苦参碱对乙醛活化的肝星状细胞CFSC-8B增殖和胶原合成的影响及机制研究
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篇名: 苦参碱对乙醛活化的肝星状细胞CFSC-8B增殖和胶原合成的影响及机制研究
TITLE: Study on the Effects and Mechanism of Matrine on Proliferation and Collagen Synthesis of Hepatic Stellate Cells CFSC-8B Activated by Acetaldehyde
摘要: 目的:研究苦参碱对乙醛活化的大鼠肝星状细胞CFSC-8B增殖和胶原合成的影响,并探讨其可能的作用机制。方法:取体外培养的CFSC-8B细胞,分为空白对照组、模型组、阳性对照组(2.5μmol/L秋水仙碱)和苦参碱低、中、高浓度组(30、60、120μmol/L)。除空白对照组外,其余各组细胞均加入200μmol/L乙醛溶液诱导活化,并同时加入相应药液(空白对照组和模型组加入等体积空白培养液),共同作用24h后,采用CCK-8法检测各组细胞的存活率。另取细胞分为空白对照组、模型组、阳性对照组(2.5μmol/L秋水仙碱)和苦参碱中、高浓度组(60、120μmol/L),同法活化和加药处理。分别采用酶消化法检测细胞培养液中羟脯氨酸(Hyp)含量;采用酶联免疫吸附法检测细胞培养液中Ⅰ型胶原蛋白(Col-Ⅰ)和Ⅲ型胶原蛋白(Col-Ⅲ)含量;采用实时荧光定量-聚合酶链式反应法检测细胞中α-平滑肌激动蛋白(α-SMA)、转化生长因子β(1TGF-β1)、TGF-β1Ⅰ型受体(TβR-Ⅰ)、TGF-β1Ⅱ型受体(TβR-Ⅱ)、Smad3、Smad4和Smad7mRNA表达水平;采用Westernblotting法检测细胞中α-SMA、TGF-β1、TβR-Ⅰ、TβR-Ⅱ、Smad3、Smad4和Smad7蛋白表达水平。结果:与空白对照组比较,模型组细胞存活率显著升高(P<0.05);细胞培养液中Hyp、Col-Ⅰ、Col-Ⅲ含量和细胞中α-SMA、TGF-β1、TβR-Ⅰ、TβR-Ⅱ、Smad3和Smad4mRNA及其蛋白表达水平均显著升高(P<0.05),细胞中Smad7mRNA及其蛋白表达水平均显著降低(P<0.05)。与模型组比较,阳性对照组和苦参碱中、高浓度组细胞存活率和细胞培养液中Hyp、Col-Ⅰ、Col-Ⅲ含量以及细胞中α-SMA、Smad4mRNA及其蛋白表达水平均显著降低(P<0.05),细胞中Smad7mRNA及其蛋白表达水平均显著升高(P<0.05);阳性对照组和苦参碱高浓度组细胞中TGF-β1、TβR-Ⅰ、TβR-Ⅱ、Smad3mRNA及其蛋白表达水平均显著降低(P<0.05)。与苦参碱中浓度组比较,苦参碱高浓度组细胞各指标水平改善程度均更显著(P<0.05)。结论:苦参碱能抑制乙醛活化的CFSC-8B细胞的增殖和胶原合成,且具有一定的浓度依赖性;其机制可能与调控TGF-β1/Smad信号通路的传导有关。
ABSTRACT: OBJECTIVE:To study the effects of matrine on proliferation and collagen synthesis of rat hepatic stellate cells CFSC-8B activated by acetaldehyde ,and to investigate its possible mechanism. METHODS :CFSC-8B cells cultured in vitro were divided into blank control group ,model group ,positive control group (2.5 μmol/L colchicine)and matrine low ,medium and high concentration groups (30,60,120 μmol/L). Except for blank control group ,other groups were activated with 200 μmol/L acetaldehyde for 24 h;medicine groups were intervened with relevant medicine for 24 h(blank control group and model group were intervened with equal volume blank medium ). Survival rate of cell was detected by CCK- 8 assay. C ells were divided into blank control group ,model group ,positive control group (2.5 μmol/L colchicine),matrine medium and high concentration groups (60,120 μmol/L),then activated and treated with same method. Hydroxyprolin (Hyp)content in cell culture solution was tested by enzyme digestion. The contents of Col- Ⅰ and Col- Ⅲ in cell culture solution were determined by ELISA. mRNA expressionss of α-SMA,TGF-β1,TβR-І,TβR-Ⅱ,Smad3,Smad4 and Smad 7 in cells were detected by RT-PCR. The protein expressions of α-SMA,TGF-β1,TβR-Ⅰ,TβR- Ⅱ,Smad3,Smad4 and Samd 7 in cells were detected by Western blotting. RESULTS :Compared with blank control group ,survival rate of cells in model group was increased significantly (P<0.05);the contents of Hyp ,Col-Ⅰ and Col- Ⅲ in cell culture solution ,mRNA and its protein expressions of α-SMA,TGF-β1,TβR-Ⅰ,TβR-Ⅱ,Smad3,Smad4 in cells were increased significantly in model group (P<0.05),while the mRNA and protein expression of Smad 7 was decreased significantly(P<0.05). Compared with model group ,survival rate of cells ,the contents of Hyp ,Col-Ⅰ and Col- Ⅲ in cell culture solution,the mRNA and protein expressions of α-SMA and Smad 4 were decreased significantly in positive control group and matrine medium and high concentration groups (P<0.05), while the mRNA and protein expression of Smad 7 was WF-0099) increased significantly (P<0.05);the mRNA and proteinexpressions of TGF-β1,TβR-Ⅰ,TβR-Ⅱ and Smad 3 were decreased significantly in positive control group and matrine high concentration group (P<0.05). Compared with matrine medium concentration group ,all above indexes were improved significantly in matrine high concentration group (P<0.05). CONCLUSIONS :Matrine can suppress the proliferation and collagen synthesis of CFSC- 8B cells activated by acetaldehyde ,with a centain concentrlation dependence ,the mechanism of which may be associated with regulating the conduction of TGFβ/Smad signal pathway.
期刊: 2020年第31卷第11期
作者: 王晓丽
AUTHORS: WANG Xiaoli
关键字: 苦参碱;肝星状细胞;增殖;胶原合成;转化生长因子β1/Smad通路;机制
KEYWORDS: Matrine;Hepatic stellate cells ;Proliferation;Collagen synthesis ;TGF-β1/Smad pathway ;Mechanism
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