菊三七致大鼠肝功能异常的氧化应激机制

Mechanism of oxidative stress induced by Gynura japonica to abnormal liver function in rats

目的探讨菊三七暴露致大鼠肝功能异常的氧化应激机制,为临床治疗及预防提供一定的理论基础。方法(1)进行动物实验,将24只雌性SD大鼠随机分为处理组和对照组,每组各12只。处理组采用经口灌胃菊三七水煎剂,剂量为15000 mg/kg,灌胃容量为10 mL/(kg·bw),1次/d;对照组纯化水灌胃,正常喂养。两组分别于第7、14 d各处死6只,腹主动脉取血,检测血生化指标包括谷草转氨酶(AST)、谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、白蛋白(ALB),检测凝血指标包括活化部分凝血活酶时间(APTT)、凝血酶原时间(PT);摘取肝脏,采用HE染色观察肝组织病理表现;用组织匀浆器处理肝脏组织,取上清液,使用Sunrise酶标仪检测丙二醛(MDA)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPX)含量。(2)进行体外实验,培养大鼠肝细胞,设空白对照组、菊三七40 mg/mL组、菊三七20 mg/mL组、菊三七10 mg/mL组和菊三七5 mg/mL组,分别在基础培养基、40 mg/mL菊三七、20 mg/mL菊三七、10 mg/mL菊三七和5 mg/mL菊三七的DMEM中孵育2 h。采用流式细胞仪检测肝细胞内活性氧水平,Mitochondria SOX探针和流式细胞仪检测肝细胞线粒体内活性氧水平。结果(1)动物实验结果示,灌胃14 d时,与对照组比较,处理组14 d时血清ALT(t=19.707,P=0.000)、AST(t=25.498,P=0.000)和ALP(t=21.452,P=0.000)升高,血清ALB(t=5.971,P=0.000)降低,血浆PT(t=4.778,P=0.004)、APTT(t=4.884,P=0.004)延长。病理结果显示处理组肝脏出现明显的炎症浸润并伴有坏死,且损伤随着时间的增长而加重。灌胃14 d时,与对照组比较,处理组14 d时肝组织匀浆中MDA含量升高(t=12.155,P=0.000)、SOD表达降低(t=17.281,P=0.000)、CAT表达降低(t=9.412,P=0.000)、GPX活力降低(t=10.294,P=0.000)。(2)体外实验结果示,菊三七10 mg/mL组、菊三七20 mg/mL组和菊三七40 mg/mL组肝细胞内及线粒体内活性氧水平均高于空白对照组(F值分别为280.841、45.780,P=0.000)。结论菊三七暴露可致大鼠肝功能损伤,其机制可能与体内氧化应激水平改变有关。

Objective To investigate the oxidative stress mechanism of liver function abnormality in rats caused by Gynura japonica exposure,so as to provide a theoretical basis for clinical treatment and prevention.Methods(i)For the animal experiments,24 female SD rats were randomly divided into 2 groups:a treatment group and a control group,each group had 12 rats.In the treatment group,Gynura japonica decoction was administered by intragastric administration at a dose of 15000 mg/kg,with a gavage volume of 10 mL/(kg·bw)for 1 time/d.The control group was gavaged with purified water and fed normally.The 2 groups were executed on the 7th and 14th days,respectively.Blood was collected from the abdominal aorta and the blood biochemical indexes,including AST,ALT,ALP and ALB,were detected.The coagulation indexes,including APTT and PT,were also detected.The livers were extracted and observed using HE staining to detect pathological manifestations of the liver tissue.Finally,a tissue homogenizer was used to observe the liver tissue.The liver tissue was processed with a tissue homogeniser,and the supernatant was extracted and tested for malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GPX)using a sunrise enzyme marker.(ii)For the in vitro experiments,the rat hepatocytes were cultured with the following groups:blank control group,40 mg/mL Gynura japonica group,20 mg/mL Gynura japonica group,10 mg/mL Gynura japonica group,and 5 mg/mL Gynura japonica group.Cells were incubated in DMEM,40 mg/mL Gynura japonica,20 mg/mL Gynura japonica,10 mg/mL Gynura japonica and 5 mg/mL Gynura japonica for 2 hours.The level of intracellular reactive oxygen species in hepatocytes was detected by flow cytometry,as well as the level of intracellular reactive oxygen species in hepatocyte mitochondria,using the Mitochondria SOX probe and flow cytometry.Results(i)The results of the animal experiments:at 14 d of gavage,compared with the control group,serum ALT(t=19.707,P=0.000),AST(t=25.498,P=0.000)and ALP(t=21.452,P=0.000)were elevated in the treated group,while serum ALB(t=5.971,P=0.000)was decreased.Additionally,plasma PT(t=4.778,P=0.004)and APTT(t=4.884,P=0.004)were prolonged.The pathological results clearly showed significant inflammatory infiltration with necrosis in the liver of the treated group,and the injury worsened with time.At 14 d of gavage,the liver tissue homogenate of the treatment group showed increased MDA content(t=12.155,P=0.000)and decreased SOD expression(t=17.281,P=0.000),decreased CAT expression(t=9.412,P=0.000),and decreased GPX activity(t=10.294,P=0.000).(ii)The in vitro experiments demonstrated that the intrahepatocyte and mitochondrial levels of reactive oxygen species were significantly elevated in the Gynura japonica 10 mg/mL and 20 mg/mL and 40 mg/mL groups(F-values of 280.841 and 45.780,respectively,with P=0.000 in both cases).Conclusion Gynura japonica exposure causes liver function impairment in rats,and the mechanism may be related to the altered level of oxidative stress in vivo.