阿托伐他汀对肝纤维化的改善作用及其机制

doi:10.3969/j.issn.1674-8115.2021.12.011

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (12): 1619-1626.doi: 10.3969/j.issn.1674-8115.2021.12.011

• 论著 · 基础研究 • 上一篇

阿托伐他汀对肝纤维化的改善作用及其机制

汪楠(), 陆晔(), 郝风节(), 王俊青()

上海交通大学医学院附属瑞金医院普外科，上海 200025

收稿日期:2021-06-29 出版日期:2021-12-28 发布日期:2022-01-28
通讯作者: 郝风节,王俊青 E-mail:wangnan2020@sjtu.edu.cn;fengjie_haochn@163.com;wangjunqingmd@hotmail.com
作者简介:汪楠（1997—），男，硕士生；电子信箱：wangnan2020@sjtu.edu.cn
汪楠（1997—），男，硕士生；电子信箱：wangnan2020@sjtu.edu.cn
基金资助:
上海市教育委员会高峰高原学科建设计划(20191901);上海市浦江人才计划(18PJD029);上海交通大学医工交叉研究基金(YG2021QN21)

Ameliorative effect of atorvastatin on hepatic fibrosis and its mechanism

Nan WANG(), Ye LU(), Feng-jie HAO(), Jun-qing WANG()

Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2021-06-29 Online:2021-12-28 Published:2022-01-28
Contact: Feng-jie HAO,Jun-qing WANG E-mail:wangnan2020@sjtu.edu.cn;fengjie_haochn@163.com;wangjunqingmd@hotmail.com
Supported by:
Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support(20191901);Shanghai Pujiang Talent Project(18PJD029);Interdisciplinary Research Foundation for Medicine and Engineering from Shanghai Jiao Tong University(YG2021QN21)

摘要/Abstract

摘要：

目的·探讨阿托伐他汀在肝纤维化患者和动物模型中的作用及其机制。方法·检索PubMed上他汀类药物治疗不同慢性肝病的文献，起止时间为2010年1月—2020年6月。对肝纤维化发生率、肝功能失代偿发生率及患者总体死亡率进行meta分析。将16只雄性C57BL/6小鼠随机分为对照组、阿托伐他汀组、四氯化碳（CCl₄）注射组、CCl₄注射联合阿托伐他汀组，每组4只。CCl₄注射组小鼠以20%体积分数的CCl₄按1 mL/kg的剂量，每周行2次腹腔注射；对照组使用同剂量玉米油进行腹腔注射；阿托伐他汀组小鼠每日以15 mg/kg剂量的阿托伐他汀灌胃1次；CCl₄注射联合阿托伐他汀组联合采用上述CCl₄和阿托伐他汀的处理方式。4周后处死小鼠，检测血清谷丙转氨酶（glutamic pyruvic transaminase，GPT）、谷草转氨酶（glutamic oxaloacetic transaminase，GOT）浓度；采用天狼星红染色检测小鼠肝内纤维化程度；采用免疫荧光染色检测肝纤维化标志物ⅠA型胶原蛋白（collagen ⅠA）的表达；采用实时荧光定量PCR检测肝组织α平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、collagen Ⅰ、collagen Ⅲ，以及白介素（interleukin，IL）-1b、IL-6、转化生长因子β（transforming growth factor-β，TGF-β）的表达；采用免疫印迹法检测肝组织α-SMA蛋白和磷酸化Smad4（pSmad4）蛋白的表达。结果·最终选取9篇文献进行meta分析，结果显示他汀类药物可降低肝病患者肝纤维化、肝功能失代偿的发生率及总体死亡率。在动物实验中，4周处理后，与对照组比较，CCl₄注射组和CCl₄注射联合阿托伐他汀组小鼠体质量均显著下降（均P=0.000）；而CCl₄注射联合阿托伐他汀组小鼠体质量与CCl₄注射组比较，下降程度较轻，组间差异有统计学意义（P=0.040）。与对照组比较，CCl₄注射组和CCl₄注射联合阿托伐他汀组小鼠GPT、GOT浓度均显著上升（均P=0.000）；而CCl₄注射联合阿托伐他汀组小鼠肝酶与CCl₄注射组比较，GPT、GOT上升幅度均较小，组间差异有统计学意义（P=0.020）。天狼星红染色显示CCl₄注射组肝内纤维过度沉积，免疫荧光染色显示collagen ⅠA在CCl₄注射组中高表达。实时荧光定量PCR、免疫印迹法检测结果显示：与对照组相比，CCl₄注射组中α-SMA蛋白表达升高，α-SMA、collagen Ⅰ、collagen Ⅲ的mRNA表达升高（均P<0.05）；而CCl₄注射联合阿托伐他汀组与CCl₄注射组比较，上述指标均有所下调，差异均有统计学意义（均P<0.05）。与对照组相比，CCl₄注射组中IL-1b、IL-6的mRNA表达轻微上升，TGF-β 的mRNA表达显著升高（均P<0.05）；而CCl₄注射联合阿托伐他汀组与CCl₄注射组比较，IL-1b、IL-6的mRNA表达显著升高，TGF-β 的mRNA表达显著降低，差异均有统计学意义（均P<0.05）。与对照组相比，CCl₄注射组pSmad4蛋白表达升高；而CCl₄注射联合阿托伐他汀组与CCl₄注射组相比，pSmad4蛋白表达较低。结论·阿托伐他汀可能通过调控TGF-β通路，延缓肝纤维化的发生及发展。

关键词: 阿托伐他汀, 肝纤维化, 炎症, 转化生长因子β, 信号通路

Abstract:

Objective·To investigate the role of atorvastatin and its mechanisms in patients and animal models of hepatic fibrosis.

Methods·The literatures in PubMed were searched for statins for different chronic liver diseases from January 2010 to June 2020. Meta analysis was performed based on the occurrence of liver fibrosis, liver failure and overall patient mortality. Sixteen male C57BL/6 mice were randomly divided into control group, atorvastatin group, CCl₄ injection group, and CCl₄ injection combined with atorvastatin group with 4 mice in each group. The mice in the CCl₄ injection group were administered intraperitoneally twice a week with 20% volume fraction of CCl₄ at a dose of 1 mL/kg; the control group was administered intraperitoneally by using the same dose of corn oil; the mice in the atorvastatin group were gavaged once daily with a dose of 15 mg/kg of atorvastatin; the mice in the CCl₄ injection combined with atorvastatin group were treated with a combination of CCl₄ and atorvastatin. After 4 weeks, the animals were euthanized, and serum glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) were detected; the degree of intrahepatic fibrosis in the mice was detected by Sirius red staining; immunofluorescence staining was used to detect the expression of collagen ⅠA; the mRNA expressions of α-smooth muscle actin (α-SMA), collagen Ⅰ, collagen Ⅲ, interleukin (IL)-1b, IL-6, and transforming growth factor-β (TGF-β) were detected by real-time fluorescence quantitative PCR; the expressions of α-SMA protein and phosphorylated Smad4 (pSmad4) protein were detected by Western blotting.

Results·Finally 9 papers were selected for meta analysis, and the results showed that statins reduced the incidence of liver fibrosis, liver failure and overall mortality in patients with liver disease. In the animal experiments, the weight of mice in both the CCl₄ injection group and the CCl₄ injection combined with atorvastatin group decreased significantly compared with that in the control group (P=0.000); while the weight of mice in the CCl₄ injection combined with atorvastatin group decreased to a lesser extent compared with that in the CCl₄ injection group (P=0.040). Compared with the control group, GPT and GOT concentrations were significantly increased in both the CCl₄ injection group and CCl₄ injection combined with atorvastatin group (P=0.000), while liver enzymes in the CCl₄ injection combined with atorvastatin group showed a smaller increase in GPT and GOT compared with that in the CCl₄ injection group (P=0.020). Sirius red staining showed excessive deposition of intrahepatic fibers in the CCl₄injection group, and immunostaining suggested high expression of collagen ⅠA in the CCl₄injection group. The results of fluorescence quantitative PCR and Western blotting suggested that α-SMA protein expression increased, and mRNA expressions of α-SMA, collagen Ⅰ, and collagen Ⅲ were upregulated in the CCl₄ injection group compared with the control group(P<0.05), whereas the expressions of these markers were downregulated in the CCl₄ injection combined with atorvastatin group compared with the CCl₄ injection group (P<0.05). The mRNA expressions of IL-1b and IL-6 were slightly upregulated and the expression of TGF-β was significantly upregulated in the CCl₄ injection group compared with the control group (P<0.05). In contrast, the mRNA expressions of IL-1b and IL-6 were significantly up-regulated and the expression of TGF-β decreased in the CCl₄ injection combined with atorvastatin group compared with the CCl₄ injection group (P<0.05). The expression of pSmad4 protein increased in the CCl₄ injection group compared with the control group, while the expression of pSmad4 protein was lower in the CCl₄ injection combined with atorvastatin group compared with the CCl₄ injection group.

Conclusion·Atorvastatin may attenuate the occurrence and development of liver fibrosis via manipulating TGF-β signaling pathway.

Key words: atorvastatin, liver fibrosis, inflammation, transforming growth factor-β, signaling pathway

中图分类号:

R575.2

汪楠, 陆晔, 郝风节, 王俊青. 阿托伐他汀对肝纤维化的改善作用及其机制[J]. 上海交通大学学报(医学版), 2021, 41(12): 1619-1626.

Nan WANG, Ye LU, Feng-jie HAO, Jun-qing WANG. Ameliorative effect of atorvastatin on hepatic fibrosis and its mechanism[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(12): 1619-1626.

图/表 8

图1 文献筛选流程

Fig 1 Flow chart of literature inclusion

表1 纳入文献的相关数据

Tab 1 Associated data of the selected literatures

No.	Author	Year	Location	Type	Time period	Patients numbers	Etiology	Outcomes of interest	Stain users	Nonusers
1	Chang	2017	Chinese Taipei	Propensity matched cohort	2000‒2013	1 350	HBV， HCV， ALD	Decompensated cirrhosis； Mortality	14%； 9%	28%； 18%
2	Huang	2016	Chinese Taipei	Propensity matched cohort	1997‒2009	13 086	HBV	Decompensated cirrhosis； Developing cirrhosis	2.6%； 0.9%	6.1%； 1.9%
3	Simon	2016	USA	Retrospective cohort	2001‒2014	9 135	HCV	Developing cirrhosis	14.0%	21.4%
4	Hsiang	2015	Chinese Hongkong	Propensity matched cohort	2000‒2012	53 513	HBV	Mortality	6.5%	14.3%
5	Mohanty	2016	USA	Propensity matched cohort	1996‒2009	2 747	HCV	Decompensated cirrhosis； Mortality	2.1%； 6.3%	3.9%； 11.1%
6	Simon	2015	USA	Retrospective cohort	2000‒2004	543	HCV	Developing cirrhosis	10%	28%
7	Yang	2015	Chinese Taipei	Propensity matched cohort	1999‒2010	84 213	HCV	Developing cirrhosis	6.0%	21.9%
8	Kumar	2014	USA	Retrospective cohort	1988‒2011	243	Mixed	Decompensated cirrhosis	30.0%	40.5%
9	Abraldes	2016	Spain	RCT	2010‒2013	147	Mixed	Mortality	9%	22%

图2 他汀类药物对于肝纤维化发生风险的影响

Fig 2 Effect of statins on the risk of hepatic fibrosis

图3 他汀类药物对于肝功能失代偿风险的影响

Fig 3 Effect of statins on the risk of liver decompensation

图4 他汀类药物对于总体死亡风险的影响

Fig 4 Effect of statins on the overall risk of death

图5 阿托伐他汀对CCl4注射导致的小鼠肝脏损伤及体质量的影响Note：A.Weight changes of mice. B. GPT levels after 4 weeks of treatment. C. GOT levels after 4 weeks of treatment.

Fig 5 Effect of atorvastatin on liver injury and weight of mice caused by CCl4 injection

图6 阿托伐他汀对CCl4注射导致的小鼠肝纤维化的缓解作用Note：A. Hepatic tissue stained with Sirius red. B. Immunofluorescence staining of collagen ⅠA. C. Protein expression of α-SMA. D. mRNA expression of α-SMA. E. mRNA expression of collagen Ⅰ. F. mRNA expression of collagen Ⅲ.

Fig 6 Mitogenic effect of atorvastatin on hepatic fibrosis in the mice after CCl4 injection

图7 阿托伐他汀对肝内炎症因子表达及TGF-β通路的影响Note：A. mRNA expression of IL-1b. B. mRNA expression of IL-6. C. mRNA expression of TGF-β. D. Protein expression of pSmad4.

Fig 7 Effect of atorvastatin on expressions of intrahepatic inflammatory factors and TGF-β pathway

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阿托伐他汀对肝纤维化的改善作用及其机制

Ameliorative effect of atorvastatin on hepatic fibrosis and its mechanism

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