吉林大学学报(医学版) ›› 2021, Vol. 47 ›› Issue (2): 257-264.doi: 10.13481/j.1671-587X.20210202

• 基础研究 • 上一篇    下一篇

骨化三醇对胆管结扎所致小鼠肝纤维化的保护作用及其机制

贾荣军1,马丽曼2,李丽华1()   

  1. 1.锦州医科大学基础医学院细胞生物学教研室,辽宁 锦州 121000
    2.台州学院医学院实验中心,浙江 台州 318000
  • 收稿日期:2020-09-17 出版日期:2021-03-28 发布日期:2021-03-25
  • 通讯作者: 李丽华 E-mail:2744900797@qq.com
  • 作者简介:贾荣军(1995-),男,河南省清丰县人,在读硕士研究生,主要从事肝纤维化基础方面的研究。
  • 基金资助:
    国家自然科学基金面上项目(31571184)

Protective effect of calcitriol on hepatic fibrosis induced by bile duct ligation in mice and its mechanism

Rongjun JIA1,Liman MA2,Lihua LI1()   

  1. 1.Department of Cell Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China
    2.Department of Clinical Medicine,School of Medical Sciences,Taizhou University,Taizhou 31800,China
  • Received:2020-09-17 Online:2021-03-28 Published:2021-03-25
  • Contact: Lihua LI E-mail:2744900797@qq.com

摘要: 目的

探讨骨化三醇对胆总管结扎(BDL)诱导的小鼠肝纤维化的保护作用,并阐述其可能的作用机制。

方法

选用45只8~10周雄性C57BL/6小鼠,随机分为假手术组、肝纤维化模型组(模型组)和骨化三醇治疗组(治疗组),每组15只。假手术组小鼠绕胆总管穿过手术线,不结扎。模型组小鼠双重结扎胆总管并离断。治疗组小鼠术后2.5 μg·kg-1腹腔注射骨化三醇,每周3次。假手术组和模型组小鼠注射等体积生理盐水。术后继续给药4周,第28天眼眶取血,取小鼠肝脏。术后第2天检测各组小鼠血清中丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)活性及总胆汁酸(TBA)、总胆红素(TBIL)和羟脯氨酸(Hyp)水平。苏木精-伊红(HE)染色和天狼猩红染色观察各组小鼠肝组织病理形态学和肝组织纤维化程度。Western blotting法检测各组小鼠肝组织中维生素D受体(VDR)细胞外信号调节蛋白激酶(ERK)、磷酸化细胞外信号调节蛋白激酶(p-ERK)、α-平滑肌蛋白(α-SMA)、转化生长因子β1(TGF-β1)、Ⅰ型胶原蛋白(Col Ⅰ)和结蛋白(Desmin)表达水平。免疫组织化学法检测各组小鼠肝组织中α-SMA和TGF-β1蛋白表达情况。

结果

与假手术组比较,模型组小鼠血清中ALT和AST活性及TBA、TBIL和Hyp水平明显升高(P<0.05);与模型组比较,治疗组小鼠血清中ALT和AST活性及TBA、TBIL和Hyp水平明显降低(P<0.05)。HE染色,假手术组小鼠肝小叶正常,组织结构较为完整,未见炎性细胞浸润;模型组小鼠肝组织出现炎性细胞浸润,坏死灶和胶原沉积较为明显;与模型组比较,治疗组小鼠肝组织坏死灶区域明显减少,炎性浸润有所改善。天狼猩红染色,假手术组小鼠肝组织仅有少量胶原沉积出现在中央静脉周围;模型组小鼠肝组织中央静脉及汇管区出现明显胶原沉积;与模型组比较,治疗组胶原沉积明显减少。Western blotting法检测,与假手术组比较,模型组小鼠肝组织中ERK、p-ERK、α-SMA、TGF-β1、Col Ⅰ和Desmin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ERK、pERK、α-SMA、TGF-β1、Col Ⅰ和Desmin蛋白表达水平明显降低(P<0.05)。免疫组织化学,模型组小鼠肝组织中α-SMA和TGF-β1蛋白表达量高于假手术组;治疗组小鼠肝组织中α-SMA和TGF-β1蛋白表达量明显低于模型组。

结论

骨化三醇通过激活维VDR,下调ERK蛋白表达,降低TGF-β1表达,减少肝星状细胞(HSCs)激活,抑制胶原在组织间沉积,从而缓解BDL诱导的小鼠肝纤维化。

关键词: 肝纤维化, 骨化三醇, 维生素D受体, 肝星状细胞

Abstract: Objective

To investigate the protective effect of calcitriol on liver fibrosis induced by common bile duct ligation (BDL) in the mice, and to explain its possible mechanism.

Methods

Forty-five male C57BL/6 mice aged 8 to 10 weeks were randomly divided into sham operation group, liver fibrosis model group (model group) and calcitriol treatment group (treatment group), with 15 mice in each group. The mice in sham operation group received the surgical line around the common bile duct without ligation; in model group, the common bile ducts of the mice were double-ligated and severed, and the mice in treatment group were injected intraperitoneally with calcitriol (2.5 μg·kg-1) 3 times a week after operation. The mice in sham operation group and model group were injected with an equal volume of saline. The administration was continued until 4 weeks after the operation. On the 28th day, blood was taken from the orbit and the liver of the mouse was taken. On the second day after the operation, the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the levels of total bile acid (TBA), total bilirubin (TBIL) and hydroxyproline (Hyp) in the serum of the mice in various groups were detected. The pathomorphology and fibrosis degrees of liver tissue were observed with hematoxylin and eosin (HE) and Sirius red staining. Western blotting method was used to detect the expression levels of vitamin D receptor(VDR),extracellular signal-regulated protein kinase (ERK),phosphorylated extracellular signal-regulated protein kinase (pERK), α-smooth muscle protein (α-SMA),transforming growth factor-β1 (TGF-β1),type Ⅰ collagen (Col Ⅰ) and Desmin in liver tissue of the mice in various groups. Immunohistochemistry method was used to detect the expressions of α-SMA and TGF-β1 in liver tissue of the mice in various groups.

Results

Compared with sham operation group, the serum activities of ALT and AST and the levels of TBA, TBIL and Hyp of the mice in model group were significantly increased (P<0.05); compared with model group, the serum activities of ALT and AST and the levels of TBA, TBIL and Hyp in treatment group were significantly reduced (P<0.05). The HE staining results showed that the liver lobules of the mice in sham operation group were normal, the tissue structure was relatively complete, and no inflammatory cell infiltration was seen; the liver tissue of the mice in model group had inflammatory cell infiltration, and necrosis and collagen deposition were more obvious; compared with model group, the necrotic area of ??liver tissue of the mice in treatment group was significantly reduced, and the inflammatory infiltration was improved. The Sirius red staining results showed that only a small amount of collagen deposition in liver tissue of the mice in sham operation group appeared around the central vein; the central vein and portal area of liver tissue of the mice in model group showed obvious collagen deposits; compared with model group, the collagen deposition in treatment group was significantly reduced. The Western blotting results showed that compared with sham operation group, the expression levels of ERK, p-ERK, α-SMA, TGF-β1, Col Ⅰ and Desmin in liver tissue of the mice in model group were significantly increased (P<0.05); compared with model group, the expression levels of ERK, p-ERK, α-SMA, TGF-β1, Col Ⅰ and Desmin in liver tissue of the mice in treatment group were significantly reduced (P<0.05). The immunohistochemical staining results showed that the expressions of α-SMA and TGF-β1 proteins in liver tissue of the mice in model group were higher than those in sham operation group, and the expressions of α-SMA and TGF-β1 proteins in treatment group were significantly lower than those in model group.

Conclusion

Calcitriol can reduce the activation of hepatic stellate cells (HSCs) by activating VDR, down-regulating the ERK protein expression, reducing the expression of TGF-β1,and inhibiting the deposition of collagen in tissue, thereby alleviate the BDL-induced liver fibrosis in the mice.

Key words: liver fibrosis, calcitriol, vitamin D receptor, hepatic stellate cells

中图分类号: 

  • R575.2