Study On The Effect Of Oxymatrine On Apoptosis Of Bone Marrow Mesenchymal Stem Cells Induced By Hydrogen Peroxide

Background: Acute myocardial infarction（AMI）is a clinically acute and critical disease.Despite the continuous development of diagnosis and treatment,the mortality of AMI has been reduced.However,infarcted cardiomyocytes can not be regenerated.After myocardial infarction,patients with myocardial infarction 5 years mortality rate is still as high as 50 percent.At present,stem cell transplantation has become a new strategy for regenerative and infarcted myocardium.Bone marrow mesenchymal stem cells（BMSCs）can be induced to differentiate into BMSCs under certain conditions because of their self-renewal and multi-directional differentiation potential.A variety of adult cell types,one of the seed cells of regenerative repair of cardiomyocytes.However,the survival rate of transplanted BMSCs in the infarct area is low and can not achieve the desired therapeutic effect.Therefore,how to improve the survival rate of stem cells in the infarct area is an important factor in the success of BMSCs transplantation.Oxymatrine（Oxymatrine,OMT）,also known as oxymatrine,is extracted from alkaloids in the roots of Sophora flavescens or plants.It has anti-virus,anti-inflammatory and antihypertensive effects.It is found that OMT can be obtained by PI3 K / AKT signaling pathway regulates cell survival and regulates Nrf2 nuclear transcription up-regulation of H0-1 expression,regulates apoptotic proteins to achieve anti-apoptotic effects,but whether oxidized matrine is protective against BMSCs under oxidative stress Does the PI3 K / AKT signaling pathway participate? Are not clear at present.Objective 1.To determine whether oxidized matrine has protective effect on BMSCs under oxidative stress.2 To investigate whether the effect of oxymatrine on BMSCs regulates Nrf2 nuclear transcription and H0-1 mediated by PI3 K / AKT signaling pathway.Method:1、The culture,passage and identification of BMSCs: BMSCs were cultured in BMSCs by BMSCs adherence method.The purity of BMSCs was determined by flow cytometry.The third generation BMSCs were followed by experiments.2、BMSCs2h was treated with different concentrations of H₂O₂（200,100,50,25,0 mol /L）,CCK8 was determined to determine the reasonable concentration of BMSCs apoptosis.3 、 The concentration of oxymatrine was explored.The pretreatment of BMSCs with different concentrations of oxymatrine（0.1mg / ml-0.07 mg / ml）was 0.1,0.09,0.08,0.07 and then treated with H₂O₂（100μM）CCK-8 method was used to detect the viability of BMSCs,and the concentration of OMT was not determined by H₂O₂ treatment in the normal group.4、The experiment is divided into three groups:blank group: cells were not treated;H₂O₂group: only H₂O₂（100μM）for 2h BMSCs were pretreated with OMT（0.1 mg / ml）for 24 h and then treated with H2 O2（100 μM）and then treated with H2 O 2（100 μM）for 2hours.Western blot was used to detect the expression of Cleaved-Caspase-3,Baxx and Bcl-2.The expression of Cleaved-Caspase-3,Bax and Bcl-2 was detected by Western blotting.5、The experiment was divided into four groups: blank group: the cells were not treated;H₂O₂ group: only H₂O₂（100 μ M）for 2h;OMT + H₂O₂ group: Pretreatment with OMT（0.1mg / ml）BMSCs were added to H₂O₂（100μM）for 2h,and H₂O₂（100μM）was added to H₂O₂（100μM）for 2h.After treated with OMT（0.1mg / ml）for 24 hours,the cells were treated with H₂O₂（100μM）for 2h.The apoptosis of each group was detected by flow cytometry（FCM）The expression of Caspase-3,Bax and Bcl-2 were detected by immunohistochemistry.6、Group with 5,The phosphorylation of AKT and P-AKT was detected by Western blotting,and the expression of HO-1 in the nucleus and outer surface of Nrf2 was compared with that of OMT.Result:1.After 48 h of primary BMSCs,the cells were observed in a circular bright spot,and the BMSCs were completely changed to spindle shape at 4-5 days.The cells were exposed to the whole bottom at 7-8 days.Primary BMSCs were subcultured to the long spindle or polygonal cells that were evenly distributed after the second to third generations.Flow cytometry was used to detect BMSCs surface markers: CD29 98.5% CD90 98.7%2.CCK8 results showed that H₂O₂ could significantly decrease the activity of BMSCs,and 100μMol / L H₂O₂ could induce the apoptosis of BMSCs（P <0.01）.3.The activity of BMSCs was significantly decreased by CCK8,and the cell viability increased with the increase of OMT concentration from small to large gradient（P <0.01）.4.Flow cytometry: OMT pretreatment significantly reduced the apoptosis of BMSCs mediated by H₂O₂,compared with H₂O₂（P <0.01）.Western blot analysis showed that the expression of Caspase-3 and Bax in OMT group（P <0.01）,and the expression of anti-apoptotic protein Bcl-2 was increased（P <0.01）.5.Flow cytometry showed that the apoptosis rate and mortality of BMSCs in OMT + H₂O₂+ blockade group were significantly increased,while the expression of Caspase-3 and Bax protein was significantly increased by Western blot,while Bcl-2 was significantly decreased（P <0.01）.Compared with the blank group,the expression of phosphorylated p-AKT protein was increased by OMT pretreatment（P <0.01）,but the phosphorylation was inhibited by OMT + H₂O₂ group（P <0.01）The protein level of p-AKT was significantly lower than that of OMT group（P <0.01）,and there was no difference compared with control group（P> 0.05）.6.Western blot analysis showed that the expression of Nrf2 in the OMT group was significantly higher than that in the H₂O₂ group（P <0.01）,and the cytoplasm was completely opposite to the nucleus.The expression of HO-1 in the OMT group was significantly higher than that in the H₂O₂ group Compared with（P <0.01）.Conclusion:1.OMT has anti-apoptotic effect on H₂O₂-induced BMSCs by inhibiting the expression of pro-apoptotic proteins Caspase-3 and Bax and up-regulating the level of anti-apoptotic protein Bcl-22.In the BMSCs,OMT may activate the PI3 K / AKT signaling pathway,initiate the Nrf2 antioxidant and promote the expression of H0-1,and then play the protective effect of OMT on the anti-oxidative stress injury.