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
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.
更多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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