胆汁酸通过p-STAT3-SOCS3途径调节下丘脑神经元食欲及抑制信号肽表达

Bile acids regulate anorexigenic neuropeptide through p-STAT3-SOCS3 signaling in mouse hypothalamic cells

目的探索胆汁酸牛磺石胆酸或鹅去氧胆酸对小鼠下丘脑GT1-7细胞食欲抑制信号肽表达的影响及可能的作用途径。方法将实验分为3组,每组3个平行样本。空白对照组:仅给予含10%胎牛血清高糖培养基培养GT1-7细胞。胆汁酸牛磺石胆酸处理组:分别采用10 nmol/L,100 nmol/L,1μmol/L和10μmol/L浓度的胆汁酸牛磺石胆酸处理GT1-7细胞12、24和48 h。鹅去氧胆酸处理组:分别采用10 nmol/L,100 nmol/L,1μmol/L和10μmol/L浓度的鹅去氧胆酸处理GT1-7细胞12、24和48 h。采用Real-time PCR检测GT1-7细胞食欲抑制信号肽阿黑皮素原(POMC)mRNA表达水平;ELISA法检测POMC剪切产物抑制食欲肽α-促黑素细胞激素水平。采用Western blot检测G蛋白偶联胆汁酸受体(TGR5)和法呢醇X受体表达以及磷酸化信号转导转录激活因子3(p-STAT3),磷酸化蛋白激酶(p-AKT),细胞因子信号传导抑制因子3(SOCS3)蛋白表达量。结果Western blot结果显示,下丘脑神经元GT1-7细胞能表达TGR5和FXR两种胆汁酸受体,且表达量受胆汁酸调节。Real-time PCR检测结果显示,10μmol/L tLCA或鹅去氧胆酸处理GT1-7细胞24 h后POMC mRNA表达水平上升,ElISA结果也发现10μmol/L胆汁酸牛磺石胆酸或鹅去氧胆酸处理GT1-7细胞24 h后具有抑制食欲作用的POMC衍生肽[α]-MSH释放增多。Western blot结果显示,胆汁酸处理可上调GT1-7细胞p-STAT3和p-AKT蛋白表达,且调控STAT3信号通路的SOCS3蛋白表达也有增加。结论胆汁酸牛磺石胆酸或鹅去氧胆酸通过GT1-7细胞胆汁酸受体TGR5和FXR促进POMC mRNA表达,增加[α]-促黑素细胞激素水平,提示胆汁酸具有通过抑制食欲信号肽表达调节食欲的潜在作用,该作用是通过p-AKT和p-STAT3-SOCS3信号通路实现。

Objective To explore the effects of taurolithocholic acid(tLCA)and chenodeoxycholic acid(CDCA)on the expression of aorexigenic neuropeptide in mouse hypothalamus GT1-7 cells.Methods Mouse hypothalamic GT1-7 cells were treated with culture medium containing 10%FBS(control group,n=3)or with 10 nmol/L,100 nmol/L,1μmol/L and 10μmol/L tLCA(tLCA group,n=3)or CDCA(CDCA group,n=3)for 12,24 or 48 h.Real-time PCR was performed to determine the expression levels of proopiomelanocortin(POMC)mRNA in the cells,and the production levels ofα-melanocyte-stimulating hormone(α-MSH)were assessed using an ELISA kit.Signal transduction and activator of transcription 3 phosphorylation(p-STAT3),threonine kinase phosphorylation(p-AKT),suppressor of cytokine signaling 3(SOCS3),G protein-coupled bile acid receptor-1(TGR5)and farnesoid X receptor(FXR)protein were detected by Western blotting.Results Western blotting results showed that mouse hypothalamic GT1-7 cells expressed two bile acid receptors,TGR5 and FXR,whose expressions were regulated by bile acids.Real-time PCR showed that the expression of POMC mRNA was significantly increased in the cells after treatment with 10μmol/L tLCA or CDCA for 24 h.POMC-derived anorexigenic peptideα-MSH increased significantly in GT1-7 cells after treatment with 10μmol/L tLCA or CDCA for 24 h.Treatment of the cells with tLCA or CDCA significantly increased the expressions of intracellular signaling proteins including p-STAT3,p-AKT and SOCS3.Conclusion Mouse hypothalamic GT1-7 cells express bile acid receptors TGR5 and FXR.Bile acids tLCA or CDCA can promote the expression of POMC mRNA and increase the production of the anorexigenic peptideα-MSH.The intracellular signaling proteins p-AKT,p-STAT3 and SOCS3 are likely involved in bile acid-induced anorexigenic peptide production.