葛根素对对乙酰氨基酚诱导的急性肝损伤的保护作用研究

doi:10.16431/j.cnki.1671-7236.2022.11.037

摘要/Abstract

摘要： 【目的】研究葛根素（puerarin，PU）对对乙酰氨基酚（acetaminophen，APAP）所致小鼠急性肝损伤的保护作用，为临床上防治APAP引起的肝损伤提供新思路。【方法】将40只4周龄体重（20±2） g的SPF级雄性昆明小鼠随机分为正常组（CON）、模型组（APAP）、葛根素低剂量组（L-PU）、葛根素高剂量组（H-PU）4组，每组10只。CON组正常饲养，L-PU和H-PU组分别以50和100 mg/kg葛根素灌胃，每天1次，连续给药7 d。最后一次给药后禁食16 h，APAP、L-PU和H-PU组均以200 mg/kg对乙酰氨基酚灌胃1次。继续禁食12 h，麻醉后经眼眶采血，颈椎脱臼处死小鼠，迅速采集肝脏，观察肝脏形态、计算肝脏指数；测定血清丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）活性，评价肝损伤情况；测定肝脏过氧化氢酶（catalase，CAT）活力及总抗氧化能力（total antioxidant capacity，T-AOC），评定肝脏氧化应激情况；测定谷胱甘肽转移酶（glutathione S-transferase，GST）及细胞色素P450家族2亚家族E成员1（cytochrome P450 2E1，CYP2E1）含量，分析APAP在肝脏的代谢情况；测定凋亡相关蛋白半胱氨酸蛋白酶-3（caspase-3）和caspase-9含量，评定肝脏细胞凋亡情况；制作肝脏病理组织切片，HE染色后观察肝脏组织学变化。【结果】与CON组相比，APAP组肝脏质地坚硬，有明显出血，肝脏组织结构受到严重破坏，肝索排列紊乱，肝小叶结构不清晰，肝细胞大量坏死，肝脏指数及血清中ALT、AST水平显著上升（P<0.05），CAT活力、T-AOC及GST水平显著下降（P<0.05），caspase-3、caspase-9及CYP2E1含量显著上升（P<0.05）。与APAP组相比，L-PU和H-PU组小鼠肝脏形态结构及组织结构基本保持正常，且H-PU组效果更显著；L-PU和H-PU组小鼠肝脏指数和血清中AST、ALT水平显著降低（P<0.05），肝脏中CAT活力、T-AOC显著提高（P<0.05），caspase-9水平显著降低（P<0.05）；此外，L-PU和H-PU组在提高小鼠GST水平的同时也显著降低了CYP2E1水平（P<0.05）。【结论】葛根素对APAP所致的小鼠肝损伤具有明显的保护作用，可能与葛根素发挥抗氧化、抗凋亡、加速APAP解毒作用有关。

关键词: 葛根素; 对乙酰氨基酚; 药物性肝损伤

Abstract: 【Objective】 The aim of this experiment was to investigate the protective effect of puerarin (PU) on acetaminophen (APAP)-induced acute liver injury in mice, and provide a new idea for the clinical prevention and treatment of liver injury caused by APAP.【Method】 Forty 4-week-old SPF-grade male Kunming mice weighing (20±2) g were randomly divided into four groups:Normal (CON), model (APAP), puerarin low-dose (L-PU), and puerarin high-dose (H-PU) groups, 10 mice in each group.Mice were kept normally in CON group, and mice in L-PU and H-PU groups were gavaged with 50 and 100 mg/kg puerarin, respectively, once daily for 7 days.After the last administration, the mice in APAP, L-PU and H-PU groups were fasted for 16 h and gavaged with 200 mg/kg acetaminophen once.Continue fasting for 12 h, blood was collected from the orbit after anesthesia, and the mice were killed after cervical dislocation.The liver was quickly collected, and the liver morphology was observed and the liver index was calculated.Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured to evaluate the liver injury.The liver catalase (CAT) activity and total antioxidant capacity (T-AOC) were determined to evaluate the liver oxidative stress.The levels of glutathione S-transferase (GST) and cytochrome P450 family 2 subfamily E member 1 (cytochrome P450 2E1, CYP2E1) were measured to analyze the metabolism of APAP in liver.The apoptosis-related protein cysteine protease caspase-3 and caspase-9 levels were determine to evaluate the apoptosis of liver cells.The pathological sections of the liver were made and the histological changes of the liver were observed after HE staining.【Result】 Compared with CON group, the liver in APAP group was hard with obvious bleeding, the liver tissue structure was severely damaged, the hepatic cords were disordered, the structure of the liver lobules was not clear, and there was massive necrosis of hepatocytes, the liver index and ALT and AST levels in serum were significantly increased (P<0.05), the activity of CAT, T-AOC and GST level were significantly decreased (P<0.05), and the contents of apoptotic proteins caspase-3, caspase-9 and CYP2E1 were significantly increased (P<0.05).Compared with APAP group, the liver morphology and tissue structure of L-PU and H-PU groups were basically normal, and the effect of H-PU group was more significant.In L-PU and H-PU groups, the liver index and the activities of AST and ALT in serum were significantly decreased (P<0.05), CAT activity and T-AOC in liver were significantly increased (P<0.05), and the level of caspase-9 was significantly decreased (P<0.05).In addition, L-PU and H-PU groups increased the level of GST and significantly decreased the level of CYP2E1 (P<0.05).【Conclusion】 The significant protective effect of puerarin on APAP-induced liver injury in mice might be related to the antioxidant, anti-apoptotic, and accelerated APAP detoxification effects of puerarin.

Key words: puerarin; acetaminophen; drug induced liver injury

中图分类号:

S853.74

蒙锦燕, 徐百昌, 司红彬. 葛根素对对乙酰氨基酚诱导的急性肝损伤的保护作用研究[J]. 中国畜牧兽医, 2022, 49(11): 4457-4465.

MENG Jinyan, XU Baichang, SI Hongbin. Study on the Protective Effect of Puerarin on Acetaminophen-induced Acute Liver Injury[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4457-4465.

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