湿生扁蕾总[口山]酮通过调控TGF-β/Smad和自噬相关通路对NCM460细胞上皮间质转化的影响

Effects of Xanthones from Gentianopsis paludos on epithelial-mesenchymal transition of NCM460 cells via TGF-β/Smad and autophagy-related pathways

目的探讨湿生扁蕾总[口山]酮介导TGF-β/Smad和自噬相关通路对NCM460细胞上皮间质转化(EMT)的影响。方法采用2.5μg/mL脂多糖(LPS)持续干预细胞14 d建立EMT模型,CCK-8法筛选最佳湿生扁蕾总[口山]酮给药剂量。设计空白组、模型组、吡菲尼酮组(200μg/mL)和湿生扁蕾总[口山]酮低、中、高剂量组(50、100、200μg/mL),采用Western blot法检测细胞ZO-1、α-SMA、Beclin-1、P62、Bcl-2、LC3B蛋白表达,RT-qPCR法检测细胞ZO-1、α-SMA、TGF-β1、Smad2、Smad3、Snail1 mRNA表达,免疫荧光观察细胞中α-SMA表达,TUNEL染色观察细胞凋亡情况,MDC染色和透射电镜分别观察细胞中自噬囊泡和自噬小体数目。结果湿生扁蕾总[口山]酮质量浓度小于200μg/mL时对细胞活率无明显影响(P>0.05),故选择50、100、200μg/mL为低、中、高剂量组。与空白组比较,模型组细胞ZO-1、Beclin-1、Bcl-2、LC3-Ⅱ/LC3-Ⅰ蛋白和ZO-1 mRNA表达降低(P<0.01),α-SMA、TGF-β1、Smad2、Smad3、Snail 1 mRNA和α-SMA、P62蛋白表达升高(P<0.01);与模型组比较,湿生扁蕾总[口山]酮组均能改善以上指标(P<0.05,P<0.01)。结论湿生扁蕾总[口山]酮能抑制NCM460细胞EMT发生进程,其机制可能与抑制TGF-β/Smad通路和增强细胞自噬有关。

AIM To investigate the effects of TGF-β/Smad and autophagy-related pathway mediated by xanthones from Gentianopsis paludos on epithelial-mesenchymal transition(EMT)of NCM460 cells.METHODS The EMT models were established by 14 days consecutive use of 2.5μg/mL lipopolysaccharide(LPS)in the NCM460 cells,and the most optimal dosage of G.paludos xanthones was determined by CCK-8 method.NCM460 cells assigned into the blank group,the model group,the pirfenidone group(200μg/mL)and G.paludos xanthones groups(50μg/mL in low-dose group,100μg/mL in medium-dose group,and 200μg/mL in high-dose group)had their protein expressions of ZO-1,α-SMA,Beclin-1,P62,Bcl-2,and LC3B detected by Western blot;their mRNA expressions of ZO-1,α-SMA,TGF-β1,Smad2,Smad3,and Snail1 detected by RT-qPCR;theirα-SMA expressions observed by immunofluorescence;their apoptosis observed by TUNEL staining;and their number of autophagy vesicles and autophagy bodies observed by MDC staining and transmission electron microscopy respectively.RESULTS G.paludos xanthones at concentrations lower than 200μg/mL made no obvious impact on the cell viability(P>0.05),therefore 50,100 and 200μg/mL were selected as the low,medium and high dosages.Compared with the blank group,the model group displayed decreased expressions of ZO-1,Beclin-1,Bcl-2,LC3-Ⅱ/LC3-Ⅰproteins and ZO-1 mRNA(P<0.01);increased expressions ofα-SMA,TGF-β1,Smad2,Smad 3 and Snail 1 mRNA andα-SMA and P62 proteins(P<0.01);increased cell apoptosis(P<0.01),and decreased number of autophagic vesicles and autophagosomes(P<0.01).Compared with the model group,G.paludos xanthones groups shared improvement in terms of all aforementioned indices levels(P<0.05,P<0.01).CONCLUSION G.paludos Xanthones can inhibit the EMT progression in NCM460 cells,and its mechanism may lie in its inhibition of TGF-β/Smad signaling pathway and enhancement of autophagy.