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王茜,成细华,刘春燕,徐文峰,张熙,张海英,王晖,唐群.六味地黄汤含药血清对CoCl2诱导HK-2细胞后E-cadherin、α-SMA表达的影响[J].湖南中医药大学学报英文版,2022,42(4):557-563.[Click to copy
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| 六味地黄汤含药血清对CoCl2诱导HK-2细胞后E-cadherin、α-SMA表达的影响 |
| 王茜,成细华,刘春燕,徐文峰,张熙,张海英,王晖,唐群 |
| (湖南中医药大学医学院, 湖南 长沙 410208;湖南中医药大学第一附属医院, 湖南 长沙 410007;湖南省脑科医院, 湖南 长沙 410021) |
| 摘要: |
| 目的 观察六味地黄汤含药血清对CoCl2缺氧后HK-2细胞中的上皮-间充质转化（epithelial-mesenchymal transition, EMT）标志物E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)的影响。方法 体外培养HK-2细胞，分为3组：正常组（10%空白血清）、模型组（10%空白血清+200 μmol·L-1 CoCl2）、含药血清组（10%含药血清+200 μmol·L-1 CoCl2）。CCK-8法检测不同浓度空白血清、含药血清（2.5%、5%、10%、20%、40%）在24、36 h对HK-2细胞存活率的影响；以及不同浓度CoCl2 （150、200、300、600 μmol·L-1）在24 h的最佳干预浓度。qRT-PCR法和Western blot法分别检测各组E-cadherin、α-SMA 的mRNA和蛋白表达水平；免疫荧光观察各组E-cadherin、α-SMA蛋白表达情况。结果 CCK-8法确定含药血清和空白血清的最佳干预浓度和干预时间分别为10%和24 h；CoCl2的最佳干预浓度为200 μmol·L-1。与正常组比较，模型组E-cadherin的mRNA和蛋白表达水平均明显降低（P<0.01）、α-SMA明显升高（P<0.05）；与模型组比较，含药血清组E-cadherin的mRNA和蛋白表达水平均明显升高（P<0.01）、α-SMA明显降低（P<0.01）。结论 缺氧能诱导HK-2细胞发生EMT，六味地黄汤含药血清能够降低α-SMA的表达，升高E-cadherin的表达，从而缓解肾小管EMT。 |
| 关键词: 六味地黄汤 缺氧 上皮-间充质转化 肾纤维化 |
| DOI：10.3969/j.issn.1674－070X.2022.04.007 |
| Received:October 28, 2021 |
| 基金项目:湖南省自然科学基金资助项目（2021JJ30506）；长沙市自然科学基金资助项目（kq2014089）。 |
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| Influence of Liuwei Dihuang Decoction durg-containing serum on expression of E-cadherin and α-SMA in HK-2 cells induced by CoCl2 |
| WANG Xi,CHENG Xihua,LIU Chunyan,XU Wenfeng,ZHANG Xi,ZHANG Haiying,WANG Hui,TANG Qun |
| (Medical College of Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;The First Affiliated Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Hunan Brain Hospital, Changsha, Hunan 410021, China) |
| Abstract: |
| Objective To observe the biomarkers E-cadherin and α-smooth muscle actin (α-SMA) of epithelial-mesenchymal transition (EMT) in HK-2 cells after CoCl2 hypoxia with Liuwei Dihuang Decoction drug-containing serum. Methods HK-2 cells were cultured in vitro and divided into 3 groups: normal group (10% blank serum), model group (10% blank serum+200 μmol·L-1 CoCl2), and drug-containing serum group (10% drug-containing serum+200 μmol·L-1 CoCl2). The CCK-8 method was used to detect the effects of different concentrations of blank serum and drug-containing serum (2.5%, 5%, 10%, 20%, 40%) on the survival rate of HK-2 cells at 24 and 36 h; and the best intervention concentration of different concentrations of CoCl2 (150, 200, 300, 600 μmol·L-1) in 24 h. qRT-PCR and Western blot method were used to detect the expression of E-cadherin and α-SMA protein and mRNA in each group. The expression of E-cadherin and α-SMA protein in each group was observed by immunofluorescence. Results CCK-8 method determined that the best intervention concentration and time of drug-containing serum and blank serum was 10% and 24 h, and the best intervention concentration of CoCl2 was 200 μmol·L-1. Compared with normal group, the mRNA and protein expression level of E-cadherin in model group were significantly decreased (P<0.01), and α-SMA expression was increased (P<0.05). Compared with model group, the mRNA and protein expression level of E-cadherin were significantly increased (P<0.01), and α-SMA expression was significantly decreased (P<0.01) in drug-containing serum group. Conclusion Hypoxia can induce EMT in HK-2 cells. Liuwei Dihuang Decoction drug-containing serum can reduce the expression of α-SMA and increase the expression of E-cadherin, thus alleviating EMT in renal tubule. |
| Key words: Liuwei Dihuang Decoction hypoxia epithelial-mesenchymal transition renal fibrosis |
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