| ObjectiveTo observe the effects of Trilobatin on oxidative stress,inflammatory reaction and apoptosis of rat cardiomyocyte H9c2 during hypoxia/reoxygenation injury;To explore the specific mechanism of the protective effect of Trilobatin on hypoxia/reoxygenation injury of H9c2 cells.MethodsRat H9c2 cells were cultured in vitro,and a hypoxia/reoxygenation model was constructed using cobalt chloride(CoCl2)and fresh culture medium.The cardiac myocytes that grew well and were in logarithmic growth phase were randomly divided into control group(Con),hypoxia/reoxygenation group(H/R),Trilobatin group(TLB+H/R),TAK-242 group(TAK-242),Trilobatin+TAK-242 group(TLB+TAK-242);The cell survival rate was measured by CCK-8 method;The level of ROS was detected by DCFH-DA fluorescence probe technique;The content or activity of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were measured by colorimetry;The apoptosis rate was detected by Annexin V-FITC/PI double staining flow cytometry;Related proteins:eNOS,TNF-α、IL-1 β、IL-6、COX-2、Bax、Bcl-2、Caspase3、TLR4、MyD88、NF-κB p65 expression level were measured by Western blot.ReaultsCCK-8 experimental results show that when the concentration of cobalt chloride(CoCl2)is 800 μmol/L,the survival rate of H9c2 cells was about 50%(IC50)after reoxygenation(H/R)after 22 hours of hypoxia;When the concentration of Trilobatin is 50 μmol/L,H9c2 cells were pretreated in vitro for 24 h,and then the H/R state was constructed.Compared with the H/R group,the cell survival rate was significantly improved(P<0.05).Using DCFH-DA fluorescence probe to detect ROS level,the results showed that the ROS level of cells in H/R group was significantly higher than that in Control group(P<0.05);Compared with H/R group,TLB group could significantly reduce ROS expression level(P<0.05);Compared with the H/R group,the ROS levels in the TAK-242 group and TLB+TAK-242 group were significantly reduced(P<0.05),and there was no significant difference between the two groups(P>0.05).The content and activity of MDA,SOD and GSH-Px were detected by colorimetry.Compared with the control group,the content of MDA in H/R group increased,and the activity of SOD and GSH-Px decreased significantly(P<0.05);Compared with H/R group,the content of MDA in TLB group was significantly decreased,and the activities of SOD and GSH-Px were significantly increased(P<0.05);Compared with the H/R group,both the TAK-242group and the TLB+TAK-242 group could significantly reduce the content of MDA and significantly increase the activity of SOD and GSH-Px(P<0.05),and there was no significant difference between the two groups(P>0.05).The expression level of eNOS protein was detected by Western blot.The results showed that the expression level of eNOS protein in the control group was lower,and the expression level of H/R group was significantly higher than that in the control group(P<0.05).Compared with the H/R group,the expression level of TLB group was significantly lower(P<0.05);Compared with the H/R group,the expression level of eNOS protein in the TAK-242 group and TLB+TAK-242 group decreased significantly(P<0.05),and there was no significant difference between the two groups(P>0.05).Western blot results showed that compared with the control group,TNF-α,IL-1β,The level of IL-6 increased significantly under H/R status(P<0.05);Compared with H/R group,TLB group can significantly reduce the expression level of the above inflammatory factors(P<0.05);Compared with H/R group,TAK-242 group can significantly reduce the expression level of the above inflammatory factors(P<0.05),and there is no statistical difference between the two groups(P>0.05).The results of Annexin V-FITC/PI double staining flow cytometry showed that the apoptosis rate of H/R group was significantly higher than that of Control group(P<0.05);The apoptosis rate of TLB group was significantly lower than that of H/R group(P<0.05);The apoptosis rate of the TAK-242 group and TLB+TAK-242 group was significantly lower than that of the H/R group(P<0.05),and there was no statistical difference between the two groups(P>0.05).Western blot test showed that the expression of Bcl-2 protein in H/R group was significantly down-regulated(P<0.05),and the expression of Bax and Caspase3 protein was significantly up-regulated(P<0.05);Compared with the H/R group,the expression of Bcl-2 protein in the TAK-242 group and TLB+TAK-242 group was significantly decreased(P<0.05),and the expression of Bax and Caspase3 protein was significantly increased(P<0.05),and there was no statistical significance between the two groups of the above proteins(P>0.05).Western blot showed that the expression of TLR4,MyD88 and NF-κB p65 protein in the control group was low;The protein expression in H/R group was significantly higher than that in Control group(P<0.05);The protein expression of TLB group was significantly lower than that of H/R group(P<0.05);The protein expression of the above groups in the TAK-242 group and TLB+TAK-242 group was significantly lower than that in the H/R group(P<0.05),and the difference was not statistically significant(P>0.05).ConclusionsTrilobatin can reduce the oxidative stress,inflammatory reaction and apoptosis of H9c2 cells in hypoxia/reoxygenation injury,and its mechanism may be related to the inhibition of TLR4/MyD88/NF-κB signal pathway. |
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