Protective Effects And Mechanisms Of Rhein On Myocardial Cell Injury Induced With H₂O₂

Objective:This study was designed to clarify the protective effects of Rhein on myocardial cell injury induced with H2O2 and to explore its possible molecular mechanisms.Method:Rat cardiomyocytes H9c2 were cultured in vitro and stimulated by H2O2(300 ?mol/L)to establish a model of cardiomyocyte injury.The experiment was randomly divided into five groups:control group,H2O2 group,Rhein groups with different concertrations(0.25 ?g/mL,0.5?g/mL,1 ?g/mL).MTT assay was performed to examine the effects of rhein on H9c2 activity.EdU assay was used to examine the effects of rhein on the proliferation of H9c2 cells.Colony formation assay was used to examine the effects of rhein on the colony formation of H9c2 cells.The effects of rhein on apoptotic level of H9c2 cells was detected by flow cytometry;mRNA expression levels of Bax,Bel-2,Caspase-3,Caspase-9 were detected by qRT-PCR,and the apoptosis-related proteins including Bax,Bel-2,Caspase-3,and Caspase-9 were evaluated with Western blot assay.In addition,the effect of rhein on the expression of caspase-3 in H9c2 cells was detected by immunofluorescence assay.Besides,ELISA assay was used to detect the effects of rhein on the expressions of inflammatory related factors in H9c2 cells such as IL-1?,IL-1?,TNF-? and IL-6.The effects of rhein on ROS expression in H9c2 cells were detected by fluorescent probe,and biochemical method was used to evaluate the effects of rhein on the expressions of CAT,GSH-Px,SOD and MDA in H9c2 cells.Finally,western blot assay was used to determine the role of rhein in the expression levels of MAPK and NF-?B signaling pathways.Excel database was established for the research data,and statistical processing was carried out.Research results:1.Establishment of oxidative damage in H9c2 cells:After H9c2 cells treated with 300?mol/L H2O2 for 2h,the cell damage was obvious,and the cell survival rate was stable and moderate,so the oxidative damage model of cardiomyocytes was established with this concentration.2.Cell viability:MTT results showed that H2O2 significantly inhibited the cell viability of H9c2 cells compared with control group,and H9c2 activity was significantly increased after rhein intervention at various concentrations in a dose-dependent manner.3.Cell proliferation ability:EdU results showed that H202 could significantly inhibit the proliferation of H9c2 cells compared with the control group.After the intervention of rhein,the proliferation of Hgc2 eells was signifieantly enhanced in a dose-dependent manner.The eolony formation resxults showed that H2O2 could significantly inhibit the colony formation of H9c2 cells compared with the control group.Force,after intervention with different concemtrations of rhein,the colony formation of H9c2 cells was significantly enhanced and showed a dose-dependent relationship.4.Cell apoptosis:The results of flow cytometry showed that H2O2 could significantly promote the apoptosis of H9c2 cells compared with the control group.The apoptotic of H9c2 cells was signifieantly reduced after the intervention of rhein with a dose-dependent relationship.RT-qPCR data suggested that H2O2 could promote the mRNA expressions of Bax,Caspase-3 and Caspase-9,and inhibit the mRNA expression of Bcl-2 in H9c2 cells compared with those in the control group,while rhein could significantly inhibit the mRNA expressions of Bax,caspase-3 and caspase-9,and promote the mRNA expression of Bcl-2.The expressions of apoptosis-related proteins in H9c2 cells were detected by Western blot assay,and the results showed that H2O2 could significantly increased Bax,Caspase-3 and Caspase-9 protein expressions and inhibited Bcl-2 protein expression in H9c2 cells compared with those in control group,while rhein treatment could obviously inhibited protein expressions of Bax,Caspase-3 and Caspase-9,and promoted the expression of Bcl-2 protein.Moreover,immunofluorescence experiments showed that H2O2 could significantly promote the expression of Caspase-3 in H9c2 cells compared with that in the control group,while rhein could significantly inhibit the expression of caspase-3.5.Inflammation response:ELISA assay was performed to detect the expression levels of inflammation-related factors including IL-1?,IL-1?,TNF-? and IL-6,and the results demonstrated that H2O2 could promote the expressions of IL-1?,IL-1?,TNF-? and IL-6 in H9c2 cells compared with the control group.After the intervention of rhein,the expression levels of IL-1?,IL-1?,TNF-? and IL-6 in H9c2 cells were decreased significantly in a dose-dependent manner.6.Oxidative stress:The results of fluorescent probe experiments showed that H2O2 could promote ROS expression in H9c2 cells compared with control group,but the ROS expression in in H9c2 cells was significantly decreased after the intervention of rhein in a dose-dependent manner.Biochemical tests showed that H2O2 could increase MDA content,and decrease the expressions of GSK-Px,CAT and SOD in H9c2 cells compared with control group,while the MDA content was significantly decreased and the expressions of GSK-Px,CAT and SOD were significantly increased in the Rhein groups at dose-dependent manner.7.Signaling pathways:Compared with the control group,H2O2 can activate MAPK and NF-?B signaling pathways in H9c2 cells,while rhein can significantly inhibit the expressions of MAPK and NF-?B signaling pathways in a dose-dependent manner.Research conclusions:1.After treated with a certain concentration of H2O2 for 2 h,the H9c2 cells were damaged obviously,and the model of H9c2 oxidative damage was established.H2O2 can significantly inhibit the viability and proliferation capacity of H9c2 cells compared with the control group.H2O2 can promote the apoptosis of H9c2 cells compared with the control group.H2O2 can promote the expressions of inflammation-related factors in H9c2 cells compared with the control group.H2O2 can promote the oxidative stress injury of H9c2 cells compared with the control group.2.Rhein can significantly enhance the viability and proliferative capacity of H9c2 cells induced by H2O2.Rhein can significantly reduce the apoptotic of Hgc2 cells induced by H2O2.3.Rhein can significantly inhibit the inflammatory response of H9c2 cells induced by H2O2.Rhein can significantly increase the ability of H9c2 cells to scavenge oxygen free radicals and reactive oxygen species.4.Rhein can alleviate H2O2-induced cardiomyocyte injury,which may be achieved by inhibiting the expressions of MAPK and NF-?B signaling pathways.5.The role of rhein in protecting cardiomyocytes has been shown to be reliable in promoting cell proliferation,inhibiting cell apoptosis,inhibiting inflammatory response and anti-oxidative stress injury.The research results provide a theoretical basis for further research on the role of Rhein and Rhubarb in myocardial protection and cardiovascular disease prevention.