对氨基水杨酸钠对锰染毒大鼠丘脑原代小胶质细胞氧化损伤及炎症反应的影响

彭东杰; 罗旖旎; 秦文霞; 梁典胤; 谢秉言; 许放; 李少军; 姜岳明

doi:10.13213/j.cnki.jeom.2018.17594

对氨基水杨酸钠对锰染毒大鼠丘脑原代小胶质细胞氧化损伤及炎症反应的影响

Effects of sodium p-aminosalicylic acid on oxidative damage and inflammatory response of thalamic primary microglia in rats exposed to manganese

摘要

摘要:
目的探讨对氨基水杨酸钠（PAS-Na）对体外染锰大鼠丘脑原代小胶质细胞氧化损伤、炎症反应的影响。

方法将提纯、鉴定后的SD大鼠丘脑小胶质细胞分别给予含不同浓度氯化锰（MnCl₂）（0、200、300、400、500 μmol/L）、PAS-Na（50、150、450 μmol/L）的完全培养基（DMEM+FBS）培养24 h，采用MTT法检测细胞存活率，根据结果选择染锰的毒性剂量并选定PAS-Na的无毒剂量。随后将细胞随机分为对照组、400 μmol/L MnCl₂组、400 μmol/L MnCl₂+（50、150、450 μmol/L）PAS-Na组、450 μmol/L PAS-Na组，共6组。采用MTT法检测各组小胶质细胞存活率；运用DCFH-DA探针标记，荧光倒置显微镜下观察小胶质细胞内活性氧（ROS）的产生情况；ELISA法测定炎症因子白介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α的蛋白分泌量和荧光定量PCR法测IL-1β，TNF-α mRNA表达水平。

结果 400、500 μmol/L MnCl₂染毒组的细胞存活率为（85.83±12.53）%、（83.30±6.33）%，低于对照组（均P < 0.05），故选择400 μmol/L作为锰的染毒剂量；与对照组比较，50、150、450 μmol/L PAS-Na组的细胞存活率无明显变化（均P>0.05）。400 μmol/L MnCl₂+（50、150、450 μmol/L）PAS-Na组的细胞存活率分别为（96.00±18.11）%、（106.13±18.32）%、（110.21±15.13）%，均比400 μmol/L MnCl₂组高（均P < 0.05）。与对照组比较，400 μmol/L MnCl₂组细胞ROS水平、IL-1β和TNF-α的蛋白及mRNA表达水平均升高（均P < 0.05）。与400μmol/L MnCl₂组比较，400μmol/L MnCl₂+（50、150、450μmol/L）PAS-Na组的ROS水平、TNF-α分泌量和IL-1β mRNA表达量均降低（均P < 0.05），400 μmol/L MnCl₂+（150、450 μmol/L）PAS-Na组的TNF-α mRNA表达量降低（均P < 0.05）。

结论 PAS-Na可减轻锰致大鼠丘脑原代小胶质细胞氧化损伤、炎症反应的程度。

Abstract:
Objective To investigate the effects of sodium p-aminosalicylic acid (PAS-Na) on oxidative damage and inflammatory response of rat thalamic primary microglia induced by manganese.

Methods Purified and identified SD rat's thalamic microglia were cultured with different concentrations of MnCl₂ (0, 200, 300, 400, and 500 μmol/L, respectively) and PAS-Na (50, 150, and 450 μmol/L, respectively) in complete mediums (DMEM+FBS) for 24 h. Cell viability was determined by MTT to identify a suitable dose of manganese exposure and non-toxic dose of PAS-Na exposure. Then cells were randomly divided into control group, 400 μmol/L MnCl₂ group, 400 μmol/L MnCl₂+PAS-Na (50, 150, and 450μmol/L, respectively) groups, and 450 μmol/L PAS-Na group. The survival rate of microglia in each group was detected by MTT assay. The level of intercellular reactive oxygen species (ROS) was detected under fluorescence inverse microscope with DCFH-DA probe mark. The protein expressions of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were detected by ELISA, and the mRNA expression levels of IL-1β and TNF-α were determined by RT-PCR.

Results The survival rates of cells treated with 400 and 500 μmol/L MnCl₂ were (85.83±12.53)% and (83.30±6.33)%, respectively, lower than that of the control group (Ps < 0.05). Therefore, the exposure dose of 400μmol/L was chosen. Compared with the control group, the survival rates of the 50, 150, and 450 μmol/L PAS-Na groups did not change (Ps>0.05). The survival rates of the 400 μmol/L MnCl₂+PAS-Na (50, 150, 450 μmol/L) groups were (96.00±18.11)%, (106.13±18.32)%, and (110.21±15.13)%, respectively, higher than that of the 400 μmol/L MnCl₂ group (Ps < 0.05). Compared with the control group, the levels of ROS, the protein and mRNA expression levels of IL-1β and TNF-α in the 400 μmol/L MnCl₂ group increased (Ps < 0.05). Compared with the 400 μmol/L MnCl₂ group, the levels of ROS, protein expression levels of TNF-α, and the mRNA expression levels of IL-1β in the 400 μmol/L MnCl₂+PAS-Na groups (50, 150, 450 μmol/L) decreased (Ps < 0.05), and the mRNA expression levels of TNF-α in the 400μmol/L MnCl₂+PAS-Na groups(150, 450μmol/L) also decreased (Ps < 0.05).

Conclusion PAS-Na may alleviate the oxidative damage and inflammatory response of thalamic primary microglia in rats induced by manganese.

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