摘要
目的:探讨桃核承气汤不同有效部位对转化生长因子-β1(TGF-β1)诱导的人肾小管上皮细胞(HK-2)中细胞外基质合成与降解的影响。方法:采用系统溶剂法萃取70%乙醇提取的桃核承气汤,得到石油醚萃取物质部位,三氯甲烷萃取物质部位,乙酸乙酯萃取物质部位,正丁醇萃取物质部位,萃余水相物质部位,多糖物质部位和芒硝物质部位。利用TGF-β1诱导HK-2细胞构建纤维化模型,以不同质量浓度(0,50,100,200,400,800 mg·L^-1)桃核承气汤各部位干预细胞,酶联免疫吸附测定(ELISA)检测细胞上清液胶原蛋白Ⅰα1(Col-Ⅰα1),纤维粘连蛋白(FN)含量,初步筛选得到主要有效物质部位进行后续实验验证。细胞增殖与毒性检测试剂盒(CCK-8)确定筛选出的有效部位的最佳浓度;蛋白免疫印迹法(Western blot)检测胶原蛋白-Ⅰ(Col-Ⅰ),胶原蛋白-Ⅲ(Col-Ⅲ),基质金属蛋白酶-2(MMP-2),基质金属蛋白酶抑制因子2(TIMP2),结缔组织生长因子(CTGF)蛋白的表达水平;免疫荧光检测α-平滑肌肌动蛋白(α-SMA)的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测纤溶酶原激活抑制剂-1(PAI-1)mRNA的表达。结果:ELISA检测结果显示,与模型组比较,乙酸乙酯、正丁醇、三氯甲烷萃取部位质量浓度为200,400 mg·L^-1时能显著降低Col-Ⅰα1和FN含量(P<0.05,P<0.01)。CCK-8检测结果显示,各组药物干预质量浓度为400,800 mg·L^-1时显著抑制细胞活性(P<0.01)。Western blot检测结果显示,与模型组比较,乙酸乙酯、三氯甲烷萃取部位均能降低Col-Ⅰ,Col-Ⅲ,TIMP2和CTGF蛋白的表达,上调MMP-2的表达(P<0.05),正丁醇萃取部位作用效果更显著(P<0.01)。免疫荧光结果显示,乙酸乙酯、三氯甲烷萃取部位均能抑制α-SMA的表达(P<0.05),正丁醇萃取部位抑制作用更显著(P<0.01)。Real-time PCR检测发现,乙酸乙酯、三氯甲烷萃取部位均能抑制PAI-1 mRNA的表达(P<0.05),正丁醇部位更显著(P<0.01)。结�
Objective:To explore the effect of different effective parts of Taohe Chengqitang on the synthesis and degradation of extracellular matrix in human kideny-2(HK-2)cells induced by transforming growth factor-β1(TGF-β1).Method:Petroleum ether extract,ethyl acetate extract,n-butanol extract,raffinate and polysaccharide extract,mirabilite extract were extracted with 70%ethanol by systematic solvent method.The HK-2 cell fibrosis model induced by TGF-β1 was built to intervene the cells in different parts of Taohe Chengqitang with different concentrations(0,50,100,200,400,800 mg·L^-1).Enzyme-linked immunosorbent assay(ELISA)kit assay was used to detect collagen(Col)-Ⅰ α1 and fibronectin(FN)in supernatant to screen out the main active parts.Cell counting kit-8(CCK-8)method was used to determine the best concentration of intervention site of bioactive components.Western blot analysis was used to detect the expression levels of Col-Ⅰ,Col-Ⅲ,matrix metalloproteinase-2(MMP-2),matrix metalloproteinase inhibitor2(TIMP2),and connective tissue growth factor(CTGF).Immunofluorescence assay was used to detect the expression ofα-smooth muscle actin(α-SMA).Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)analysis was used to detect the mRNA expression of plasminogen activator inhibitor-1(PAI-1).Result:ELISA kit assay demonstrated that compared with the model group,ethyl acetate extract,n-butanol extract and chloroform extract significantly reduced the Col-Ⅰ α1 and FN content at the concentrations of 200 and 400 mg·L^-1(P<0.05,P<0.01).CCK-8 assay showed that the cells viability was significantly inhibited with drug intervention at the concentrations of 400 and 800 mg·L-1(P<0.01).Western blot demonstrated that compared with the model group,ethyl acetate extract,n-butanol extract and chloroform extract decreased the expression levels of Col-Ⅰ,Col-Ⅲ,TIMP2 and CTGF in HK-2 cells induced by TGF-β1,and increased the expression of MMP-2(P<0.05),with more significant effect in n-butanol extract(
作者
周珊珊
艾中柱
李伟男
巴元明
ZHOU Shan-shan;AI Zhong-zhu;LI Wei-nan;BA Yuan-ming(Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Provincial Hospital of Traditional Chinese Medicine(TCM),Wuhan 430061,China;Hubei Provincial Academy of TCM,Wuhan 430074,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第19期127-134,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
武汉市科技计划项目(2017060201010223)
鄂卫生计生通项目(〔2017〕20号)。
关键词
肾脏纤维化
桃核承气汤
有效部位
细胞外基质
renal fibrosis
Taohe Chengqitang
effective parts
extracellular matrix(ECM)
