白藜芦醇通过调控肝脏生物钟改善CCl4诱导的小鼠肝纤维化

Resveratrol improves CCl4 induced liver fibrosis in mice by regulating liver circadian clock

关键词:  白藜芦醇  肝纤维化  四氯化碳  生物钟

Key words:  resveratrol  hepatic fibrosis  carbon tetrachloride  circadian clock

摘要:

[目的] 观察白藜芦醇（Res）对四氯化碳（CCl4）诱导的小鼠肝纤维化（HF）的干预作用及其对肝脏生物钟的调节效应。[方法] 本实验采用雄性C57BL/6小鼠，将小鼠按体质量随机分成对照组、模型组、白藜芦醇组，每组10只。模型组及白藜芦醇组按2.5mL/kg腹腔注射体积分数为20% CCl₄油剂溶液进行造模，一周两次，连续4周，对照组按同等剂量同等频次给予腹腔注射橄榄油。白藜芦醇以生理盐水稀释呈终浓度为30mg/kg进行腹腔注射干预，每日1次，连续4周，对照组及模型组给予等量生理盐水按同等频次腹腔注射。4周后处死小鼠并收集血清及肝组织样本。采用病理组织切片苏木精-伊红（HE）染色、马松（Masson）染色、天狼星红（Sirius Red）染色观察肝纤维化情况，全自动生化分析仪测定小鼠血清丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、乳酸脱氢酶（LDH）活性，免疫组织化学检测肝脏α-平滑肌肌动蛋白（α-SMA）、I型胶原（COL-1）评估肝纤维化程度，实时聚合酶链反应（RT-PCR）、蛋白质印迹法（Westernblot）检测肝脏生物钟基因Clock、脑和肌肉芳香烃受体核转运样蛋白1（Bmal1）、周期基因1（Per1）周期基因2（Per2）、隐花色素1（Cry1）、隐花色素2（Cry2）的表达。[结果] 模型组与对照组相比，肝功能指标如ALT、AST、LDH等均显著升高；病理切片HE染色提示模型组汇管区纤维组织增生，肝小叶间胆管明显增生，部分分割包绕肝小叶使其结构紊乱，Masson染色及Sirius Red染色提示模型组汇管区胶原沉积显著增多，免疫组化显示α-SMA、COL-1阳性面积显著增多；RT-PCR及Western lot结果显示肝脏生物钟基因Clock、Bmal1、Per1、Per2、Cry1、Cry2的mRNA及蛋白表达均显著下降。白藜芦醇干预后可显著逆转上述指标变化。[结论] 白藜芦醇可显著减轻CCl₄诱导的小鼠肝纤维化，及机制可能与调控肝脏生物钟基因有关。

Abstract:

[Objective] To observe the intervention effect of resveratrol (Res) on carbon tetrachloride (CCL₄)-induced hepatic fibrosis (HF) in mice and its regulatory effect on liver circadian clock. [Methods] Male C57BL/6 mice were randomly divided into control group,model group and resveratrol group,10 mouse in each group. The model group and resveratrol group were made by intraperitoneal injection of 20% CCl₄ oil solution at a volume fraction of 2.5mL/kg,twice a week for 4 weeks. The control group was given intraperitoneal injection of olive oil at the same dose and frequency. Resveratrol was diluted with normal saline and the final concentration was 30 mg/kg for intraperitoneal injection intervention,once a day for 4 weeks. The control group and model group were given the same amount of normal saline and intraperitoneal injection at the same frequency. After 4 weeks,the mice were killed and serum and liver tissue samples were collected. The liver fibrosis was observed by HE staining,Masson staining and Sirius red staining. The activities of serum alanine aminotransferase (ALT),aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured by automatic biochemical analyzer,and the liver was detected by immunohistochemistry α-Smooth muscle actin (α-SMA) and collagen I (COL-1) were used to evaluate the degree of liver fibrosis. The expressions of clock gene in liver,including clock,brain and muscle aromatic hydrocarbon receptor nuclear like protein 1 (BMAL1),period 1 (Per1),period 2 (Per2),cryptochrome 1 (Cry1) and cryptochrome 2 (Cry2) were detected by real-time PCR and Western blot. [Results] Compared with the control group,the indexes of liver function such as ALT,AST and LDH in the model group were significantly increased. HE staining of pathological sections showed that the fibrous tissue in the portal area of the model group was proliferated,and the bile duct between hepatic lobules was significantly proliferated. Some segments wrapped around the hepatic lobules made its structure disordered. Masson staining and Sirius red staining showed that the collagen deposition in the portal area of the model group was significantly increased,and immunohistochemistry showed that the positive areas of α-SMA and COL-1 increased significantly. The results of real-time PCR and Western blot showed that the mRNA and protein expressions of clock, Bmal1,Per1,Per2,Cry1 and Cry2 decreased significantly. Resveratrol intervention can significantly reverse the changes of the above indicators. [Conclusion] Resveratrol can significantly reduce CCl₄ induced liver fibrosis in mice,and the mechanism may be related to the regulation of liver circadian clock genes.