兰州大学机构库 >公共卫生学院
枸杞多糖对酒精性肝损伤的保护作用及机制研究
Alternative TitleProtective effect and mechanism of Lycium barbarum polysaccharides on alcoholic liver injury
张铃浩
Thesis Advisor刘晔玮
2019-05-25
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Degree Discipline公共卫生
Keyword枸杞多糖 氧化应激 酒精性肝损伤 L-02 细胞凋亡 Nrf2/HO-1
Abstract目的:本文通过动物实验和细胞实验研究枸杞多糖(LBP)对酒精性肝损伤的预防和修复作用及其抗氧化机制,为LBP的高值化利用提供实验及理论依据。方法:1. LBP对小鼠酒精性肝损伤保护作用的研究。
将小鼠随机分为8组,分别为对照组、模型组、LBP低剂量组、LBP中剂量组、LBP高剂量组及阳性对照低剂量组、阳性对照中剂量组和阳性对照高剂量组,建立LBP保护酒精性肝损伤动物模型,检测小鼠肝脏指标及血清转氨酶含量。
2. LBP对酒精性肝细胞损伤预防及修复作用的机制研究。
(1)培养L-02细胞,采用MTT法筛选乙醇和LBP的作用浓度及时间,建立酒精性肝细胞损伤模型。将肝细胞随机分为6组,分别为正常对照组、模型组、阳性对照+乙醇预防组、LBP+乙醇预防组、乙醇+阳性对照修复组、乙醇+LBP修复组。
(2)用流式细胞法检测LBP对细胞凋亡的影响,激光共聚焦显微镜及荧光分光光度法检测细胞内ROS水平;化学法检测细胞中AST、 ALT和GSH含量、MDA水平,SOD活性和GSH-Px活性,抗O2-能力及NO含量。
(3)Western blot法检测细胞中胞浆胞核Nrf2的表达及总蛋白中HO-1、NQO1、GCLC的表达。结果:1. 阳性对照中剂量组及高剂量组、LBP低剂量组及中剂量组均可显著降低小鼠酒精性肝损伤的肝脏指数,LBP高剂量组及阳性对照低剂量组小鼠肝脏指数与模型组比较无统计学意义(P>0.05)。LBP各剂量组均可显著降低酒精性肝损伤小鼠血清ALT含量,其中LBP低剂量组及中剂量组血清AST含量也降低;与模型组相比,阳性对照各剂量组均可显著降低小鼠血清中ALT含量,其中阳性对照低剂量组和中剂量组血清AST含量也降低。 2.(1)建立酒精损伤L-02细胞模型并优选出LBP对酒精性肝细胞损伤预防和修复浓度及时间。最终以终浓度为5%(V/V)的无水乙醇损伤4h建立酒精损伤肝细胞模型,质量浓度为24μg/mL的LBP干预24h为干预条件。(2) LBP的预防和修复作用可以减少酒精损伤L-02细胞后的细胞凋亡率。与正常对照组对比,模型组中细胞早期+晚期凋亡率显著增加(P<0.05);与模型组相比,LBP预防及修复组中细胞凋亡率降低(P<0.05)。与正常对照组比较,模型组细胞中SOD、GSH-Px活性及抗O2-能力降低且GSH生成量明显减少(P<0.05),细胞中MDA水平及NO含量升高(P<0.05);与模型组比较,LBP预防组及修复组中细胞的GSH-Px活性升高且GSH生成量增加,MDA及NO含量均降低(P<0.05),其中修复组中细胞的SOD活性及抗O2-能力增强。预防组中细胞SOD活性及抗O2-能力与模型组相比无统计学意义(P>0.05)。(3)与正常对照组相比,模型组肝细胞胞核中Nrf2蛋白表达水平及其下游的抗氧化蛋白HO-1、NQO1和GCLC的表达量均显著降低(P<0.05),但LBP干预可增加Nrf2蛋白的入核表达,明显上调胞核中Nrf2蛋白及其下游蛋白HO-1、NQO1和GCLC的表达(P<0.05)。结论:LBP对小鼠酒精性肝损伤具有一定的保护作用。其机制与调控Nrf2/HO-1信号通路、清除活性氧自由基和活性氮的水平以及抗凋亡有关。
Other AbstractObjective:The purpose of this study is to study the prevention and repair effect of Lycium barbarum polysaccharide (LBP) on alcoholic liver injury and its antioxidant mechanism through in vivo experiment and in vitro experiment, so as to provide experimental and theoretical basis for the high-value utilization of LBP.Methods:1. Study on the protective effect of LBP on alcoholic liver injury in mice.
Mice were randomly divided into eight groups: control group, model group, LBP low-dose group, LBP middle-dose group, LBP high-dose group and positive control low-dose group, positive control middle-dose group and positive control high-dose group. An animal model of alcoholic liver injury protected by LBP was established, and liver indexes and serum transaminase content of mice were detected.
2. Mechanism of LBP on prevention and repair of alcoholic hepatocyte injury.
(1) L-02 cells were cultured and MTT method was used to screen the concentration and time of ethanol and LBP to establish alcoholic liver cell injury model. Hepatocytes were randomly divided into six groups: normal control group, model group, positive control group+ethanol prevention group, LBP+ethanol prevention group, ethanol+positive control repair group, ethanol+LBP repair group.
(2) The effect of LBP on apoptosis was detected by flow cytometry, and the intracellular ROS level was detected by laser confocal microscopy and fluorescence spectrophotometry. The content of ALT and AST, the level of GSH and MDA, the activity of SOD and GSH-Px, the ability of anti-O2-and the content of NO were determined by chemical method.
(3) Western blot was used to detect the expression of cytoplasm and nucleus Nrf2 in cells and the expression of HO-1, NQO1 and GCLC in total proteins.Results:1. The positive control middle-dose group and high-dose group, LBP low-dose group and middle-dose group can significantly reduce the liver index of alcoholic liver injury in mice. The liver index of high-dose group was not statistically different from the model group. LBP doses significantly reduced serum ALT levels in mice with alcohol-induced liver injury. The serum AST levels in the low-dose and middle-dose LBP groups also decreased. Compared with the model group, the positive control doses significantly reduced the ALT content in mice. The positive control low-dose group and the middle-dose group also decreased serum AST content. 2. (1) The model of ethanol-induced hepatocyte injury. Finally, an ethanol-induced hepatocyte model was established after 4 hours of absolute ethanol concentration of 5% (V/V). Intervention with LBP at a concentration of 24 μg/ml for 24 h. (2) The prevention and repair effect of LBP can reduce the apoptosis rate of L-02 cells damaged by alcohol. Compared with the normal control group, the early+late apoptosis rate in the model group was significantly increased (P<0.05). Compared with the model group, the apoptosis rate of the LBP prevention and repair group were decreased (P<0.05). Compared with the normal control group, the activity of SOD and GSH-Px and content of GSH in the model group is significantly reduced (P<0.05), MDA and NO content were increased (P<0.05), and the ability of anti-O2-was weakened in cells. Compared with the model group, GSH-Px activity and GSH production were increased in the LBP prevention group and the repair group, and the amount of MDA and NO decreased (P<0.05), increased the level of superoxide anion radical in the repair group. The SOD activity and the ability of anti-O2-of the prevention group was no statistical difference compared with the model group in cells. (3) Compared with the normal control group, the expression level of Nrf2 protein in the nucleus of hepatocytes and the protein expression levels of downstream antioxidant genes HO-1, NQO1 and GCLC were significantly lower in the ethanol-induced model group (P<0.05). However, LBP intervention can increase the nuclear expression of Nrf2 protein and significantly up-regulate the expression levels of Nrf2 protein and its downstream genes HO-1, NQO1 and GCLC in the cell nucleus (P<0.05).Conclusion:LBP has a protective effect on alcoholic liver injury in mice. Its mechanism is related to the regulation of Nrf2/HO-1 signaling pathway, scavenging of reactive oxygen species and reactive nitrogen levels, and anti-apoptosis.
Pages59
URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/339366
Collection公共卫生学院
Affiliation公共卫生学院
First Author AffilicationSchool of Public health
Recommended Citation
GB/T 7714
张铃浩. 枸杞多糖对酒精性肝损伤的保护作用及机制研究[D]. 兰州. 兰州大学,2019.
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