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张海英,王晖,潘佳俊,高海波,赵欣然,刘春燕,唐群.六味地黄汤通过miR-210/HIF-1α信号通路对CoCl2诱导的HK-2细胞上皮间质转化的影响和机制研究[J].湖南中医药大学学报英文版,2023,43(4):619-626.[Click to copy
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| 六味地黄汤通过miR-210/HIF-1α信号通路对CoCl2诱导的HK-2细胞上皮间质转化的影响和机制研究 |
| 张海英,王晖,潘佳俊,高海波,赵欣然,刘春燕,唐群 |
| (湖南中医药大学医学院, 湖南 长沙 410208) |
| 摘要: |
| 目的 观察六味地黄汤（Liuwei Dihuang Decoction, LWDHD）调控miR-210/HIF-1α信号通路对CoCl2诱导的HK-2细胞上皮间质转化（epithelial mesenchymal transdifferentiation, EMT）的作用和机制。方法 体外培养HK-2细胞，分对照组（N组）、模型组（M组）、LWDHD血清组（LW组）、空白血清+miR-210过表达组（LV-miR-210组）、空白血清+miR-210沉默组（LV-anti-miR-210组）、空白血清+miR-210阴性对照组（LV-miR-210 NC组）、LWDHD血清+miR-210过表达组（LW+LV-miR-210组）、LWDHD血清+miR-210沉默组（LW+LV-anti-miR-210组）、LWDHD血清+miR-210阴性对照组（LW+LV-miR-210 NC组），除N组外，其他各组加入CoCl2处理。CCK-8法检测不同浓度LWDHD、CoCl2在24 h后对HK-2细胞活性的影响并选择最佳干预浓度。细胞免疫荧光和Western blot法检测各组HK-2细胞中缺氧诱导因子-1α（hypoxia-inducible factor-1α, HIF-1α）、β-联蛋白（β-catenin）、波形蛋白（Vimentin）、上皮钙黏素（E-cadherin）蛋白表达情况；qPCR法检测miR-210、HIF-1α、β-catenin、Vimentin mRNA表达情况。通过慢病毒感染HK-2细胞，实现miR-210的过表达和抑制，并加入CoCl2，观察miR-210、HIF-1α、β-catenin、Vimentin蛋白和mRNA表达以及LWDHD的干预作用。结果 CCK-8结果显示，LWDHD、CoCl2最佳干预浓度分别为10%、200 μmol/L。与N组相比，M组细胞形态由铺路石样向长梭形改变，HIF-1α、β-catenin、Vimentin蛋白和mRNA表达升高（P＜0.05，P＜0.01），miR-210 mRNA表达升高（P＜0.01），E-cadherin蛋白表达降低（P＜0.01）；与M组相比，LW组HIF-1α、β-catenin、Vimentin蛋白和mRNA表达降低（P＜0.05，P＜0.01），miR-210 mRNA表达降低（P＜0.05），E-cadherin蛋白表达升高（P＜0.01）。与LV-miR-210 NC组相比，LV-anti-miR-210组HIF-1α、β-catenin、Vimentin蛋白和mRNA表达降低（P＜0.05，P＜0.01）；与LV-miR-210 NC组相比，LV-miR-210组HIF-1α、β-catenin、Vimentin蛋白和mRNA表达升高（P＜0.05，P＜0.01）。在慢病毒转染各组的基础上，加入LWDHD处理后，LWDHD可降低各组HIF-1α、β-catenin、Vimentin蛋白和mRNA表达（P＜0.05，P＜0.01）。结论 LWDHD抗纤维化作用机制与其下调miR-210/HIF-1α信号通路，抑制肾小管EMT有关。 |
| 关键词: 肾纤维化 六味地黄汤 上皮间质转化 miR-210 缺氧诱导因子1α HK-2细胞 |
| DOI：10.3969/j.issn.1674-070X.2023.04.007 |
| Received:September 02, 2022 |
| 基金项目:湖南省自然科学基金项目（2021JJ30506）；湖南省教育厅项目（21A0242，20C1398）；长沙市自然科学基金项目（kq2014089）；湖南中医药大学研究生科研创新项目（2021CX32）。 |
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| Effects and mechanism of Liuwei Dihuang Decoction on CoCl2-induced epithelial-mesenchymal transition of HK-2 cells through the miR-210/HIF-1α signaling pathway |
| ZHANG Haiying,WANG Hui,PAN Jiajun,GAO Haibo,ZHAO Xinran,LIU Chunyan,TANG Qun |
| (Medical College, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To observe the effects and mechanism of Liuwei Dihuang Decoction (LWDHD) regulating epithelial mesenchymal transition (EMT) of HK-2 cells induced by CoCl2 through miR-210/HIF-1α signaling pathway.Methods HK-2 cells were cultured in vitro and divided into normal group (N group), model group (M group), LWDHD medicated serum group (LW group), blank serum+over-espressed miR-210 group (LV-miR-210 group), blank serum+silent miR-210 group (LV-anti-miR-210 group), blank serum+miR-210 negative group (LV-miR-210 NC group), LWDHD medicated serum+over-expressed miR-210 group (LW+LV-miR-210 group), LWDHD medicated serum+silent miR-210 group (LW+LV-anti-miR-210 group) and LWDHD medicated serum+miR-210 negative group (LW+LV-miR-210 NC group). Except for N group, the other groups were treated with CoCl2. CCK-8 was used to detect the effect of different concentrations of LWDHD and CoCl2 on the activity of HK-2 cells after 24 hours, and the best intervention concentration was selected. Cell immunofluorescence and Western blot were used to determine hypoxia-inducible factor-1α (HIF-1), β-catenin, Vimentin, and E-cadherin protein expression levels. miR-210, HIF-1α, β-catenin and Vimentin mRNA expression levels were measured by qPCR. By lentivirus transfection of HK-2 cells, miR-210 was overexpressed and inhibited. Then CoCl2 was added to the medium. The miR-210, HIF-1α , β-catenin, Vimentin protein and mRNA expression of the regulation effect of LWDHD were observed.Results The CCK-8 showed that the optimal intervention concentrations of LWDHD and CoCl2 were 10% and 200 μmol/L, respectively. Compared with N group, the morphology of cells in group M changed from the shape of paving stone to the shape of long spindle. HIF-1α, β-catenin, Vimentin protein and mRNA expression increased (P<0.05, P<0.01) while miR-210 mRNA expression increased (P＜0.01) and E-cadherin protein expression decreased (P<0.01). Compared with M group, LW group can significantly reduce the expression of HIF-1α, β-catenin, Vimentin protein and mRNA (P<0.05, P<0.01), down-regulate the expression of miR-210 mRNA (P＜0.05) and up-regulate the expression of E-cadherin protein (P<0.01). Compared with the LV-miR-210 NC group, LV-anti-miR-210 group showed lower HIF-1α, β-catenin, Vimentin protein and mRNA (P<0.05, P<0.01), and LV-miR-210 group showed higher HIF-α, β-catenin, Vimentin protein and mRNA (P＜0.05, P＜0.01). After LWDHD administration to each group of lentivirus transfection, HIF-1α, β-catenin, Vimentin protein and mRNA expression decreased (P<0.05, P<0.01).Conclusion The anti-fibrosis mechanism of LWDHD is related to its down-regulation of miR-210/HIF-1α signaling pathway, which is related to the inhibition of renal tubular EMT. |
| Key words: renal fibrosis Liuwei Dihuang Decoction epithelial-mesenchymal transition miR-210 hypoxia inducible factor-1α HK-2 cells HK-2 cells |
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