Annexin V-FITC/PI apoptosis detection kit.4.The mRNA expression levels of autophagy-related genes Beclin-1 and LC3b and apoptotic-related genes Bcl-2 and Bax in HUVEC were detected by q PCR.5.The protein expressions of Beclin-1,LC3b,Bcl-2,Bax and autophagy related pathways Akt,p-Akt,mTOR and p-mTOR in HUVEC cells were detected by Western blot.Results Part I:1.CCK-8 results showed that the cell activity decreased significantly after treated with 400μM H₂O₂for 2 h（P<0.01）,which was determined as the damage condition for establishing HUVEC oxidative stress model.2.0.5g/kg ZBP serum recovered HUVEC cell viability to the maximum（P<0.01）,which was established as the subsequent experimental conditions.3.Compared with H₂O₂group,H₂O₂+ZBP group could significantly reduce the level of apoptosis（P<0.05）,LDH（P<0.01）,ROS production（P<0.05）,and maintain the level of MMP（P<0.01）.The autophagy level was observed under a confocal microscope.Compared with the normal control group,the LC3fluorescence in the H₂O₂group and the H₂O₂+CMC-Na group was significantly enhanced.Compared with the H₂O₂group,the LC3 fluorescence in the H₂O₂+ZBP group and the H₂O₂+ZBP+3-MA group was significantly decreased.2.The number of autophagosomes was observed under electron microscopy.Compared with the normal control group,the number of typical autophagosomes in the H₂O₂group and the H₂O₂+CMC-Na group was significantly increased,and the number of autophagosomes in the H₂O₂+ZBP group and the H₂O₂+ZBP+3-MA group was significantly decreased compared with the H₂O₂group.3.The apoptosis rate in H₂O₂+ZBP+3-MA group was further decreased compared with that in H₂O₂+ZBP group（P<0.05）.4.Compared with the H₂O₂group,the mRNA expression of Beclin-1 and LC3 in H₂O₂+ZBP group was inhibited（P<0.01）,the mRNA expression of Bax was decreased（P<0.05）,and the mRNA expression of Bcl-2 was increased（P<0.01）;Compared with H₂O₂+ZBP group,H2O2+ZBP+3-MA group promoted the expression of Bcl-2 mRNA（P<0.01）and inhibited the expression of Bax mRNA（P<0.05）.5.Compared with the H₂O₂group,the expression of Beclin-1 and LC3-II proteins in the H₂O₂+ZBP group was inhibited（P<0.01）,the expression of Bcl-2 was enhanced（P<0.01）,and the expression of Bax protein was decreased（P<0.01）.Meanwhile,compared with the H₂O₂+ZBP group,the expression of Bax protein in the H₂O₂+ZBP+3-MA group was further inhibited（P<0.05）,and the expression of Bcl-2 was further enhanced,but there was no statistical significance.6.Compared with the H₂O₂group,the expression of p-Akt protein and p-mTOR protein in H₂O₂+ZBP group was increased（P<0.01）,and the expression of p-mTOR protein was increased（P<0.05）.Conclusion ZBP can protect against HUVEC oxidative stress injury induced by H₂O₂.ZBP may protect against oxidative stress injury to vascular endothelial cells through anti-apoptosis and anti-autophagy effects,and may play a role by activating PI3K/Akt/mTOR pathway and its upstream and downstream.The protective effect of ZBP on cerebral ischemia reperfusion injury has not been previously explored from this perspective,and our results provide a new idea for the research on the target of action of ZBP.