摘要
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15 000 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.
Abstract
Objective To investigate the oxidative stress mechanism of liver function abnormality in rats caused by Gynu-ra japonica exposure,so as to provide a theoretical basis for clinical treatment and prevention.Methods(i)For the ani-mal 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 15 000 mg/kg,with a gavage volume of 10 mL/(kg·bw)for 1 time/d.The control group was gavaged with puri-fied 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 coagula-tion indexes,including APTT and PT,were also detected.The livers were extracted and observed using HE staining to de-tect 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 glu-tathione 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 japon-ica group.Cells were incubated in DMEM,40 mg/mL Gynura japonica,20 mg/mL Gynura japonica,10 mg/mL Gynura ja-ponica and 5 mg/mL Gynura japonica for 2 hours.The level of intracellular reactive oxygen species in hepatocytes was de-tected 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 expres-sion(t=9.412,P=0.000),and decreased GPX activity(t=10.294,P=0.000).(ii)The in vitro experiments demon-strated that the intrahepatocyte and mitochondrial levels of reactive oxygen species were significantly elevated in the Gynu-ra 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.
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