黄芪提取物调节TRPM2表达的抗急性药物性肝损伤作用研究

Effects of the extract of Astragalus on acute drug-induced liver injury by regulating the expression of TRPM2

目的 探讨黄芪提取物(AR)调节TRPM2表达的抗急性DILI作用机制。方法 雄性小鼠39只,随机分为对照组(n=7),对乙酰氨基酚(APAP)模型组,AR低、高剂量组及N-乙酰半胱氨酸(NAC)治疗组,每组各8只。其中AR低、高剂量组分别以50、75 mg/kg的AR灌胃,NAC组以100 mg/kg灌胃,每日1次,共3 d。第4天,除对照组外,其余各组以350 mg/kg剂量APAP灌胃,12 h后处死全部小鼠,收集血清及肝组织。检测血清ALT、AST活性;HE染色观察肝组织病理学变化;免疫组化染色观察肝组织髓过氧化物酶(MPO)、瞬时受体电位M2型离子通道(TRPM2)表达。体外实验以不同剂量APAP(0、5、10、20 mM)孵育AML12细胞12、24 h,筛选出APAP肝细胞损伤最佳浓度和作用时间,并动态检测肝细胞TRPM2基因及蛋白表达。肝细胞分为对照组,模型组,AR低、高剂量组及NAC对照组,除对照组外,其余各组以20 mM APAP诱导损伤,并分别以125、250μg/mL的AR、50 mM NAC孵育24 h。采用CCK8检测细胞活力,qRT-PCR检测肝细胞TRPM2基因表达、Western Blot检测蛋白表达。结果 与对照组比较,APAP可诱导急性DILI,血清ALT、AST活性明显升高(F=35.717、F=18.997,P值均<0.01),肝组织结构破坏严重、肝细胞大块/亚大块坏死、中央静脉淤血;与模型组相比,AR能显著降低模型小鼠血清ALT、AST活性,减轻肝细胞坏死、肝组织MPO及TRPM2表达,具有明显的保肝作用,且AR高剂量组与NAC疗效相当。5 mM APAP孵育AML12细胞24 h明显诱导肝细胞损伤,且肝细胞TRPM2表达随着时间的延长而增加(F=25.408,P<0.01);与模型组相比,AR可提高肝细胞活力、降低肝细胞TRPM2表达。结论 黄芪提取物具有良好的保护APAP诱导急性DILI作用,其机制与抑制TRPM2表达相关。

Objective To observe the mechanism of the extract of Astragalus(EA) regulating the expression of transient receptor potential melastatin 2(TRPM2) on acetaminophen(APAP)-induced acute liver injury(ALI). Methods In vivo experient: thirty-nine male mice were randomly divided into control group(n=7) and study groups(n=8 each). The study groups were divided into the following groups at random: the APAP model group, the low-dose EA group(50mg/kg), the high-dose EA group(75 mg/kg) and the N-acetylcysteine(NAC) treatment group(100mg/kg). Both of EA and NAC were administered daily through oral gavage once a day for 3 consecutive days. On the 4th day, the mice in the study groups were given 350 mg/kg APAP by gavage. After 12h of APAP treatment, samples were collected. Blood was collected to detect the serum levels of ALT and AST;Liver tissue slices were subjected to hematoxylin and eosin(HE) staining for observing the pathological changes, while immunohistochemical staining was performed to observe the expression of myeloperoxidase(MPO) and TRPM2. In vitro experiment: The AML12 mouse hepatocyte line was cultured in vitro and was stimulated by APAP(0, 5, 10, 20 mM) for 12 or 24 h. After the optimal stimulation concentration and duration of action were screened out, RT-PCR and Western blot were used to dynamically measure TRPM2 in Hepatocytes. The ALI cell model was established with 5 mM APAP and divided into control group, APAP model group, low-dose EA group(125 μg/mL), high-dose EA group(250 μg/mL) and NAC control group. Except the control group, the other groups were incubated with NAC for 24 h. The CCK-8 assay was used to measure the viability of hepatocytes. RT-PCR and Western blot were used to measure TRPM2 in hepatocytes. Results In vivo experiment, compared with the normal group, the levels of serum ALT and AST in the APAP model group significantly increased(P<0.01). The liver tissue showed severe destruction of liver tissue structure, massive/submassive necrosis of hepatocytes, and central venous congestion. Compared with the APAP model group, EA had obvious hepatoprotective effects, including lowering the levels of serum ALT and AST in model mice, alleviating the necrosis of hepatocytes and down-regulated the expression of MPO and TRPM2 in liver tissue. While the high-dose EA group had the same efficacy as NAC. In vitro experiment: 5mM APAP incubation could significantly induce hepatocyte damage(F=25.408, P<0.01), and the expression of TRPM2 in hepatocytes increased with time;compared with the APAP model group, EA could increase the viability of hepatocytes and reduce the expression of TRPM2 in hepatocytes. Conclusion EA has a good protective effect on APAP drug-induced liver injury, which may be related to down-regulation of TRPM2 expression.