Effect of Shenfu Injection(参附注射液)on SIRT1 Deacetylation-modified Regulation of HMGB1/TLR4/NF-κB Pathway in Isoproterenol-induced Cardiomyocyte Injury Model
Objective To investigate the effect and possible mechanism of Shenfu Injection(参附注射液)on rat cardiomyocyte injury induced by isoproterenol from the perspective of regulating the high mobility group protein B1(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor κB(NF-κB)pathway through the deacetylation modification of silent information regulator 1(SIRT1).Methods The optimal concentration and intervention duration of isoprotere-nol hydrochloride and the optimal intervention concentration of Shenfu Injection were screened out by CCK-8 method.Log-arithmically growing H9c2 rat cardiomyocytes were taken at 5×104 cells/well and divided into normal group,model group,Shenfu Injection group,and SIRT1 inhibitor group,with 3 replicates in each group.Except for the normal group,the cells in the other groups were induced by isoproterenol hydrochloride to establish a chronic heart failure cell model.After modeling,the Shenfu Injection group was given Shenfu Injection at the optimal intervention concentration,and the SIRT1 inhibitor group was given 1 µmol/L of SIRT1 inhibitor EX-527,for optimal intervention duration.CCK-8 assay was used to detect the cell activity and calculate the inhibitory rate.ELISA assay was used to detect the nicotin-amide adenine dinucleotide oxidation state/nicotinamide adenine dinucleotide reduction state(NAD+/NADH)in car-diomyocytes.Immunofluorescence was used to detect the immunofluorescence localization of HMGB1 and SIRT1 in cardiomyocytes.Western blotting was used to detect the protein expression of acetylated HMGB1 in cardiomyocytes,HMGB1 in the nucleus and cytoplasm,and SIRT1,TLR4,myeloid differentiation factor 88(MYD88)and NF-κB p65 in cardiomyocytes.RT-qPCR was used to detect the mRNA expression of SIRT1,HMGB1,TLR4,MYD88 and NF-κB p65 in cardiomyocytes.Results The optimal intervention concentration of isoproterenol hydrochloride was 300 µmol/L,and the intervention duration was 48 hours;8％was the optimal intervention concentration of Shenfu Injection.Compared to those in the normal group,the cell activity,NAD+/NADH value,nuclear HMGB1 protein ex-pression,cardiomyocyte SIRT1 protein and mRNA expression in the model group decreased,while the cell inhibition rate,cardiomyocyte acetylated HMGB1 and cytoplasmic HMGB1 protein expression,cardiomyocyte TLR4,MYD88,NF-κB p65 protein and mRNA expression all increased(P＜O.05);fluorescence localization showed that the content of HMGB1 in cardiomyocytes in the model group increased and was localized in both the nucleus and cytoplasm.Com-pared to the model group and the SIRT1 inhibitor group,the Shenfu Injection group showed significant improvements in all the above indicators(P＜O.05);fluorescence localization showed that the SIRT1 content increased in the Shenfu injection group,while the HMGB1 content decreased,and was mainly located in the nucleus.Conclusion Shenfu Injection can improve myocardial cell damage by increasing SIRT1 expression to reduce the acetylation level of HMGB 1,regulating the HMGB1/TLR4/NF-κB pathway and inhibiting the nuclear translocation of HMGB1.
chronic heart failuremyocardial cellsShenfu Injection(参附注射液)silent information regulator 1deacetylationhigh mobility group protein B1
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