Effects Of Lingguizhugan Decoction-containing Serum On Myocardial Cell Injury Induced By H₂O₂ And Its Effect On Rho/rock Signaling Pathway

Background and Objective chronic heart failure（CHF）is an end-stage clinical syndrome caused by all disorders of the heart tissue and circulatory system,whose initial cause is usually myocardial remodeling（MR）,and its main pathological basis are often myocardial fibrosis（MF）and myocardial hypertrophy.However,such pathological changes mostly start from the damage of cardiomyocytes caused by various reasons.Linggui Zhugan Decoction（LGZGD）is a classic traditional Chinese medicine prescription of warming Yang to promote urination,which has been found more and more relevant biological and pharmacological evidences for alleviation of clinical symptoms of CHF in recent years,such as anti-inflammation,anti-oxidation,inhibition of excessive activation of cytokines,inhibition of myocardial remodeling,and protection of myocardial cells.However,the related molecular mechanisms and pathways need to be further studied.This study was to explore the effect of LGZGD drug containing serum on cell viability,oxidative stress,cell cycle arrest,myocardial fibrosis,Rho molecules and its downstream related coiled coil kinase（Rho-associated coiled helix kinase,ROCK）protein expression in the process of H9c2 myocardial cell injury induced by H₂O₂.To further explore whether LGZGD drug containing serum has a protective effect on oxidative stress injury in H9c2 myocardial cell by regulating Rho/ROCK,and to provide a new laboratory basis for its anti-heart failure effect.Methods H9c2 myocardial cell were selected as the research object.（1）H9c2 myocardial cell cultured in vitro were inoculated into 6-well plates and divided into normal control group and model group,with three holes in each group.After the myocardial cell was induced successfully by 100mM H₂O₂for 0.5h,and detecting its morphological observation and cell viability.（2）The oxidative stress model cells were treated with 20%LGZGD drug containing serum and 20%blank serum respectively,and seeded in 96-well plates at a certain concentration.Detecting the cell viability by CCK8,and hydroxylamine method was used to detect the activity of superoxide dismutase（SOD）.TBA method was used to detect malondialdehyde（MDA）,Western blot was used to detect RhoA and ROCK protein expression,flow cytometry was used to detect cell cycle.（3）The oxidative stress model cells were incubated with 20%LGZGD drug containing serum,20%blank serum and FAS 0.1mM for 24h,respectively.CCK8 method was used to detect the cell viability after Rho/ROCK pathway inhibition combined with drug treatment,and hydroxylamine method was used to detect the activity of SOD.TBA was used to detect MDA content.Western blot was used to detect the protein expression of RhoA and ROCK in the Rho/ROCK signaling pathway.Results The morphological evidence showed that after treatment with 100mM H₂O₂for0.5h,the growth shape of some cells changed from long spindle shape to irregular shape,and the cell volume increased.However,the condition of LGZGD cells treated with drug-containing serum was better than that of the model group,and the volume was reduced.After co-treatment with FAS and LGZGD drug containing serum,the number of cells from long spindle shape to irregular shape was significantly reduced,and the cell volume was not significantly increased.（2）The cell viability and SOD activity of the model group were significantly decreased,and the G0/G1 phase cell arrest was increased（P<0.01,P<0.05）,the oxidative stress marker MDA content and the expression of Rho and ROCK proteins were increased（P<0.01）;However,LGZGD drug containing serum and 0.1mM FAS could inhibit H₂O₂-induced oxidative stress in H9c2cardiomyocytes to varying degrees,and the cell viability and SOD activity were increased（P<0.05）,and the proportion of cells in G0/G1 phase was significantly decreased while that in G2/M phase was increased（P<0.01）,MDA content was decreased and the expression of Rho and ROCK proteins was inhibited（P<0.01）;（3）After treatment with FAS inhibitor,the cell activity and SOD activity of negative control and observation 2 group were enhanced compared with model group and observation group 1（P<0.05,P<0.01）,the cell arrest of G0/G1 phase was alleviated while in G2/M phase was increased（P<0.01,P<0.05）.The content of MDA and the expression of Rho and ROCK protein were inhibited（P<0.01）.Conclusions（1）LGZGD drug containing serum can protect H9c2 cells from H₂O₂-induced oxidative stress injury by increasing SOD activity and reducing MDA content and scavenging oxygen free radicals effectively.It can also inhibit and reduce cell apoptosis,cell cycle arrest and fibrosis caused by oxidative stress,thereby improving myocardial cell function and delaying the progression of heart failure.（2）Both LGZGD drug containing serum and Rho/ROCK pathway inhibitor FAS can prevent the activation of Rho/ROCK signaling pathway by inhibiting the protein expression of RhoA molecule and its downstream substrate ROCK,suggesting that LGZGD drug containing serum can be used as a new Rho kinase inhibitor of traditional Chinese medicine to protect myocardial cell,delay myocardial remodeling and fight heart failure.