[1]王彩娥,杨鹿奎,李桂芳,等.沙利度胺对四氯化碳致小鼠肝纤维化的影响[J].新乡医学院学报,2021,38(8):706-713.[doi:10.7683/xxyxyxb.2021.08.002]
　WANG Caie,YANG Lukui,LI Guifang,et al.Effect of thalidomide on liver fibrosis induced by carbon tetrachloride in mice[J].Journal of Xinxiang Medical University,2021,38(8):706-713.[doi:10.7683/xxyxyxb.2021.08.002]
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沙利度胺对四氯化碳致小鼠肝纤维化的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
38
期数:
2021年8
页码:
706-713
栏目:
基础研究
出版日期:
2021-08-05

文章信息/Info

Title:
Effect of thalidomide on liver fibrosis induced by carbon tetrachloride in mice
作者:
王彩娥; 杨鹿奎; 李桂芳; 曹永建; 高文芳
（河南科技大学第一附属医院药学部，河南科技大学临床医学院，河南　洛阳　471003）
Author(s):
WANG Cai′e; YANG Lukui; LI Guifang; CAO Yongjian; GAO Wenfang
(Department of Pharmacy,the First Affiliated Hospital of Henan University of Science and Technology,Clinical Medical College of Henan University of Science and Technology,Luoyang 471003,Henan Province,China)
关键词:
沙利度胺; 肝纤维化; 巨噬细胞极化; M1/M2巨噬细胞
Keywords:
thalidomideliver fibrosismacrophage polarizationM1/M2 macrophages
分类号:
R575.2
DOI:
10.7683/xxyxyxb.2021.08.002
文献标志码:
A
摘要:
目的　探讨沙利度胺对四氯化碳（CCl4）诱导的小鼠肝纤维化的保护作用及机制。方法　将24只雄性C57BL/6小鼠按随机数字表法分为正常组、模型组、复方甘草酸苷片组（阳性对照组）、沙利度胺组，每组6只。每周一和周四，正常组小鼠腹腔注射橄榄油10 μL·g-1，模型组、阳性对照组、沙利度胺组小鼠腹腔注射体积分数0.6 % CCl4（10 μL·g-1），连续6周；造模第5周开始，阳性对照组小鼠给予3.5 g·L-1复方甘草酸苷片混悬液灌胃（35 mg·kg-1·d-1）；沙利度胺组小鼠给予3.0 g·L-1沙利度胺混悬液灌胃（30 mg·kg-1·d-1），正常组和模型组小鼠给予生理盐水灌胃（10 μL·g-1），各组小鼠每日灌胃1次，连续2周；各组小鼠均于每周一测体质量；第6周末次给药2 h后，使用毛细管眼眶取血0.6 mL，采用速率法检测血清丙氨酸转氨酶（ALT）和天门冬氨酸氨基转移酶（AST）水平；取血后采用过量七氟烷麻醉处死小鼠，取肝组织，计算小鼠肝指数（肝指数=肝质量/体质量×100%）；采用苏木精-伊红（HE）染色观察小鼠肝细胞状态，Masson染色观察小鼠肝组织结构，酶联免疫吸附试验法检测肝组织中肿瘤坏死因子α（TNF-α）水平，石蜡切片免疫荧光双标法检测小鼠肝组织中M1型巨噬细胞标志物CD68和M2型巨噬细胞标志物CD163表达。结果　造模第0、1、2、3、4、5、6周各组小鼠体质量比较差异无统计学意义（P>0.05）。第6周末，模型组小鼠肝质量及肝指数均显著高于正常组（P<0.01），阳性对照组和沙利度胺组小鼠肝质量及肝指数均显著低于模型组（P<0.01），沙利度胺组与阳性对照组小鼠肝质量及肝指数比较差异无统计学意义（P>0.05）；模型组、阳性对照组及沙利度胺组小鼠血清ALT、AST水平显著高于正常组（P<0.05），阳性对照组及沙利度胺组小鼠血清ALT、AST水平显著低于模型组（P<0.05），沙利度胺组与阳性对照组小鼠血清ALT、AST水平比较差异无统计学意义（P>0.05）；模型组小鼠肝组织中TNF-α水平显著高于正常组（P<0.05），阳性对照组及沙利度胺组与正常组小鼠肝组织中TNF-α水平比较差异无统计学意义（P>0.05），阳性对照组及沙利度胺组小鼠肝组织中TNF-α水平显著低于模型组（P<0.05），沙利度胺组与阳性对照组小鼠肝组织中TNF-α水平比较差异无统计学意义（P>0.05）。HE染色显示，正常组小鼠肝细胞结构完整，排列规则，肝小叶结构清晰；模型组小鼠肝组织汇管区与门静脉周围肝细胞大量空泡样坏死，排列紊乱，肝小叶结构不完整；阳性对照组和沙利度胺组小鼠肝组织汇管区与门静脉周围肝细胞有少量空泡样坏死，排列基本规则。Masson染色显示，正常组小鼠肝组织无明显胶原纤维增生；模型组小鼠肝汇管区门静脉与中央静脉周围大量胶原纤维增生，且有少量桥接形成；阳性对照组和沙利度胺组小鼠肝组织局部汇管区和中央静脉周围有少量胶原纤维增生。模型组小鼠肝组织Masson评分显著高于正常组（P<0.05），阳性对照组和沙利度胺组小鼠肝组织Masson评分显著低于模型组（P<0.05），阳性对照组与沙利度胺组小鼠肝组织Masson评分比较差异无统计学意义（P>0.05）。免疫荧光双标检测显示正常组小鼠肝组织中存在少量CD68和CD163阳性表达；与正常组比较，模型组小鼠肝组织中CD68阳性表达相对增强， CD163阳性表达相对减弱；与模型组比较，沙利度胺组小鼠肝组织中CD68阳性表达相对较弱，CD163阳性表达相对较强；与阳性对照组相比，沙利度胺组小鼠肝组织中CD68阳性表达相似，CD163阳性表达较强。结论　沙利度胺可有效改善CCl4诱导的小鼠肝纤维化，显著减轻肝损伤，其作用机制可能与影响巨噬细胞极化有关。
Abstract:
Objective　To explore the protective effect and mechanism of thalidomide on carbon tetrachloride (CCl4)-induced liver fibrosis in mice.Methods　Twenty-four male C57BL/6 mice were randomly divided into normal group,model group,compound glycyrrhizin tablet group (positive control group) and thalidomide group,with 6 mice in each group.On every monday and thursday,the mice in the normal group were injected intraperitoneally with olive oil 10 μL·g-1,and the mice in the model group,positive control group and thalidomide group were injected intraperitoneally with volume fraction of 0.6% CCl4 (10 μL·g-1),continuing for 6 weekson the fifth week of modeling,the mice in the positive control group were given 3.5 g·L-1 compound glycyrrhizin tablets suspension (35 mg·kg-1·d-1) by intragastric administrationthe mice in the thalidomide group were given 3.0 g·L-1 thalidomide suspension (30 mg·kg-1·d-1) by intragastric administration,the mice in the normal group and model group were given normal saline (10 μL·g-1 ) by intragastric administration,the mice in each group were given intragastric administration once a day for consecutive 2 weeksthe mice in each group were weighted on every mondayafter 2 hours of administration on the sixth week,0.6 mL of blood was taken from the orbit by using capillary.The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by rate methodthe mice were sacrificed with excessive sevoflurance after blood collection,liver tissues were taken,and the liver index of the mice was calculated (liver index=liver mass/body mass×100%)the state of liver cells of mice were observed by hematoxylin-eosin (HE) staining,the structure of liver tissue of mice was observed by Masson staining.The content of tumor necrosis facto-α(TNF-α) in liver tissue was detected by enzyme-linked immunosorbent assay.The expressions of M1 macrophage marker CD68 and M2 macrophage marker CD163 in liver tissue were detected by paraffin section immunofluorescence double labeling.Results　At 0,1,2,3,4,5 and 6 weeks after modeling,there was no significant difference in the body weight of mice among each group (P>0.05).At the end of the sixth week,the liver quality and liver index of mice in the model group were significantly higher than those in the normal group (P<0.01)the liver quality and liver index of mice in the positive control group and thalidomide group were significantly lower than those in the model group (P<0.01)there was no significant difference in liver quality and liver index of mice between the thalidomide group and the positive control group (P>0.05).The serum ALT and AST levels of mice in the model group,positive control group and thalidomide group were significantly higher than those in the normal group (P<0.05)the serum ALT and AST levels of mice in the positive control group and the thalidomide group were significantly lower than those in the model group (P<0.05)there was no significant difference in serum ALT and AST levels of mice between the thalidomide group and the positive control group (P>0.05).The content of TNF-α in the liver tissue of mice in the model group was significantly higher than that in the normal group (P<0.05)there was no significant difference in the content of TNF-α in the liver tissue of mice among the positive control group,thalidomide group and normal group(P>0.05)the content of TNF-α in the liver tissue of mice in the positive control group and thalidomide group was significantly lower than that in the model group (P<0.05)there was no significant difference in the content of TNF-α in the liver tissue of mice between the thalidomide group and the positive control group (P>0.05).The HE staining showed that the liver cells in the normal group were structure intact and arranged regularly,and the structure of liver lobules was clearlythere was a large number of vacuole-like necrosis,disordered arrangement and incomplete liver lobule structure of liver cells in the portal area of liver tissue and portal vein of mice in the model groupthere was a small amount of vacuolar necrosis around the portal area and portal vein in the liver tissue of the mice in the positive control group and thalidomide group,and the liver cells were arranged regularly.Masson staining showed that there was no obvious collagen fiber proliferation of the liver tissue of mice in the normal groupthere was a large number of collagen fibers proliferated around the portal vein and the central vein of mice in the model group,and a small amount of bridging was formedthere was a small amount of collagen fiber proliferation in the local portal area and central vein of the liver tissue in the positive control group and thalidomide group.The Masson score of the liver tissue of mice in the model group was significantly higher than that in the normal group (P<0.05)the Masson score of the liver tissue of the mice in the positive control group and thalidomide group was significantly lower than that in the model group (P<0.05)there was no significant difference in Masson score in liver tissues of mice between the positive control group and thalidomide group (P>0.05).Immunofluorescence double-labeled detection showed that there were a small amount of CD68 and CD163 fluorescent positive expression in the liver tissue of mice in the normal groupcompared with the normal group,the positive expression of CD68 in the liver tissue of mice in the model group was increased,and the positive expression of CD163 was weakenedcompared with the model group,the positive expression of CD68 in the liver tissue of mice in the thalidomide group was weaked,and the positive expression of CD163 was strongercompared with the positive control group,the positive expression of CD68 in the liver tissue of mice in the thalidomide group was similar,and the positive expression of CD163 was stronger.Conclusion　Thalidomide can effectively improve liver fibrosis of mice induced by CCl4 and significantly alleviate liver injury in model mice.Its mechanism of action may be related to influencing macrophage polarization.

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更新日期/Last Update: 2021-08-05