Effect And Mechanism Of Conditioned Medium From Hypoxia-Preconditioned Human WJMSC On Myocardial Ischemia/Reperfusion Injury

Background:Myocardial ischemia/reperfusion injury（I/RI）with recanalization after myocardial infarction is an important clinical problem that needs to be solved urgently.Wharton’s jelly derived mesenchymal stem cell（WJMSC）is one of the most important seed cells among many clinical stem cells.In vivo transplantation of WJMSC can reduce myocardial I/RI and improve cardiac function after myocardial infarction.Objective:To investigate the effect and possible mechanism of conditioned media from hypoxia-preconditioned WJMSC on the apoptosis of H9C2 cells induced by myocardial I/RI.Methods:The second-generation human WJMSC frozen in liquid nitrogen tanks were resuscitated and subcultured after confluence to 80%-90%.The expression of surface markers of the third-generation human WJMSCs was detected by flow cytometry.WJMSC was divided into normoxic culture and hypoxic pretreatment,and the culture supernatants were collected to obtain normoxic and hypoxic conditioned medium,respectively.Rat H9C2 cells were divided into four groups:normoxic culture（control）group,model（H/R）group,normoxic conditioned medium（N-CM）group and hypoxic conditioned medium（H-CM）group.H9C2 cells were cultured in 1%O₂ and serum-free for 6 h,then cultured in 21%O₂ and complete medium for 24 h to mimic myocardial I/RI in vitro.After reperfusion,H9C2 cells were intervened for 24 h with conditioned medium.The morphology of H9C2 cells at each stage was observed under an electron microscope,the lactate dehydrogenase activity in the culture medium of each group was detected by lactate dehydrogenase commercial kit,and the apoptosis rate of H9C2 cells in each group was detected by flow cytometry.The expression of apoptosis-related proteins（BAX,Bcl-2）and autophagy-related proteins（p62,Beclin-1,LC3）was detected by Western Blot.Results:（1）Flow cytometry results show that human WJMSC highly express CD44,CD105,CD73,CD90,but did not express CD45,CD34,and HLA-DR;（2）There was no significant change in cell morphology after hypoxia pretreatment of WJMSC under electron microscope;（3）The LDH activity in the supernatant of the H/R group was significantly higher than that of the control group,and lower after treatment with N-CM and H-CM;（4）H9C2 cells at each stage showed irregular shape and no significant change in morphology under electron microscope;（5）The apoptosis of H9C2 in the H/R group was significantly higher than that in the control group as detected by flow cytometry（P<0.05）,and went down after treatment with N-CM and H-CM,and the decrease in H-CM group was more significant;（6）Western Blot results showed that the expression of protein Bax was significantly increased,the Bcl-2 was significantly decreased in the H/R group vs control group;however,the expression of Bax in the H-CM group was significantly decreased,the Bcl-2 was increased vs H/R group;（7）Western Blot results showed that compared with control group,the expression of p62 in H/R group was decreased,and the Beclin-1 and LC3II/LC3I were increased;Compared with H/R group,the p62 in the H-CM group was increased,Beclin-1 and LC3II/LC3I were significantly decreased.Conclusion:The conditioned medium of human WJMSC can reduce apoptosis after myocardial ischemia/reperfusion injury,and this protective effect may be related to the inhibition of intracellular autophagy by the conditioned medium of WJMSC,and the protective effect of H-CM was even stronger.