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姜黄素衍生物C66对转化生长因子-β刺激的大鼠肝星状细胞增殖和活化的研究

The effects of curcumin derivatives C66 on proliferation and activation of rat hepatic stellate cells induced by transforming growth factor-β

摘要:

目的 观察姜黄素衍生物C66对TGF-β刺激的大鼠肝星状细胞(HSC)增殖及α-平滑肌动蛋白(α-SMA)、Ⅰ型胶原表达的影响及其与大麻素受体(CB)1的关系.方法 体外培养HSC-T6细胞,分为对照组、C66不同浓度组(1、2、5、10和20 μmol/L),细胞计数试剂盒-8检测其吸光度(A)值.选取最佳C66作用浓度及时间,分为对照组、TGF-β干预组、TGF-β+CB1拮抗剂组、TGF-β+-C66干预组、TGF-β-+-CB1拮抗剂+C66联合干预组,分别培养48 h,以蛋白质印迹法、实时荧光定量PCR检测CB1、α-SMA、Ⅰ型胶原蛋白及mRNA的表达,以蛋白质印迹法检测磷酸化c-jun氨基末端激酶(p-JNK)、c-jun氨基末端激酶(JNK)表达水平.多样本均数方差齐性者采用LSD法进行两两比较,方差不齐者采用Dunnett T3检验,组间比较采用单因素方差分析.结果 C66能有效抑制HSC T6增殖,呈现浓度和时间依赖性,其中10 μmol/L C66作用48 h对HSC-T6的抑制效果最明显,抑制率为54%.与对照组相比,单独TGF-β1干预组CB1、Ⅰ型胶原、α-SMA蛋白表达明显升高,差异均有统计学意义(t值分别为6.188、3.48和20.64,均P<0.05).与对照组相比,单独TGF-β1干预组CB1、Ⅰ型胶原、α-SMA mRNA相对表达显著升高,差异均有统计学意义(t值分别为4.705、9.492和38.27,均P<0.05).与对照相组比,TGF-β1干预组p-JNK水平显著升高,差异有统计学意义(t=9.567,P<0.05).结论 C66能有效抑制HSC-T6细胞的增殖及胶原合成,且联合使用CB1拮抗剂效果更明显,该过程可能涉及JNK磷酸化水平的调节,有助于理解肝纤维化发生、发展的机制及为治疗肝纤维化寻找新的靶点.

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abstracts:

Objective To investigate the effects of curcumin derivatives (C66) on proliferation and expressions of α-smooth muscle (α SMA) and Collagen Ⅰ in rat hepatic stellate cells (HSC) induced by transforming growth factor-β (TGF-β) in vitro and the relationship with cannabinoid receptor type 1 (CB1).Methods To determine the optimum time and concentration of C66,HSC-T6 cell line was cultured in vitro and divided into control group and groups with different doses of C66 (1 μmol/L,2 μmol/L,5 μmol/L,10 μmol/L,20 μmol/L).Cell proliferation was detected by Cell Counting Kit-8 assay.Then,according to the time and concentration of C66 above,cells were divided into 5 groups including control group,TGF-β only group,TGF-β combined with CB1 antagonist group,TGF-β combined with C66 group and TGF β combined with CB1 antagonist plus C66 group.Quantitative realtime polymerase chain reaction and western blot were used to assess the expressions of α SMA,Collagen Ⅰ,CB1,JNK and phosphorylation of JNK (p-JNK).The variance homogeneity of multiple samples was compared by LSD method.The variance was compared with Dunnett T3 test.One-way analysis of variance was performed to compare the mean values among the groups.Results The inhibitory effect of C66 on HSC-T6 proliferation was dose and time dependent.The optimum time and concentration were 48h and 10 μmol/L,respectively,with the inhibition rate of 54%.Compared with control group,expressions of α-SMA,collagen Ⅰ and CB1 were significantly elevated in TGF-β group (t=6.188,3.48 and 20.64,respectively,all P<0.05).TGF-β1 could increase the relative mRNA expressions of CB1,collagen Ⅰ and α-SMA with significant differences (t =4.705,9.492 and 38.27,respectively,all P< 0.05).Compared with control group,p-JNK expression was significantly elevated in TGF-β group (t=9.567,P<0.05).Conclusions C66 could inhibit the proliferation and collagen synthesis in HSC-T6 induced by TGF-β and the effect is strengthened when combined with CB1 antagonist,which may involve JNK phosphorylation.Our study provides a better understanding on the mechanism and a new target for treatment of liver fibrosis.

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作者: 陈泽 [1] 陈达之 [1] 萧云蕾 [1] 肖茜茜 [1] 王永林 [1] 许烂漫 [1] 陈永平 [1]
期刊: 《中华传染病杂志》2017年35卷8期 492-497页 ISTICCSCD
栏目名称: 论著
DOI: 10.3760/cma.j.issn.1000-6680.2017.08.009
发布时间: 2017-11-29
基金项目:
National Natural Science Foundation of China Natural Science Foundation of Zhejiang Province of China Public Welfare Social Development (Medical and Health) Science and Technology Projects of Municipal Science and Technology Brueau of Wenzhou of China The Medical Award Fund of Beijing of China The End Stage Liver Disease Early Warning and Intervention Treatment Technology Innovation Team of Wenzhou of China Wang Baoen Liver Fibrosis Research Fund Chinese Academy of Medical Science Reseach Project (15030230611)国家自然科学基金 浙江省自然科学基金 温州市科技局公益性社会发展(医疗卫生)科技项目 北京医学奖励基金 温州市终末期肝病的预警与干预治疗科技创新团队 王宝恩肝纤维化研究基金 中华医学会科研项目
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