异甘草酸镁对丙基硫氧嘧啶诱导人HepG2细胞损伤的保护研究

发布时间：2019-02-17 19:28

【摘要】：目的在细胞水平研究丙基硫氧嘧啶(PTU)对T4干预下人Hep G2细胞(模拟在甲亢环境下)的损伤机制,并探讨异甘草酸镁(Mg IG)对丙基硫氧嘧啶诱导的人Hep G2细胞损伤的保护作用。方法1、体外培养Hep G2细胞于100n M浓度的T4干预环境中,在含有PTU 0、75、150、300、600、1200、2400μg/ml一系列浓度培养基分别孵育不同的时间,利用过氧化氢(H2O2)体外诱导肝细胞损伤作为阳性对照,采用MTT比色法及LDH法检测细胞增殖及细胞毒性情况,并测定谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性,得到最佳PTU诱导Hep G2细胞损伤浓度,建立PTU诱导的体外甲亢肝细胞损伤模型。2、将Hep G2细胞分成对照组、阳性对照组、PTU损伤组、Mg IG保护组,Hep G2细胞贴壁后,阳性对照组用H2O2诱导损伤,PTU损伤组和保护组则以PTU最佳损伤浓度预处理,8h后弃原培养液,Mg IG保护组加入5.0mg/ml Mg IG培养液中孵育24小时,其他组别补充等体积培养液。采用生化分析仪检测天冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)活性,试剂盒测定细胞胞浆中超氧化物歧化酶(SOD)的活性和谷胱甘肽(GSH)、丙二醛(MDA)的含量,探讨异甘草酸镁对PTU致肝损伤的保护作用。3、以PTU损伤模型作为研究对象,利用免疫印记法(Western blot)检测细胞中凋亡相关蛋白Bax蛋白和Bcl-2蛋白的表达,c-Jun氨基末端激酶(JNK)、P38丝裂原活化蛋白激酶(P38)及其磷酸化形式蛋白的表达。结果1、PTU对Hep G2细胞的损伤作用随剂量的增加和时间的增长而增强,在PTU浓度为600μg/ml,作用时间8h时对Hep G2细胞的抑制率为55.35±0.45%,死亡率为49.16±0.49%,培养液上清中ALT和AST水平较正常组明显升高(P0.05)。2、异甘草酸镁预处理后,与损伤组相比,异甘草酸镁能显著提高细胞生存率;降低Hep G-2细胞培养液上清中ALT和AST水平(P0.05),;降低细胞内MDA水平(P0.05),同时增高细胞内SOD的活性和GSH的含量(P0.05)。3、Western blot结果显示,PTU能升高Hep G-2细胞JNK、P38及其磷酸化蛋白的表达,与损伤组相比,异甘草酸镁保护组中的Bax蛋白表达降低,Bcl-2蛋白表达显著升高(P0.05)。结论1.建立PTU致甲亢Hep G2细胞损伤模型的最佳干预浓度和时间分别为600μg/ml,8h;2.PTU致肝损伤的机制可能与JNK、p38MAPK通路的激活有关;3.异甘草酸镁对PTU致Hep G2细胞损伤具有明显的保护作用。
[Abstract]:Objective to study the damage mechanism of propylthiouracil (PTU) on human Hep G2 cells (simulated in hyperthyroidism) induced by T4 at the cellular level. To investigate the protective effect of magnesium isoglycyrrhizinate (Mg IG) on human Hep G2 cells induced by propyl thiouracil. Methods 1. Hep G2 cells cultured in vitro were incubated with T4 at 100nM concentration for different time on a series of culture medium containing PTU 075, 150, 300, 200 and 2 400 渭 g/ml. The hepatocyte injury induced by hydrogen peroxide (H2O2) in vitro was used as positive control. The cell proliferation and cytotoxicity were detected by MTT colorimetric assay and LDH assay, and the activities of (ALT) and (AST) were determined. The optimal concentration of PTU induced Hep G2 injury was obtained. PTU induced hyperthyroidism hepatocyte injury model was established in vitro. 2. Hep G2 cells were divided into three groups: control group, positive control group, Mg IG protection group, Hep G2 cell adhesion group, H2O2 induced injury in positive control group. The PTU injury group and the protection group were pretreated with the optimal concentration of PTU. After 8 hours, the, Mg IG group was incubated in the 5.0mg/ml Mg IG medium for 24 hours, and the other groups were supplemented with the same volume culture medium. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were detected by biochemical analyzer. The activities of superoxide dismutase (SOD) and glutathione (GSH), (GSH),) in the cytoplasm of cells were measured by kit. The content of malondialdehyde (MDA) and the protective effect of magnesium isoglycyrrhizinate on liver injury induced by PTU. 3. PTU damage model was used as the research object. The expression of apoptosis-related protein (Bax) and Bcl-2 protein and the expression of (JNK), P38 mitogen-activated protein kinase (P38) and its phosphorylated form protein (P38) were detected by (Western blot). Results 1the damage of Hep G2 cells was increased with the increase of dose and time. The inhibition rate of Hep G2 cells was 55.35 卤0.45 and the mortality was 49.16 卤0.49 when the concentration of PTU was 600 渭 g / ml and the exposure time was 8 h. The levels of ALT and AST in the supernatant of culture medium were significantly higher than those in the normal group (P0.05). After pretreatment with magnesium isoglycyrrhizinate, magnesium isoglycyrrhizinate could significantly improve the cell survival rate compared with the injured group. Decrease the levels of ALT and AST in the supernatant of Hep G-2 cells (P0.05),;) The results of Western blot showed that PTU could increase the expression of JNK,P38 and its phosphorylated protein in Hep G-2 cells. The expression of Bax protein decreased and the expression of Bcl-2 protein increased significantly in magnesium isoglycyrrhizinate group (P0.05). Conclusion 1. The optimal intervention concentration and time of PTU induced hyperthyroidism Hep G2 cell injury model were 600 渭 g / ml ~ (8) h ~ (-1) respectively. 2. The mechanism of PTU-induced liver injury may be related to the activation of JNK,p38MAPK pathway. 3. Magnesium isoglycyrrhizinate has obvious protective effect on Hep G2 cell injury induced by PTU.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R581.1

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