Effects Of Cinnamaldehyde Pretreatment On Autophagy Of H9C2 Cardiomyocytes After Hypoxic Reoxygenation

Objective To investigate whether Cinnamaldehyde(CA) pretreatment can reduce the damage of H9C2 cardiomyocytes induced by hypoxia and reoxygenation through autophagy pathway.Methods In the experiment,rat H9C2 cardiomyocytes were routinely cultured in vitro,and cells in logarithmic growth phase were treated with hypoxia 2h/reoxygenation 4h to simulate myocardial ischemia/reperfusion(I/R) to establish hypoxia Hypoxia/Reoxygenation(H/R) damage model.Select the concentration of 0 μM-100μM cinnamaldehyde to pre-treat the cells for 2h,4h and 6h respectively,and use cell counting kit(CCK-8)to detect the cell viability of each group after H/R,and determine the best administrat ion concentration and the time of administration which were used in subsequent experiments under this condition.Enzyme standard method was used to detect the effect of CA on lactate dehydrogenase(LDH) leakage rate in H/R cardiomyocyte culture fluid.Methyl adenine(3-MA) was an autophagy inhibitor and rapamycin(Rapa)is an autophagy enhancer.The cells were randomly divide d into five groups: normal control group(Sham group),Hypoxia /reoxygenation group(H/R group),Cinnamaldehyde group(H/R+CA group),3-MA group(H/R+3-MA,group),Rapa group(H/R+Rapa group);Monodansylcadaverine(MDC)fluorescence staining kit was used to observe the H/R induced autophagosome aggregation in each group of cells,and the average fluorescence intensity of each group of cells was detected;Hoechst 33342 fluorescence staining method was used to observe H/R the morphological changes of cell nucleus in each group and then the apoptosis rate were calculated;Western Blot detected protein the expression of Becli n-1,LC3 II/LC3 I,P62 and Bax/Bcl-2.Results1.Effect of CA pretreatment on myocardial cell H/R injury Compared with the Sham group,the LDH level in the cell supernatant was increased(P<0.01);compared with the H/R group,the cell viability was increased to different degrees after 0 μM-100 μM cinnamaldehyde treatment for 2h,4h,and 6h.And when the pretreatment time was 6 hours,the administration concentration reached a peak of 60 μM.The LDH test results showed that after 6hours of 60 μM cinnamaldehyde pretreatment,the LDH release in the supernatant was significantly lower than that in the H/R group(P<0.01).2.Results of MDC autophagy fluorescence detection and Hoechst33342 fluorescence detection.Compared with the Sham group,the autophagy fluorescence intensity of the H/R group increased(P<0.01),and the apoptosis rate increased(P<0.01).Compared with the H/R group,In the H/R+CA group the fluorescence average intensity of cells were significantly reduced(P<0.05),and the rate of apoptosis was reduced(P<0.01).Compared with the H/R group,the average fluorescence intensity of the cells in the H/R+3-MA group was significantly reduced(P<0.01),and the cells Apoptosis rate decreased(P<0.01);compared with the H/R group,the average fluorescence intensity of cells in the H/R+Rapa group increased(P<0.05),and the rate of apoptosis increased(P<0.05).3.Results of Westernblot detection related protein.Compared with the Sham group,Beclin-1 protein expression increased in the H/R group(P<0.01),LC3 II/LC3 I protein expression increased(P<0.01),P62 protein expression decreased(P<0.01),and Bax/Bcl-2 protein ratio Increased(P<0.01);compared with the H/R group,Beclin-1 protein expression decreased in the H/R+CA group(P<0.05),LC3 II/LC3 I protein expression decreased(P<0.01),and P62 protein levels increased High(P<0.01),Bax/Bcl-2 protein ratio decreased(P<0.01);compared with H/R group,Beclin-1 protein expression decreased in H/R+3-MA group(P<0.01),LC3 II/LC3 I protein expression decreased(P<0.01),P62 protein expression increased(P<0.01),Bax/Bcl-2 protein ratio decreased significantly(P<0.01);compared with H/R group,In the H/R+Rapa group,Beclin-1protein expression increased(P<0.05),LC3 II/LC3 I protein expression increased(P<0.05),P62 level further decreased(P<0.05),and Bax/Bcl-2 protein ratio increased(P<0.05).Conclusions The cinnamaldehyde pretreatment can improve cell viability after H/R,reduce LHD level and the damage of H9C2 cardiomyocyte caused by H/R,and inhibit excessive autophagy induced by H/R,reduce apoptosis.