Investigation Of Antagonistic Effect And Mechanism Of Curcumin On H9C2 Cardiomyocytes And Mouse Heart Injury Induced By Inorganic Arsenic

ObjectiveAs an environmental pollutant,arsenic（As）can enter the human body through drinking water and cause damage to the cardiovascular system.Some studies have shown that sodium arsenite（NaAsO₂）will lead to cell apoptosis,oxidative damage,and changes the function of heart,However,few studies focused on studying the bioactive substances that can protect against cardiotoxicity induced by sodium arsenite.Curcumin is a polyphenol compound extracted from turmeric,which has a variety of biological activities such as anti-oxidant and scavenging oxygen free radicals,Curcumin is widely used in the protection of heart and cancer diseases.In this study,C57BL/6N mouse,H9C2 cells and HEH2 cells were treated to observe their cell proliferation,apoptosis,oxidative stress injury,cardiac function indicators,and changes in genes such as Yes associated protein（YAP）、Tumor Necrosis Factor-α（TNF-α）、Transforming Growth Factor-β1（TGF-β1）and Matrix Metallopeptidase 2（MMP2）related to apoptosis myocardial remodelingt o explore whether curcumin has an antagonize effect on the cardiac toxicity of sodium arsenite.MethodsFirstly,H9C2 cardiomyocytes and HEH2 human embryonic cardiac fibroblasts were chosen as two cell models in vitro.CCK8 kit was used to detect cell activity to select the appropriate treatment and concentration of curcumin and sodium arsenite.H9C2 cells were divided into four groups:control group,curcumin group,sodium arsenite group,and the combined treatment group of curcumin and sodium arsenite.Caspase3/7 apoptosis kit was used to detect the apoptosis level of H9C2 cells.Oxidative stress damage of cells was detected by Glutathione（GSH）and Superoxide dismutase（SOD）oxidative stress kits.The expression levels of YAP,TGF-β1 and MMP2 genes were measured by RT-PCR and the protein expression levels of YAP,TGF-β1 and TNF-αwere determined by Western-blot.Secondly,in vivo experiments:32 healthy C57BL/6N mice were randomly divided into 4 groups,including control group（deionized water）,curcumin group（200mg/kg）,sodium arsenite group（50mg/L）,curcumin group（200mg/kg）and sodium arsenite combined treatment group（50mg/L）.Sodium arsenite was exposed to free drinking water.The mice were exposed to sodium arsenite in drinking water for 75 days.After the cardiac function of the mice was detected by echocardiography,HE staining was performed on the hearts of the mice and then the RNA was extracted from heart tissue and the oxidative stress indexes of glutathione peroxidase（GSH-Px）,GSH,SOD and hydrogen peroxide（H₂O₂）were detected.The expressions of YAP,TGF-β1,MMP2 and TNF-αgenes were detected by RT-PCR,and Western-blot was used to detect changes in the expression levels of YAP,TGF-β1 and TNF-αproteins.Results1.Cell experiment in vitro results have shown that sodium arsenite had a damaging effect on the activity of H9C2 cardiomyocytes,which was correlated with the concentration positively.However,The effect of curcumin on the viability of H9C2cardiomyocytes was increased at low concentration and inhibited at high concentration.In the experiment of combined treatment with curcumin and sodium arsenite,10μmol/L curcumin could alleviate the damage of cell viability induced by 15μmol/L sodium arsenite（P<0.05）.Caspase3/7 experiment results showed that curcumin pretreatment for 6h alleviated apoptosis induced by sodium arsenite treatment for 8h（P<0.05）.But this effect disappeared with the prolonged treatment time of sodium arsenite.Detection results of oxidative stress in H9C2 cells showed that curcumin treatment can increase the content of GSH in H9C2 cells（P<0.05）.Real time PCR results showed that the expression of TGF-β1 and MMP2 genes in H9C2 cells was reduced after treatment with sodium arsenite compared with the control group（P<0.05）.Compared with the sodium arsenite group,the expression of TGF-β1 and MMP2 genes increased after treatment with curcumin（P<0.05）.However,the combined treatment of curcumin and sodium arsenite could not alleviate the decreasing trend of TGF-β1 and MMP2 genes.Compared with the control group,curcumin treatment can increase the expression of YAP gene in H9C2 cardiomyocytes（P<0.05）.Protein detection results showed that compared with the control group,the expression of TGF-β1 protein after curcumin treatment had increased significantly（P<0.05）,while the expression of TNF-αprotein had a tendency to increase（P>0.05）.There was no significant difference between the combined treatment group of curcumin and sodium arsenite and the sodium arsenite group in YAP,TGF-β1 and TNF-α.2.The results of in vivo animal experiments showed that after 75 days of exposure,there were no significant difference in mouse body weight,heart weight,and organ coefficient between the groups.The results of echocardiography in mouse showed that the cardiac function index of mouse after sodium arsenite treatment showed a downward tendency,which was manifested by a decrease in the left ventricular ejection and the left ventricular shortening fraction compared with the control group.Compared with the sodium arsenite group,the LVEF and LVFS of the mouse treated with curcumin and sodium arsenite and curcumin group were increased（P>0.05）.These results indicated that curcumin can alleviate the decline of heart function caused by sodium arsenite.The hematoxylin-eosin staining results of cardiac histopathology showed that the morphology of cardiomyocytes was normal and there was no inflammatory cell infiltration after curcumin treatment.There were inflammatory cell infiltration,myocardial hypertrophy and loose arrangement can be observed in the sodium arsenite group,and a few inflammatory cells were infiltrated in the group treated with sodium arsenite and curcumin.Results of biochemical indicators showed that in the hearts of mouse the SOD activity was significantly reduced（P<0.05）and the content of GSH was decreased（P>0.05）and the content of H₂O₂ was increased（P>0.05）after treatment with sodium arsenite.There was no significant change in the activity of GSH-Px,ACP and GOTafter treatment with sodium arsenite.Compared with the sodium arsenite group,the SOD activity was significantly increased and ACP activity significantly decreased after treatment with curcumin and sodium arsenite（P<0.05）.Real time PCR results showed that compared with the control group sodium arsenite decreased the expression of TNF-αgene（P<0.05）,but had no significant effects on the expression of YAP,TGF-β1 and MMP2 genes.Curcumin can promote the expression of the YAP gene（P<0.05）,and decrease the expression of MMP2 and TNF-α（P<0.05）.Compared with the sodium arsenite group,curcumin can promote the expression of the YAP gene（P<0.05）,and decrease the expression of MMP2 gene.In the protein detection experiment,there were no significant changes in YAP protein and TNF-αprotein.Conclusion1.Under our experimental conditions,curcumin can antagonize the damage of H9C2 cardiomyocytes viability and apoptosis caused by sodium arsenite2.Curcumin can antagonize the damage of sodium arsenite to H9C2cardiomyocytes by increasing the content of GSH and the expression of YAP gene.3.In vivo experiments had shown that sodium arsenite can reduce heart function,and its cardiotoxicity was related to oxidative stress damage.Curcumin can improve heart function and relieve oxidative stress damage.Its antagonism may be related to the increase of myocardial regeneration-related gene YAP mRNA expression.