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（R-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／,tool／L），细胞计数试剂盒一8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48h，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DunnettT3检验，组间比较采用单因素方差分析。结果C66能有效抑制HSCT6增殖，呈现浓度和时间依赖性，其中10μmol／LC66作用48h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（￡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、Ⅰ-SMAmRNA相对表达显著升高，差异均有统计学意义（￡值分别为4．705、9．492和38．27，均P〈0．05）。与对照相组比，TGF—B1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。

Objective To investigate the effects of curcumin derivatives （C66） on proliferation and expressions of α smooth muscle （a 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 /lmol/L, 5 /,mol/L, 10 /,mol/L, 20 μmol/L）. Cell proliferation was detected by Cell Counting Kit-8 assay. Then, according to the time and concentration of C66 above, ceils were divided into 5 groups including control group, TGF-α only group, TGF-α combined with CB1 antagonist group, TGF-α combined with C66 group and TGF-α combined with CB1 antagonist plus C66 group. Quantitative real time polymerase chain reaction and western blot were used to assess the expressions of a-SMA, Collagen I , CB1, JNK and phosphorylation of JNK （p-JNK）. The variance homogeneity of multiple samples was com, ared 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-α 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 I-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 TGFβ 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.