Aristolochic acid-induced liver injury in rats and its effects on autophagy factors

CHEN Jialiang; XU Jiaruo; ZHOU Yuehua; YAO Guangtao

doi:10.16306/j.1008-861x.2021.05.008

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Aristolochic acid-induced liver injury in rats and its effects on autophagy factors

Experiment Research | 更新时间：2022-09-09

- Aristolochic acid-induced liver injury in rats and its effects on autophagy factors
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 35, Issue 5, Pages: 44-51(2021)
- 作者机构：
  
  1.上海中医药大学（上海　201203）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2021.05.008
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CHEN Jialiang, XU Jiaruo, ZHOU Yuehua, et al. Aristolochic acid-induced liver injury in rats and its effects on autophagy factors. [J]. Academic Journal of Shanghai University of Traditional Chinese Medicine 35(5):44-51(2021)
DOI：

CHEN Jialiang, XU Jiaruo, ZHOU Yuehua, et al. Aristolochic acid-induced liver injury in rats and its effects on autophagy factors. [J]. Academic Journal of Shanghai University of Traditional Chinese Medicine 35(5):44-51(2021) DOI： 10.16306/j.1008-861x.2021.05.008.

摘要

目的：,2,观察马兜铃酸Ⅰ（AAⅠ）致大鼠肝脏损伤的情况，并探讨其对自噬相关因素的影响。,方法：,2,雄性Wistar大鼠随机分为正常组、自噬抑制剂组、AAⅠ低剂量（20 mg/kg）组、AAⅠ高剂量（40 mg/kg）组、自噬抑制剂+AAⅠ低剂量组、自噬抑制剂+AAⅠ高剂量组，每组5只。自噬抑制剂干预组大鼠预先腹腔注射3-甲基腺嘌呤（3-MA，20 mg/kg）；抑制剂干预1 h后，AAⅠ干预组腹腔注射给予相应剂量的AAⅠ。药物干预连续9 d。末次给药后，取血、分离肝脏。生化分化仪检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平，并计算比值；光镜和透射电镜下观察肝组织形态学变化。PCR检测肝组织,Beclin 1,、,LC3,、,p53,的mRNA表达水平；免疫组化检测Beclin 1、LC3、p53的蛋白表达水平。,结果：,2,①与正常组相比，AAⅠ低剂量组大鼠血清AST水平显著升高（,P,<,0.05），AAⅠ高、低剂量组AST/ALT比值明显升高（,P,<,0.05，,P,<,0.01）；与自噬抑制剂组相比，自噬抑制剂+AAⅠ高剂量组与自噬抑制剂+AAⅠ低剂量组AST/ALT比值均明显升高（,P,<,0.01）。②HE染色观察显示，AAⅠ高、低剂量组可见肝细胞肿胀，且AAⅠ高剂量组的损伤程度更为显著；自噬抑制剂+AAⅠ高剂量组出现肝细胞出血性坏死，损伤程度较AAⅠ高剂量组明显加重。透射电镜观察显示，AAⅠ高、低剂量组线粒体肿胀，AAⅠ高剂量组亦出现脂肪变性；自噬抑制剂+AAⅠ低剂量组线粒体肿胀；自噬抑制剂+AAⅠ高剂量组可见内质网肿胀，隐见自噬小体。③与正常组相比，AAⅠ高、低剂量组,p53, mRNA表达量显著上调（,P,<,0.05）；与自噬抑制剂组相比，自噬抑制剂+AAⅠ低剂量组,LC3, mRNA表达量明显下调（,P,<,0.05），自噬抑制剂+AAⅠ高剂量组,Beclin 1,和,LC3,的mRNA表达量显著降低（,P,<,0.01）；自噬抑制剂+AAⅠ高剂量组,Beclin 1,、,LC3,、,p53,的mRNA表达量明显低于自噬抑制剂+AAⅠ低剂量组（,P,<,0.05，,P,<,0.01）。④与正常组相比，AAⅠ高、低剂量组Beclin 1、LC3、p53蛋白表达均明显上调（,P,<,0.01），且AAⅠ高剂量组3种蛋白表达明显高于AAⅠ低剂量组（,P,<,0.05，,P,<,0.01）；与自噬抑制剂组相比，自噬抑制剂与不同剂量AAⅠ协同作用后3种蛋白表达均明显上调（,P,<,0.01）；与自噬抑制剂+AAⅠ低剂量组相比，自噬抑制剂+AAⅠ高剂量组Beclin 1、LC3蛋白表达明显降低（,P,<,0.05，,P,<,0.01），p53蛋白表达明显升高（,P,<,0.01）。,结论：,2,AAⅠ能够诱导大鼠肝脏损伤，且自噬因素可促进AAⅠ肝损伤，其作用机制可能与调节p53蛋白表达、诱导自噬相关。

Abstract

Objective:,2,To observe the aristolochic acid Ⅰ(AA Ⅰ) -induced liver injury in rats and investigate its influence on autophagy-related factors.,Methods:,2,Male Wistar rats were randomly divided into the normal group, autophagy inhibitor group, AA Ⅰ low-dose(20 mg/kg) group, AA Ⅰ high-dose(40 mg/kg) group, autophagy inhibitor + AA Ⅰ low-dose group and autophagy inhibitor + AA Ⅰ high-dose group, five rats in each group.Rats in the autophagy inhibitor treatment groups were intraperitoneally injected with 3-methyladenine(3-MA, 20 mg/kg) in advance.One hour after the inhibitor intervention, the AA Ⅰ treatment groups were intraperitoneally injected with AA Ⅰ at the corresponding dose.Drug intervention lasted for 9 days.After the last administration, blood was taken and liver was isolated.The serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected by biochemical analyzer, and the ratio was calculated.The morphological changes of liver tissue were observed under light and transmission electron microscope.The mRNA expression levels of ,Beclin 1,LC3, and ,p53, were detected by PCR.The protein expression levels of Beclin 1, LC3 and p53 were detected by immunohistochemistry.,Results:,2,①Compared with the normal group, the serum AST level in the AA Ⅰ low-dose group was increased significantly(,P,<,0.05), and the AST/ALT ratio was obviously increased in the AA Ⅰ low-dose and high-dose groups(,P,<,0.05,P,<,0.01) .Compared with the autophagy inhibitor group, the AST/ALT ratio was increased significantly in the autophagy inhibitor + AAⅠ high-dose group and autophagy inhibitor + AA Ⅰ low-dose group(,P,<,0.01) .② After HE staining, hepatocyte swelling was observed in the AA Ⅰ low-dose and high-dose groups, and the degree of injury was more significant in the AA Ⅰ high-dose group. Hepatocyte hemorrhagic necrosis was found in the autophagy inhibitor + AA Ⅰ high-dose group, and the degree of injury was significantly worse than that in the AA Ⅰ high-dose group. Transmission electron microscopy showed the mitochondria swelling in the AA Ⅰ low-dose and high-dose groups, and the steatosis was also observed in the AA Ⅰ high-dose group.Mitochondrion swelling was found in the autophagy inhibitor + AA Ⅰ low-dose group, as well as the endoplasmic reticulum swelling and implicit autophagy bodies in the autophagy inhibitor + AA Ⅰ high-dose group.③Compared with the normal group, the expression of ,p53, mRNA was significantly up-regulated in the AA Ⅰ low-dose and high-dose groups(,P,<,0.05) .Compared with the autophagy inhibitor group, the expression of ,LC3, mRNA was significantly decreased in the autophagy inhibitor + AA Ⅰ low-dose group(,P,<,0.05), and the expressions of ,Beclin 1, and ,LC3, mRNA were significantly decreased in the autophagy inhibitor + AA Ⅰ high-dose group(,P,<,0.01) .The mRNA expressions of ,Beclin 1,LC3, and ,p53, in the autophagy inhibitor + AA Ⅰ high-dose group were significantly lower than those in the autophagy inhibitor + AA Ⅰ low-dose group(,P,<,0.05,P,<,0.01) .④Compared with the normal group, the protein expressions of Beclin 1, LC3 and p53 were significantly up-regulated in the AA Ⅰ low-dose and high-dose groups(,P,<,0.01), and the expressions of these three proteins in the AA Ⅰ high-dose group were significantly higher than those in the AA Ⅰ low-dose group(,P,<,0.05,P,<,0.01) .Compared with the autophagy inhibitor group, after the co-treatment of autophagy inhibitor and AA Ⅰ with different doses, the expressions of these three proteins were significantly up-regulated(,P,<,0.01) .Compared with the autophagy inhibitor + AA Ⅰ low-dose group, the expressions of Beclin 1 and LC3 proteins were decreased significantly(,P,<,0.05,P,<,0.01) and the expression of p53 protein was increased significantly(,P,<,0.01) in the autophagy inhibitor + AA Ⅰ high-dose group.,Conclusion:,2,AA Ⅰ can induce liver injury in rats, and autophagy factors promote AA Ⅰ liver injury.Its mechanism may be related to regulating p53 protein expression and inducing autophagy.

关键词

马兜铃酸Ⅰ肝损伤自噬大鼠

Keywords

aristolochic acid Ⅰliver injuryautophagyrat

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