维生素D受体激活对小鼠胆管结扎所致肝纤维化的影响及其机制

doi:10.13481/j.1671-587x.20200409

摘要/Abstract

摘要： 目的：探讨维生素D受体（VDR）激活对胆总管结扎（BDL）诱导小鼠肝纤维化的保护作用，并阐明其作用机制。方法：将60只C57BL/6小鼠随机分为假手术组、假手术后给药组、模型组和治疗组，每组15只。假手术组小鼠开腹但不结扎胆总管，模型组小鼠双线结扎胆总管并中间离断，假手术后给药组和治疗组小鼠手术前3 d腹腔注射VDR激动剂帕立骨化醇（200ng·kg^-1，每周3次），术后继续给药5 d。术后第5天取小鼠肝脏，采用HE染色和天狼猩红染色观察小鼠肝组织病理形态表现和肝纤维化程度。二氢乙啶（DHE）染色检测小鼠肝组织活性氧（ROS）水平。Western blotting法检测小鼠肝组织中沉默交配型信息调节3（Sirt3）、8-羟基鸟嘌呤DNA糖苷酶1（OGG1）、α-平滑肌蛋白（α-SMA）和Ⅰ型胶原蛋白（Col Ⅰ）表达水平。结果：HE染色，假手术组和假手术后给药组小鼠肝细胞形态结构完整，无炎性细胞浸润；模型组小鼠组织中出现大量浸润的炎性细胞、点状样灶状坏死区，在肝小叶之间出现条索状的胶原沉积；与模型组比较，治疗组小鼠肝组织坏死灶面积明显缩小，炎症细胞浸润明显改善。天狼猩红染色，假手术组和假手术后给药组小鼠肝组织中仅大胆管周围存在少量的纤维化；模型组小鼠肝组织汇管区胶原沉积明显；与模型组比较，治疗组小鼠肝组织中胆管周围胶原沉积减少。DHE染色，假手术组和假手术后给药组小鼠肝组织中，仅在汇管区存在少量的红色荧光；模型组小鼠肝组织中，汇管区及大胆管周围呈现出明显增多的红色荧光；与模型组比较，治疗组小鼠红色荧光的强度降低。Western blotting法检测，与假手术组和假手术后给药组比较，模型组小鼠肝组织中Sirt3蛋白表达水平明显降低（P<0.05），OGG1、α-SMA和Col Ⅰ蛋白表达水平明显升高（P<0.05）；与模型组比较，治疗组小鼠肝组织中Sirt3蛋白表达水平明显升高（P<0.05），OGG1、α-SMA和Col Ⅰ蛋白表达水平明显降低（P<0.05）。结论：VDR激活通过上调Sirt3蛋白降低氧化应激导致的肝星状细胞（HSC）激活，缓解胆汁淤积性肝纤维化。

关键词: 肝纤维化, 维生素D受体, 沉默交配型信息调节3, 氧化应激, 肝星状细胞

Abstract: Objective: To investigate the protective effect of vitamin D receptor (VDR) activation on the bile duct ligation (BDL)-induced liver fibrosis in the mice, and to elucidate its possible mechanism. Methods: Sixty male C57BL/6 mice were randomly divided into sham operation group, sham operation+ paricalcitol group, model group (BDL) and treatment group (BDL+paricalcitol) (n=15).In sham operation, the abdominal cavities were opened but the bile duct was not ligated. In model group, the bile ducts of the mice were ligated with double lines and the middle of the bile duct was severed. The mice in sham operation+paricalcitol group and treatment group were intraperitoneally injected with the VDR agonist paricalcitol (200 ng·kg^-1, three time a week) 3 d before the operation and continued treatment for 5 d. The livers of mice were removed on the 5th day after bile duct ligation,and the morphology of liver tissue and the fibrosis degree were observed by HE staining and Sirus red staining. The levels of reactive oxygen species (ROS) in the liver tissue of the mice were detected by dihydroethidine (DHE) staining. Western blotting method was used to detect the expression levels of silence mating type information regulation 3 (Sirt3), 8-hydroxy guanine DNA glycosidase 1(OGG1), alpha-smooth muscle (α-SMA)and collagen Ⅰ (Col Ⅰ) in the liver tissue of the mice. Results: The HE staining results showed that the structure of liver cells was complete, no inflammatory cell infiltration; in model group, a large number of infiltrated inflammatory cells and spot-like focal necrotic areas were observed in the liver of the mice; compared with model group, the area of liver tissue necrosis in treatment group was significantly reduced and the infiltration of inflammatory cells was significantly decreased. The Sirus red staining results showed that there was only a small amount of fibrosis around the big bile duct in the liver tissue of the mice in sham operation group and sham operation+paricalcitol group; collagen deposition was obvious in the portal area of the liver tissue of the mice in model group;the collagen deposition around the bile duct of the mice in treatment group was reduced compared with model group.The DHE staining results showed that there was only a small amount of red fluorescence in the portal area in the liver tissue of the mice in sham operation group and sham operation+paricalcitol group; in model group, there were significantly more red fluorescence in the portal area and the area around the bile duct; the intensity of red fluorescence in level treatment group was lower than that in model group.The Western blotting results showed that the expression level of Sirt3 protein in liver tissue of the mice in model group was significantly lower than sham operation group and sham operation+paricalcitol group (P<0.05);the expression levels of OGG1, α-SMA and Col Ⅰ proteins were increased significantly(P<0.05);compared with model group, the expression level of Sirt3 protein in liver tissue of the mice in treatment group was significantly increased(P<0.05),and the expression levels of OGG1, α-SMA and Col Ⅰ proteins were increased obviously (P<0.05). Conclusion: VDR activation can reduces oxidative stress-induced hepatic stellate cell activation by increasing the Sirt3 protein; thereby reduces the BDL-induced liver fibrosis in the mice.

Key words: liver fibrosis, vitamin D receptor, silence mating type information regulation 3, oxidative stress, hepatic stellate cell

中图分类号:

R575

杨帆, 李丽华. 维生素D受体激活对小鼠胆管结扎所致肝纤维化的影响及其机制[J]. 吉林大学学报(医学版), 2020, 46(04): 722-727.

YANG Fan, LI Lihua. Effect of vitamin D receptor activation on hepatic fibrosis induced by bile duct ligation in mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 722-727.

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