摘要
目的:探究内皮素-1(endothelin-1,ET-1)对大鼠肝星状细胞HSC-T6内磷酸化肌球蛋白轻链Ⅱ(phosphorylated myosin light chainⅡ,p-MLCⅡ),肌球蛋白轻链Ⅱ(myosin light chainⅡ,MLCⅡ)蛋白表达的影响及当归芍药散(Danggui Shaoyao San)含药血清对其的干预作用。方法:将HSC-T6细胞种板后,各组每孔加入DMEM和终体积分数分别为2. 5%,5%,10%,15%,20%的空白大鼠血清,采用噻唑蓝(MTT)比色法测定HSC-T6细胞的活力,筛选出适合的大鼠血清浓度范围;将细胞分为空白血清组(5%,10%,15%)和当归芍药散含药血清组(5%,10%,15%),酶联免疫吸附测定(ELISA)检测基础状态下细胞培养上清液中ET-1的含量;细胞分为空白血清组(10%),当归芍药散含药血清低、中、高剂量组(5%,10%,15%),实时荧光定量聚合酶链式反应(Real-time PCR)检测基础状态下细胞培养上清液中ET-1 mRNA的水平;将细胞分为空白血清组(10%),模型组(10%),当归芍药散含药血清低、中、高剂量组(5%,10%,15%),Y-27632抑制剂组(100μmol·L^-1),除空白血清组外,其余各组均加入10 nmol·L^-1ET-1诱导HSC-T6细胞,蛋白免疫印迹法(Western blot)检测ET-1诱导的HSC-T6细胞中p-MLCⅡ,MLCⅡ蛋白表达。结果:选用血清浓度为5%,10%,15%作为含药血清浓度。与空白血清组比较,当归芍药散含药血清组明显降低基础状态下ET-1含量,ET-1 mRNA相对含量(P <0. 05,P <0. 01)。与空白血清组比较,模型组细胞内p-MLCⅡ,MLCⅡ蛋白表达水平均显著升高(P <0. 01);与模型组比较,当归芍药散含药血清各剂量组及Y-27632抑制剂组均可明显下调p-MLCⅡ,MLCⅡ蛋白表达(P <0. 05,P <0. 01)。结论:当归芍药散含药血清可能通过下调ET-1的含量,抑制ET-1的自分泌,从而下调p-MLCⅡ,MLCⅡ蛋白表达。
Objective: To investigate the effect of endothelin-1( ET-1) on the expression of phosphorylated myosin light chain Ⅱ( p-MLC Ⅱ) and myosin light chain Ⅱ( MLC Ⅱ) protein in rat hepatic stellate cells HSC-T6 and explore the intervention effect of Danggui Shaoyao San( DSS) drug-containing serum.Method: After HSC-T6 cells were seeded,DMEM and blank rat serum with final concentrations of 2. 5%,5%,10%,15% and 20% were added to each well. The viability of HSC-T6 cells was determined by methyl thiazolyl tetrazolium( MTT) assay to screen the suitable serum concentration range. The cells were divided into blank serum control group( 5%,10%,15%) and DSS drug-containing serum group( 5%,10%,15%). ELISA was used to detect the content of ET-1 in cell culture supernatant under basic state. The cells were divided into blank serum control group( 10%),DSS drug-containing serum low( 5%),medium( 10%) and high dose( 15%) groups.Real-time fluorescent quantitative polymerase chain reaction( Real-time PCR) was used to detect the level of ET-1 mRNA in cell culture supernatant under basic state. The cells were divided into blank serum control group( 10%),model group( 10%),DSS drug-containing serum low( 5%),medium( 10%),high dose( 15%)groups and Y-27632 inhibitor group( 100 μmol·L^-1. Except the blank serum control group,the other groups all received 10 nmol·L^-1ET-1 to induce HSC-T6 cells. Western blot was used to detect the expression of p-MLCⅡand MLCⅡ in HSC-T6 cells induced by ET-1. Result: Serum concentrations of 5%,10% and 15% were used as drug-containing serum concentrations. As compared with the blank serum control group,the DSS drug-containing serum group significantly reduced the relative content of ET-1 and ET-1 mRNA in the basic state( P < 0. 05,P <0. 01). As compared with the blank serum control group,the expression of p-MLCⅡ and MLCⅡ protein in the model group was significantly increased( P < 0. 01);DSS drug-containing serum groups and Y-27632 inhibitor group can significantly down-regulate p-MLC Ⅱ and MLC Ⅱ protein expression( P < 0. 05, P < 0. 01).Conclusion: DSS drug-containing serum may down-regulate the expression of p-MLC Ⅱ and MLC Ⅱ by downregulating the content of ET-1 and inhibiting the autocrine of ET-1.
作者
蒋沙莎
潘永福
杨沫
王运来
尹丹丹
许钒
JIANG Sha-sha;PAN Yong-fu;YANG Mo;WANG Yun-lai;YIN Dan-dan;XU Fan(School of Pharmacy,Anhui University of Chinese Medicine,Key Laboratory of Chinese Medicine Formula of Anhui Province,Hefei 230012,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2019年第2期14-19,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81573720)
