丁胱亚磺酰亚胺对正常肝细胞的毒性作用及其机制探讨

Toxic effects of buthionine sulfoximine on human hepatocytes L-02 through ROS/p38 pathway

目的探讨丁胱亚磺酰亚胺(BSO)对人胚正常肝细胞株L-02的毒性及其机制。方法 (1)用0、10、25、50、100和200μmol/L的BSO孵育L-02细胞48 h,用MTT法测算细胞存活率。(2)用0、10、25、50、100、200μmol/L的BSO培养L-02细胞48 h,用DTNB法检测细胞内还原型谷胱甘肽(GSH)水平。(3)用200μmol/L的BSO培养L-02细胞0、2、12、24、36、48 h,用Western blotting法测算细胞p38磷酸化水平。(4)用200μmol/L的BSO培养L-02细胞0、2、6、12、24、48 h,用荧光探针H2DCFDA法测算细胞活性氧簇(ROS)水平。(5)L-02细胞分为对照组、BSO组、SB203580组和BSO+SB203580组。对照组不加任何药物;BSO组加入终浓度为200μmol/L的BSO;SB203580组加入终浓度为20μmol/L的SB203580;BSO+SB203580组先加入终浓度为20μmol/L的SB203580孵育15 min后加入终浓度为200μmol/L的BSO。24 h后采用MTT法测算各组细胞存活率,采用荧光探针H2DCFDA法检测ROS水平。结果 (1)从50μmol/L开始,BSO可以剂量相关性地降低L-02细胞的存活率(P均<0.05)。(2)BSO可剂量相关性地降低L-02细胞GSH含量,BSO浓度为25μmol/L时达到最低值。(3)BSO呈时间依赖性增加L-02细胞内p38磷酸化水平(P均<0.05)。(4)加入BSO后L-02细胞ROS水平均较对照组提高(P均<0.05),培养24 h时达到高峰。(5)BSO组L-02细胞ROS水平高于对照组(P<0.05),细胞存活率低于对照组(P<0.05);SB203580组L-02细胞ROS水平和细胞存活率与对照组相比P均>0.05;BSO+SB203580组L-02细胞ROS水平低于BSO组(P<0.05),细胞存活率高于BSO组(P<0.05)。结论 BSO对人正常肝细胞具有毒性作用,其机制可能是通过促进p38磷酸化和ROS生成,降低GSH水平实现的。

Objective To study the toxic effects and mechanism of buthionine sulfoximine （BSO） on human normal hepatocytes L-02 and its mechanism. Methods （1） Human normal hepatocytes L-02 were incubated with 0, 10, 25, 50, 100, and 200 μmol/L BSO for 48 h, the cell viability was measured by MTF and trypan blue staining. （2） L-O2 cells were incubated with 0, 10, 25, 50, 100, and 200 μmol/L BSO for 48 h, the content of glutathione （GSH） was detected by DTNB method. （3） L-02 cells were incubated with 200 μmol/L BSO for 0, 2, 12, 24, 36, and 48 h, Western blotting was applied to detect the phosphorylation of p38. （4） L-02 ceils were incubated with 200 μmol/L BSO for 0, 2, 12, 24, 36, and 48 h, fluorescence probe H2DCFDA was used to observe the production of ROS in L-02 cells. （5） L-02 cells were di- vided into the control group,. BSO group, SB203580 group, and BSO ＋ SB203580 group. No drugs were added to the con- trol group, BSO group was given 200 tLmol/L BSO, while the SB203580 group was added with 20 μmol/L SB203550, and the BSO ＋ SB203580 group was first incubated with 20 μmol/L SB203580 for 15 min, then was added with 200 μmol/L BSO. After 24 h, the survival rate of each group was measured by MTF method, and the level of ROS was detected by fluo- rescence probe H2DCFDA. Results （1） BSO reduced the survival rate of L-02 cells in a dose-dependent manner, and the minimum toxic dose was 50 μmol/L （P 〈 0.05 ）. （2） BSO could reduce the GSH content of L-if2 ceils in a dose-dependent manner, and it reached the minimum value since the concentration of BSO was 25μmol/L. （3） BSO increased the phosphorylation level of p38 in L-02 cells in a time-dependent manner （ P 〈 0.05 ）. （~ Compared with control group, the level of ROS increased in L-02 cells when treated with BSO, and reached its peak at 24 h. （5） The ROS level in the BSO group was higher and the cell survival rate was lower than those of the control group （ both P 〈 O. 05 ）. There was no statistically sig- nificant difference in the ROS level and cell survival rate between the contro! group and SB203580 group （ P 〉 0.05 ）. The ROS level in the BSO ＋ SB203580 group was lower and the cell survival rate was higher than those of the BSO group （ both P 〈 0.05）. Conclusion BSO has certain cytotoxicity to human normal liver cells by increasing the phosphorylation level of p38 and ROS production, and decreasing the content of GSH.