Inhibitory Effects Of Oxymatrine On Transdifferentiation Of Cardiac Fibroblast To Myofibroblast Induced By Aldosterone Via Mediating The Keap1/Nrf2 Signaling Pathways In Vitro

Objective: To investigate the inhibitory effects of oxymatrine(OMT)on transdifferentiation of neonatal Sprague Dawley rat cardiac fibroblasts to myofibroblast induced by aldosterone(ALD)in vitro,and then explore the underlying mechanisms.Methods: The heart tissue of 1-2 d SD rats digested with 0.08% trypsin,and the CFs was obtained for primary culture after 1.5 h differential adherent.The primary cellular morphology was observed by inverted microscope.The CFs was identified by vimentin immunocytochemistry.Nrf2 agonist curcumin and Nrf2 siRNA were used to further investigate the relationship between the inhibitory effects of OMT on transdifferentiation of cardiac fibroblasts into myofibroblasts induced by ALD and Nrf2.The optimal dose-effect and time-effect of ALD induced CFs proliferation were determined by MTT,and the effects of different dose of OMT,spirolactone(Spiro)and Curcumin on ALD induced CFs proliferation were also determined by MTT.The experiment was divided into 6 groups: Control group(Control,serum free DMEM),ALD group(ALD,0.1 μmol/L),OMT low dose group(OMT-L,OMT 18.9μmol/L+0.1μmol/L ALD),OMT high dose group(OMT-H,OMT 37.8μmol/L+0.1μmol/L ALD),Spironolactone group(Sprio 1μmol/L+0.1μmol/L ALD),Curcumin(Curcumin 10 μmol/L+0.1μmol/L ALD).The CFs were transfected with Nrf2 siRNA with Lipofectamine 2000(Invitrogen)were randomly divided into 6 groups as following: Control group(Control,serum free DMEM),negative control(NC),ALD group(ALD,0.1 μmol/L),OMT-H(OMT 37.8μmol/L+0.1μmol/L ALD),Transfection group(Nrf2 siRNA+0.1μmol/L ALD),Transfection + OMT-H group(Nrf2 siRNA +0.1μmol/L ALD +37.8μmol/L OMT-H).Gimesa staining was performed to evaluate morphological changes of CFs.scratch analysis was used to assess CFs migration capacity.Cell cycle assay was carried out by flow cytometry analysis.The level of hydroxyproline(Hyp)in supernatant of CFs was determined using a commercial Hyp detection kit,the protein expressions of Collagen I,Collagen III,fibronectin(FN),connective tissue growth factor(CTGF),a-smooth muscle actin(a-SMA),mineralocorticoid receptor(MR),Nrf2 in nucleus,Nrf2 in cytoplasm,Keap1 in cytoplasm and Heme oxygenase-1(HO-1)was detected by Western blotting.Immunofluorescence staining was used to observe the expression of a-SMA protein.The mRNA expression of Nrf2 was detected by qRT-PCR.Nrf2 siRNA was used to explore the role of Nrf2 in OMT-treated CFs.GSH,SOD,and MDA levels were measured by colorimetric assay with commercial kits.The DCFH-DA fluorescent probe was used to assess cellular ROS levels.Results: Under inverted microscope,the primary CFs morphological characteristics were following: closely arrang,fusiform and polygonal,and no spontaneous beats.The purity of cultured CFs was more than 95% by vimentin immunocytochemistry.MTT assay showed that the optimal ALD concentration and treatment time was determined as 0.1μmol/L for 24 hours.The results confirmed that pre-incubation OMT and MR antagonist Spiro and Nrf2 agonist curcumin inhibited CFs proliferation effect induced by aldosterone.Gimsa staining showed that the violet of nucleus and light pink of cytoplasm of CFs,compared with the control group,ALD exposure remarkably increased the number of CFs and closely arranged,which was attenuated by pre-incubation with OMT,spironolactone and curcumin.The scratch results showed that ALD exposure enhanced migration ability of CFs compared to the control group.Pre-incubation with OMT,spironolactone and curcumin could significantly alleviate the migration ability of CFs induced by ALD.Flow cytometry showed that ALD-induced greater increase of CFs in S phase compared with the control group.Pretreatment with OMT,spironolactone and curcumin reduced the number of CFs in the S phase of the cell cycle following ALD-stimulation.After exposure to ALD,the Hydroxyproline content was significantly increased in medium compared with the control group.Compared with ALD group,OMT,spironolactone and curcumin significantly alleviated the ALD-induced Hydroxyproline secretion.Western blot results showed that the fibrosis associated protein of Collagen I,Collagen III,FN,a-SMA,CTGF and MR expression levels were remarkably increased induced by ALD compared to the control group,OMT attenuated the protein of ALD-induced Collagen I,Collagen III,FN,a-SMA,CTGF and MR,as well as spironolactone and curcumin.A similar trend was confirmed in immunofluorescent staining for α-SMA by fluorescence microscope.Compared with control group,ALD exposed to CFs,the expression of Nrf2 in nucleus and HO-1 were significantly decreased,and the protein of Nrf2 and Keap1 in cytoplasm expression levels were remarkably increased in the ALD group compared with control group by western blot assay.Pretreatment with OMT,spironolactone and curcumin significantly decreased the protein levels of ALD-induced Nrf2 and Keap1 protein in cytoplasm increasing and enhanced ALD-reduced the protein of Nrf2 in nucleus and HO-1 expression levels.The Nrf2 protein in cytoplasm,Keap1 in cytoplasm,HO-1,and Nrf2 in nucleus exhibited no significant difference between curcumin group and OMT + curcumin group.RT-PCR results showed that ALD induced Nrf2 mRNA decreased in CFs.Compared with ALD group,pretreatment with OMT,spironolactone and curcumin also reversed the ALD-induced reduced Nrf2 mRNA expression.The transfection efficiency of CFs transfected with Nrf2 siRNA was 60.15%.OMT significantly decreased ALD-induced increased protein of α-SMA,CTGF,collagen I and collagen III expression levels.However,the attenuation effect of OMT was reversed by Nrf2 siRNA.The α-SMA,CTGF,collagen I and collagen III protein expression exhibited no significant difference between Nrf2 siRNA groups and OMT + Nrf2 siRNA groups.A similar trend was confirmed in immunofluorescent staining for α-SMA by fluorescence microscope.The results of fluorescence probe DCFH-DA showed that ALD significantly increased ROS level in CFs.Pretreatment with OMT,spironolactone and curcumin decreased ALD-induced ROS level,as well ROS scavenger N-acetylcysteine(NAC).According to the results of Kit,ALD exposed to CFBs,the level of MDA was significantly increased compared with control group,the GSH and SOD activity of CFs was significantly decreased in ALD stimulation.Pretreatment with OMT,spironolactone and curcumin significantly decreased ALD-induced MDA increasing and enhanced ALD-reduced GSH level and SOD activity.Conclusion: OMT alleviating transdifferentiation of cardiac fibroblast to myofibroblast induced by aldosterone and its mechanism may be involved in activating the Nrf2/Keap1 pathway.