Myostatin通过ERK1/2-PPAR-γ通路抑制猪皮下脂肪前体细胞成脂分化的研究

Myostatin inhibits adipogenic differentiation via the ERK1/2-PPAR-γsignaling pathway in porcine subcutaneous preadipocytes

旨在探讨细胞外信号调节激酶1/2-过氧化物酶体增殖物激活受体-γ(ERK1/2-PPAR-γ)信号通路在肌肉生长抑制素(MSTN)抑制猪皮下脂肪前体细胞(PSPAs)成脂分化中的作用。PSPAs在诱导分化培养液(DIM)处理同时,随机分3组:对照组(0 ng/mL MSTN)、100 ng/mL MSTN组(100 ng/mL MSTN)以及ERK1/2特异性抑制剂PD98059和100 ng/mL MSTN联合组(PD98059+100 ng/mL MSTN)。采用细胞计数试剂盒-8(CCK-8)检测细胞增殖活力,油红O染色法及甘油三酯(TG)定量分析成脂分化,使用荧光定量PCR(RT-qPCR)和Western blot法检测过氧化物酶体增殖物激活受体γ(PPAR-γ)、CCAAT/增强子结合蛋白-α(C/EBP-α)、脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC)、细胞外信号调节激酶(ERK)及其磷酸化ERK(p-ERK)表达。结果表明:细胞增殖活力在3组间无显著性差异(P>0.05)。与对照组相比,100 ng/mL MSTN处理48 h后细胞内脂滴沉积量、油红O阳性细胞数及TG含量均显著降低(P<0.05),PPAR-γ、C/EBP-α、FAS和ACC的mRNA表达量均显著下降(P<0.05),p-ERK/ERK比值显著增加(P<0.05);与100 ng/mL MSTN组相比,PD98059+100 ng/mL MSTN组p-ERK/ERK比值显著降低(P<0.05),而PPAR-γ、C/EBP-α、FAS和ACC的表达均显著升高(P<0.05),与此同时,细胞内脂滴沉积量、油红O阳性细胞数及TG含量均显著升高(P<0.05)。综上,ERK1/2-PPAR-γ信号通路可能介导了MSTN对PSPAs成脂分化的调控,结果为将MSTN作为抑制猪皮下脂肪沉积的分子靶点提供了新的理论依据。

This study was to explore the effect of the extracellular signal-regulated kinase 1/2-peroxidase proliferative activation receptor-γ(ERK1/2-PPAR-γ)signal pathway in myostatin(MSTN)inhibiting adipogenic differentiation of porcine subcutaneous preadipocytes(PSPAs).During the treatment of the differentiation induced media(DIM),the PSPAs were randomly divided into 3 groups:the control group(DIM only,0 ng/mL MSTN),the 100 ng/mL MSTN administrated group(100 ng/mL MSTN),and the ERK1/2 specific inhibitor PD98059 and 100 ng/mL MSTN co-treated group(PD98059+100 ng/mL MSTN).Cell counting kit-8(CCK-8)was used to detect the cell viability,while the adipogenic differentiation was analyzed by oil-red O staining and triglyceride(TG)quantification.RT-qPCR and Western blot were used to analyze the expression of adipogenic differentiation key transcription factors PPAR-γ,CCAAT/enhancer binding protein-α(C/EBP-α),fat synthesis rate-limiting enzymes fatty acid synthase(FAS),acetyl-CoA carboxylase(ACC),ERK and phos-phorylated ERK(p-ERK).The results showed that no significant changes in the cell viability could be detected among the three groups(P>0.05).However,compared with the control group,100 ng/mL MSTN treatment for 48 h significantly reduced the amount of intracellular lipid droplets deposition,the number of oil-red O staining positive cells and the intracellular TG content(P<0.05).The mRNA expressions of PPAR-γ,C/EBP-α,FAS and ACC,and the PPAR-γprotein level were all significantly reduced,but the p-ERK/ERK ratio was significantly increased(P<0.05).However,compared with the cells treated with 100 ng/mL MSTN alone,the PD98059+100 ng/mL MSTN group showed significantly reduced p-ERK/ERK ratio,but significantly increased PPAR-γ,C/EBP-α,FAS and ACC mRNA expressions and the PPAR-γprotein level(P<0.05).Meanwhile,the amount of lipid deposition,the number of oil-red O staining positive cells and the intracellular TG content were all significantly increased(P<0.05).In summary,the ERK1/2-PPAR-γsignaling pathway might be involved in regulation of MSTN inhibiting the adipogenic differentiation of PSPAs(P<0.05).The above results provided new theoretical basis for considering MSTN as an effective inhibitor of porcine subcutaneous fat deposition.