YAP对高糖培养大鼠心脏成纤维细胞活化作用的影响及其机制

Function and mechanism of Yes-associated protein in regulating the activation of rat cardiac fibroblasts cultured with high glucose

目的探究Yes相关蛋白(YAP)对高糖诱导的大鼠心脏成纤维细胞(CFs)增殖和转分化的影响及其作用机制。方法分离培养1~3日龄SD大鼠CFs,用40.0 mmol/L葡萄糖诱导CFs,构建糖尿病心肌病(DCM)细胞模型,24 h后检测细胞YAP、α平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(collagenⅠ)、Ⅲ型胶原蛋白(collagenⅢ)、结缔组织生长因子(CTGF)的表达水平。取原代大鼠CFs,分别设置正常糖(5.5 mmol/L D-Glucose,NG)组、高糖(40.0 mmol/LD-Glucose,HG)组、NG+维替泊芬(NG+VP)组、HG+VP组。VP为YAP抑制剂,剂量为0.5μmol/L。各组处理CFs24 h后,采用波形蛋白免疫荧光法鉴定CFs,Ki-67免疫荧光染色检测各组细胞增殖情况,Westernblotting检测各组YAP、α-SMA、collagenⅠ、collagenⅢ、CTGF蛋白表达水平。结果免疫荧光法鉴定波形蛋白阳性,提示原代培养的细胞为大鼠CFs。Ki-67免疫荧光检测结果显示,与NG组比较,HG组CFs的Ki-67阳性率(%)明显升高(67.33±5.14 vs.22.94±4.88,P<0.05);YAP抑制剂VP处理后,CFs的Ki-67阳性率(%)降低(46.83±3.86 vs.67.33±5.14,P<0.05)。Western blotting检测结果显示,与NG组比较,HG组高糖干预24 h后,CFs中α-SMA、collagenⅠ、collagenⅢ的相对表达水平均明显升高(1.43±0.98 vs.0.93±0.06,1.80±0.09 vs.1.08±0.09,1.43±0.09 vs.0.88±0.10,P<0.05),YAP和CTGF蛋白相对表达水平也升高(1.93±0.15 vs.1.17±0.09,1.80±0.18 vs.1.23±0.16,P<0.05);与HG组比较,HG+VP组的α-SMA、collagen I、collagenⅢ、CTGF蛋白相对表达水平均明显降低(1.27±0.06vs.1.71±0.12,2.05±0.23vs.3.03±0.17,1.10±0.12vs.1.82±0.18,1.31±0.16 vs.1.57±0.03,P<0.05);与NG组比较,HG+VP组中α-SMA、collagenⅠ、CTGF蛋白相对表达水平均较高(P<0.05)。结论高糖可刺激SD大鼠乳鼠CFs增殖及活化,合成过多的胶原蛋白等细胞外基质,其作用机制可能与YAP/TEAD/CTGF传导通路有关。

Objective To investigate the effect and mechanism of Yes-associated protein(YAP)on rat cardiac fibroblasts(CFs)proliferation and transdifferentiation induced by high glucose.Methods The CFs cells were isolated and cultured of newborn SD rats aged 1-3 days,then treated with 40 mmol/L D-glucose for concentration of diabetes cardiomyopathy model(DCM),and the relative expression levels were detected with Western blotting of YAP,α-smooth muscle actin(α-SMA),collagenⅠ,collagenⅢand connective tissue growth factor(CTGF),respectively,at the time points 24 hours.The CFs cells were divided into normal glucose(NG)control group(5.5 mmol/L D-glucose),high glucose(HG)group(40.0 mmol/L D-glucose),normal glucose+0.5μmol/L verteporfin(NG+VP)group and high glucose+0.5μmol/L verteporfin(HG+VP)group.Verteporfin is an inhibitor of YAP,which blockades the interaction between YAP and TEAD.Immunofluorescence of vimentin was used to identify CFs.Immunofluorescence of Ki-67 was used to detect the proliferation activities of CFs in various groups.Western blotting was performed to detect the levels ofα-SMA,collagenⅠand collagenⅢ,YAP and CTGF proteins in CFs of various groups.Results The positive result of vimentin immunofluorescence prompted that the primary cultured cells were rat?s CFs.Immunofluorescence of Ki-67 showed that,the positive rate(%)of Ki-67 in CFs was obviously higher in HG group than in NG group(67.33±5.14vs.22.94±4.88,P<0.05);After treatment with VP,the positive rate(%)of Ki-67 in CFs decreased markedly(46.83±3.86 vs.67.33±5.14,P<0.05).Western blotting showed that,compared with NG group,the relative expression levels ofα-SMA,collagenⅠand collagenⅢin CFs increased significantly in HG group(1.43±0.98 vs.0.93±0.06,1.80±0.09 vs.1.08±0.09,1.43±0.09vs.0.88±0.10,P<0.05),and the relative expression levels of VAP and CTGF protein also increased(1.93±0.15 vs.1.17±0.09,1.80±0.18 vs.1.23±0.16,P<0.05).Compared with the HG group,the relative expression levels ofα-SMA,collagen I,collagenⅢand CTGF proteins decreased significantly in HG+VP group(1.27±0.06 vs.1.71±0.12,2.05±0.23 vs.3.03±0.17,1.10±0.12vs.1.82±0.18,1.31±0.16 vs.1.57±0.03,P<0.05),while compared with NG group,the relative expression levels ofα-SMA,collagenⅠand CTGF proteins were higher in HG+VP group(P<0.05).Conclusion High glucose promotes the proliferation and activation of neonatal rats CFs and excessive synthesis of extracellular matrix proteins such as collagen by regulating the YAP/TEAD/CTGF signaling pathway.