右归丸含药血清通过下调瘦素表达抑制RLE-6TN细胞上皮间质转化的机制研究  

Mechanism of Yougui Pills containing serum on inhibiting epithelial-mesenchymal transition in RLE-6TN cells by down-regulating leptin expression

作　　者：萧闵[1,2] 邓朝阳 江晓翠 郑岚 XIAO Min;DENG Chaoyang;JIANG Xiaocui;ZHENG Lan(Laboratory Animal Center,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Shizhen Laboratory,Wuhan 430065,China;College of Basic Medical Sciences,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Hospital of Traditional Chinese Medicine Affiliated Hospital of Hubei University of Chinese Medicine,Wuhan 430061,China)

机构地区：[1]湖北中医药大学实验动物中心,湖北武汉430065 [2]湖北时珍实验室,湖北武汉430065 [3]湖北中医药大学基础医学院,湖北武汉430065 [4]湖北中医药大学附属湖北省中医院,湖北武汉430061

出　　处：《中草药》2024年第18期6250-6260,共11页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金青年项目(82104825)。

摘　　要：目的研究右归丸含药血清抑制香烟烟雾提取物(cigarette smoke extract,CSE)+转化生长因子-β1(transforming growth factor-β1,TGF-β1)诱导的大鼠II型肺泡上皮细胞(RLE-6TN)上皮间质转化(epithelial-mesenchymal transition,EMT)的作用机制。方法RLE-6TN细胞经5%CSE处理12 h,再给予10 mg/L TGF-β1处理72 h建立EMT模型,给予5%、10%、15%、20%右归丸含药血清进行干预,采用CCK-8法测定细胞活力;采用免疫荧光法检测瘦素(leptin,LEP)表达;采用Western blotting检测LEP、E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达。构建100 ng/mL LEP干预RLE-6TN细胞模型,给予10%右归丸含药血清进行干预,采用CCK-8法测定细胞活力;采用划痕实验测定细胞迁移能力;采用免疫荧光法检测E-cadherin、α-SMA表达;采用qRT-PCR法检测E-cadherin、α-SMA表达;采用Western blotting检测E-cadherin、α-SMA及Janus激酶2(Janus kinase 2,JAK2)/信号传导及转录激活因子3(signal transducer and activator of transcription 3,STAT3)信号通路相关蛋白表达。结果RLE-6TN细胞经5%CSE+10 mg/L TGF-β1干预后,LEP、α-SMA表达显著升高(P<0.01),E-cadherin表达显著降低(P<0.01);与模型组比较,给予右归丸含药血清干预后,LEP、α-SMA表达显著降低(P<0.05、0.01),E-cadherin表达显著升高(P<0.05、0.01)。RLE-6TN细胞经100 ng/mL LEP干预后,细胞迁移率显著降低(P<0.01),α-SMA蛋白及mRNA的表达显著升高(P<0.01),E-cadherin蛋白及mRNA的表达显著降低(P<0.01),p-JAK2/JAK2、p-STAT3/STAT3表达显著升高(P<0.05、0.01);与模型组比较,给予10%右归丸含药血清干预后,细胞迁移率显著升高(P<0.01),α-SMA蛋白及mRNA的表达显著降低(P<0.05、0.01),E-cadherin蛋白及mRNA的表达显著升高(P<0.05、0.01),p-JAK2/JAK2、p-STAT3/STAT3表达显著降低(P<0.05、0.01)。结论右归丸含药血清能够通过降低LEP表达,抑制JAK2/STAT3信号通路,从而抑制RLE-6TN细胞EMT进程。Objective To study the mechanism of Yougui Pills(右归丸)containing serum on inhibiting epithelial-mesenchymal transition(EMT)induced by cigarette smoke extract(CSE)and transforming growth factor-β1(TGF-β1)in rat type II alveolar epithelial cells(RLE-6TN).Methods RLE-6TN cells were treated with 5%CSE for 12 h,followed by treatment with 10 mg/L TGF-β1 for 72 h to establish an EMT model.5%,10%,15%and 20%Yougui Pills containing serum was administered for intervention,and cell viability was measured using CCK-8 method.Immunofluorescence method was used to detect the expression of leptin(LEP).Western blotting was used to detect the expressions of LEP,E-cadherin andα-smooth muscle actin(α-SMA).A model of RLE-6TN cells treated with 100 ng/mL LEP was established,followed by treatment with 10%Yougui Pills containing serum,and cell viability was measured using CCK-8 method,and measure cell viability using CCK-8 method.Scratch test was used to determine cell migration ability.Immunofluorescence assay was used to detect the expressions of E-cadherin andα-SMA.qRT-PCR was used to detect the expressions of E-cadherin andα-SMA.Western blotting was used to detect the expressions of E-cadherin,α-SMA and Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway related proteins.Results After intervention with 5%CSE+10 mg/L TGF-β1 in RLE-6TN cells,the expressions of LEP andα-SMA were significantly increased(P<0.01),while the expression of E-cadherin was significantly decreased(P<0.01).Compared with model group,after intervention with Yougui Pills containing serum,the expressions of LEP andα-SMA were significantly reduced(P<0.05,0.01),and the expression of E-cadherin was significantly increased(P<0.05,0.01).After intervention with 100 ng/mL LEP,the cell migration rate of RLE-6TN cells was significantly reduced(P<0.01),the expressions ofα-SMA protein and mRNA were significantly increased(P<0.01),the expressions of E-cadherin protein and mRNA were significantly reduced(P<0.01),the exp

关 键 词：右归丸 瘦素 上皮间质转化 RLE-6TN细胞 马钱苷 JAK2/STAT3信号通路 

分 类 号：R285.5[医药卫生—中药学]