摘要
目的探讨硫酸吲哚酚(IS)通过有机阴离子转运蛋白-3(OAT-3)及氧化应激诱导心肌肥大及纤维化的作用。方法对大鼠心肌细胞(H9C2)进行传代培养,并分为四组:Control组、IS组、小干扰RNA(siRNA)阴性对照组(siOAT-3)及siOAT-3+IS组。Control组常规培养,不进行IS刺激;IS组加入含250μmol浓度的IS刺激;siRNA阴性对照组加入20 nmol/L OAT-3 siRNA;siOAT-3+IS组加入20 nmol/L OAT-3 siRNA 48 h后,加入IS刺激24 h。采用RT-PCR方法检测并分析各组细胞中OAT-3、NADPH氧化酶-4(Nox-4)、抗氧化蛋白[核因子E2相关因子2(Nrf-2)、血红素加氧酶1(HO-1)]、心肌肥大指标[心房钠尿肽(ANP)、脑钠肽(BNP)、β-肌球蛋白重链基因(β-MHC)]及纤维化指标[转化生长因子-β1(TGF-β1)、Smad同源物3(Smad-3)、Ⅰ型胶原蛋白(CollagenⅠ)]的mRNA表达水平。H9C2细胞进一步分为Control组、IS组以及N-乙酰半胱氨酸(NAC)组,NAC组以抗氧化剂NAC预处理后加入250μmol浓度的IS刺激24 h,采用RT-PCR试验方法检测上述指标的mRNA表达水平。结果IS组H9C2细胞中OAT-3的mRNA表达水平显著高于Control组,siOAT-3组及siOAT-3+IS组H9C2细胞中OAT-3的mRNA表达水平显著低于IS组(P<0.001)。相较于Control组,IS组H9C2细胞中Nox-4、ANP、BNP、β-MHC、TGF-β1、Smad-3以及CollagenⅠ的mRNA表达水平明显升高(均P<0.001),Nrf-2、HO-1的mRNA表达水平明显降低(均P<0.001)。相较于IS组,siOAT-3组及siOAT-3+IS组H9C2细胞中Nox-4、ANP、BNP、β-MHC、TGF-β1、Smad-3以及CollagenⅠ的mRNA表达水平明显降低(均P<0.001),Nrf-2、HO-1的mRNA表达水平明显升高(均P<0.001)。以NAC预处理H9C2细胞,能显著抑制IS诱导的Nox-4、ANP、BNP、β-MHC、TGF-β1、Smad-3以及CollagenⅠmRNA的高表达(均P<0.001),显著增高Nrf-2、HO-1 mRNA表达水平(均P<0.01)。结论IS在H9C2细胞中通过OAT-3和氧化应激诱导心肌肥大和纤维化因子高表达。
Objective To investigate the role of indoxyl sulfate(IS)in myocardial hypertrophy and fibrosis through organic anion transporter-3(OAT-3)and oxidative stress in H9C2 cells.Methods Rat myocardial cells(H9C2)were cultured and divided into four groups:Control group,IS group,siRNA negative control group(siOAT-3),and siOAT-3+IS group.The control group was cultured routinely without IS stimulation.The IS group was stimulated with 250μmol IS.The siRNA negative control group was transfected with 20 nmol/L OAT-3 siRNA,and the siOAT-3+IS group was transfected with OAT-3 siRNA 48 hours later,then stimulated with IS for 24 hours.Real-time polymerase chain reaction(RT-PCR)was used to detect and analyze the mRNA expression levels of OAT-3,NADPH oxidase-4(Nox-4),antioxidant proteins[nuclear factor E2 related factor 2(Nrf-2),heme oxygenase 1(HO-1)],myocardial hypertrophy markers[atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),β-myosin heavy chain gene(β-MHC)],and fibrosis markers[transforming growth factorβ1(TGF-β1),Smad homologous protein 3(Smad-3),collagen typeⅠ(CollagenⅠ)]in each group.H9C2 cells were further divided into three groups:Control group,IS group,and N-acetylcysteine(NAC)group.The NAC group was pre-treated with the antioxidant NAC before stimulation with 250μmol IS for 24 hours.RT-PCR was used to detect the mRNA expression levels of the above indicators.Results The mRNA expression level of OAT-3 in the IS group was significantly higher than that in the control group,while the mRNA expression levels of OAT-3 in the siOAT-3 group and the siOAT-3+IS group were significantly lower than those in the IS group(all P<0.001).Compared with the Control group,the mRNA relative expression levels of Nox-4,ANP,BNP,β-MHC,TGF-β1,Smad-3,and CollagenⅠin H9C2 cells of the IS group were significantly increased(all P<0.001),while the mRNA expression levels of Nrf-2 and HO-1 were significantly decreased(all P<0.001).Compared with the IS group,the mRNA relative expression levels of Nox-4,ANP,BNP,β-MHC,TGF-β1,Smad-3,and CollagenⅠin H9C2 cells of the siOAT-3 group and the siOAT-3+IS group were significantly lower(all P<0.001),while the mRNA expression levels of Nrf-2 and HO-1 were significantly increased(all P<0.001).Pre-treatment with NAC significantly inhibited the high expression of Nox-4,ANP,BNP,β-MHC,TGF-β1,Smad-3,and CollagenⅠmRNA induced by IS(all P<0.001),while significantly increasing the mRNA expression levels of Nrf-2 and HO-1(all P<0.01).Conclusions IS promotes myocardial hypertrophy and fibrotic factor overexpression in H9C2 cells through OAT-3 and oxidative stress.
作者
买买提艾力·艾则孜
杜宇奎
买买提·依斯热依力
邵劲杰
刘筠
张总刚
Maimaitiaili Aizezi;Du Yukui;Maimaiti Yisireyili;Shao Jinjie;Liu Jun;Zhang Zonggang(Department of Cardiac Surgery,People′s Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China;Medical Research and Transformation Center,People′s Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China)
出处
《中国医师杂志》
CAS
2023年第10期1484-1489,共6页
Journal of Chinese Physician
基金
新疆维吾尔自治区自然科学基金(2020D01C090,2021D01C175)
