Antagonistic Effects And Mechanisms Of PhG-RE On H₂O₂-Induced ERS Damage In H9c2 Cells

Phenylethanoid glycoside-rich extract from Cistanche deserticola,（PhG-RE）is a kind of chemical component extracted from herba cistanche,a medicinal plant of Orobanchaceae.Previous studies showed that PhG-RE plays a good protective effect on myocardial apoptosis caused by ischemia reperfusion.However,it is unclear whether its protective effect on myocardial cells is related to the regulation mechanism of endoplasmic reticulum stress（ERS）damage.In this experiment,ERS damage model is established by inducing H9c2 myocardial cells with H₂O₂ to investigate the protective effect of PhG-RE pretreatment on ERS damage of myocardial cells and the related molecular mechanism.The research contents are as follows.1.Experimental methods:PhG-RE of the concentrations of 50,100,150,200,250 and 300μg·mL^-11 are respectively used to treat H9c2 cells for 24h.The effects of different concentrations of PhG-RE on cell survival are tested by CCK-8 assay to determine the safe range of drug concentration,which is then based to set the drug concentration gradients.Next,PhG-RE of the concentrations of 50,100,150,200 and250μg·mL^-1 are respectively used to treat the cells for 24 h.Then 100μmol·mL^-1H₂O₂ is added to the cells to induce cell damage.The cell survival rate is measured by CCK-8 assay to determine the optimal administration concentration of PhG-RE.Then required by the experiment,the cells are divided into control group,PhG-RE group,ERS damage model group（H₂O₂ group）,（PhG-RE）-H₂O₂ group,ERS inhibitor（4-PBA）-H₂O₂ group,ERS activator TG group and（PhG-RE）-TG group.The release amount of LDH is detected to assess the degree of cell damage.After that,Annexin V/PI double staining flow cytometry is employed to identify apoptotic cells at different phases and detect cell apoptosis.RT-q PCR is applied to detect the expression levels of GRP78,CHOP,JNK and Caspase-12 in the groups.Meanwhile,the expressions of proteins such as GRP78,CHOP,p-JNK and Caspase-12 in the groups are detected by Western Blot.2.Experimental results:when the administration concentration of PhG-RE is≤250μg·mL^-1,the cell survival rate is not affected by the drug（P>0.05）,and this concentration range is the safe drug concentration range,based on which the drug concentration gradients are established to treat the cells.Similar to H₂O₂ group,when the concentration of PhG-RE in PhG-RE group is 150 or 200μg·mL^-1,cell apoptosis can be effectively reduced（P<0.01）,and the comparison between groups showed no significant difference in the inhibiting effect of apoptosis between the two groups（P>0.05）.So 150μg·mL^-1 is selected as the administration concentration for follow-up experiment.According to the need of the experiment,grouping comparison is made and the results show that the cells pretreated with PhG-RE and ERS inhibitor 4-PBA for 24 h can significantly inhibit cell damage induced by H₂O₂（P<0.01）,reduce the release of LDH（P<0.01）and down-regulate the expression of mRNA of ERS damage and apoptosis mechanism related genes,including GRP78,CHOP and Caspase-12（P<0.01）,PhG-RE and 4-PBA have similar roles;PhG-RE can reduce the expression of mRNA of genes such as GRP78,JNK and Caspase-12,which is induced by ERS activator TG（P<0.01）.The results of Western blot test showed that PhG-RE and4-PBA play a similar role of significantly reducing the expression of proteins including GRP78,CHOP,p-JNK and Caspase-12 in the cell ERS damage process induced by H₂O₂ and ERS activator TG（P<0.01）,indicating that PhG-RE can antagonize cell damage and apoptosis induced by ERS by inhibiting the expression of proteins related to the apoptotic mechanism of ERS.3.Conclusion:The experimental results confirm that PhG-RE can enhance cell survival ability,reduce LDH release amount after H₂O₂-induced cell damage,and effectively reduce apoptosis rate.Meanwhile,by regulating the expression of mRNA of GRP78,Caspase-12 and CHOP as well as GRP78,CHOP,Caspase-12,p-JNK and other proteins in the apoptotic mechanism of ERS,PhG-RE plays a similar cell protective effect to ERS inhibitor 4-PBA and antagonizes the damage and apoptosis of cardiac myocytes caused by ERS.