麦冬多糖对转化生长因子-β1诱导的人胚胎肺成纤维细胞表型转化的影响

Ophiopogon Japonicus polysaccharide hinders phenotypic transformation of human embryonic lung fibroblasts through attenuation of transformation growth factor-β1 production

目的观察麦冬多糖(OJP)对转化生长因子-β1(TGF-β1)诱导的人胚胎肺成纤维(HEL)细胞表型转化的影响,探讨其分子机制。方法将经6ng/mL诱导TGF-β1的HEL细胞作为模型组,加入25、50、100μg/mL OJP继续培养的HEL细胞作为OJP干预组。通过电镜观察细胞的超微结构;实时荧光定量RT-PCR检测α-SMA mRNA和COLⅠmRNA表达;Western blot检测α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(COLⅠ)、Smad2蛋白和p-Smad2蛋白表达。结果模型组及25、50、100μg/mL OJP干预组细胞的存活率分别为(99.65±1.55)%,(88.39±1.68)%,(82.77±1.96)%和(79.48±1.74)%,OJP干预组存活率低于模型组且随浓度的增加呈剂量依赖趋势,差异有统计学意义(P均<0.05);100μg/mL OJP干预组细胞表面微绒毛减少、微丝变短,胞浆内线粒体数目下降,粗面内质网减少;模型组和100μg/mL OJP干预组α-SMA mRNA表达量为(1.00±0.09)和(0.69±0.05),COLⅠmRNA表达量为(1.02±0.11)和(0.86±0.09),100μg/mL OJP干预组细胞α-SMA和COLⅠmRNA表达较模型组下调(P均<0.05);与模型组比较,100μg/mL OJP干预组α-SMA、COLⅠ、Smad2和p-Smad2蛋白表达明显下降(P均<0.01)[α-SMA蛋白:(0.48±0.03)比(1.56±0.02);COLⅠ蛋白:(0.41±0.02)比(1.66±0.02);Smad2蛋白:(0.71±0.01)比(1.34±0.01);p-Smad2蛋白:(0.69±0.01)比(1.52±0.01),P均<0.05]。结论麦冬多糖可能通过干预TGF-β/Smads信号通路,在一定程度上抑制成纤维细胞向肌成纤维细胞的转化。

Objective To investigate the effect of Ophiopogon Japonicus polysaccharides(OJP)on the phenotypic transformation of human embryonic lung fibroblasts induced by transforming growth factor-β1(TGF-β1)and to explore the underlying molecular mechanism.Methods Human embryonic lung fibroblasts(HEL)were incubated with(model group)or without(control group)6ng/ml TGF-β1.Cells in the model group were further treated with different concentrations of OJP(25,50 and 100μg/mL,intervention groups).For functional analyses,only 100μg/mL OJP was used in the intervention group.The ultrastructure of cells was observed using electron microscope,mRNA expressions ofα-smooth muscle actin(α-SMA)and typeⅠcollagen(COL I)were analyzed by quantitative RT-PCR,protein expressions ofα-SMA and COLⅠ,Smad2 and p-Smad2 were determined by Western blot.Results The cell viability in the model group and the intervention groups cultured with 25,50,or 100μg/ml OJP showed a dose-dependent inverse trend(99.65±1.55 vs.88.39±1.68 vs.82.77±1.96 vs.79.48±1.74%;all P<0.05).Compared to the model group,the intervention group with 100μg/ml OJP had fewer microvilli on the cell surface,shortened microfilaments,and reduced numbers of intracytoplasmic,mitochondria and rough endoplasmic reticulum;lower mRNA levels ofα-SMA(1.00±0.09 vs.0.69±0.05,P<0.05)and COLⅠ(1.02±0.11 vs.0.86±0.09,P<0.05);lower protein levels ofα-SMA,COL I,Smad2 and p-Smad2(0.48±0.03 vs.1.56±0.02;0.41±0.02 vs.1.66±0.02;0.71±0.01 vs.1.34±0.01;and 0.69±0.01 vs.1.52±0.01,respectively;all P<0.05).Conclusion OJP could partly inhibit the transformation of fibroblasts to myofibroblasts by interfering with TGF-β/Smads signaling pathway.