miR-124-3p和自噬在LPS致小胶质细胞死亡中的作用机制研究
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摘要
目的:探讨miR-124-3p和细胞自噬在脂多糖(lipopolysaccharides, LPS)引起小胶质细胞死亡中的作用,以及miR-124-3p与自噬的相互关系。方法 :离体条件下,使用1μg/mL LPS处理BV2细胞,采用噻唑蓝检测BV2细胞活力,实时定量荧光PCR检测miR-124-3p表达水平,Western Blot检测LC3B蛋白表达水平。应用慢病毒载体过表达miR-124-3p,观察miR-124-3p对LPS引起BV2细胞活力以及自噬的影响。0.1μmol/L雷帕霉素预处理BV2细胞,观察自噬对LPS引起BV2细胞活力及miR-124-3p水平变化的影响。结果:LPS处理BV2细胞后,BV2细胞活力显著降低,且miR-124-3p表达下调,细胞自噬水平显著上升。过表达miR-124-3p能有效抑制由LPS引起的BV2细胞活力降低,并有效抑制LPS引起的细胞自噬。雷帕霉素预处理BV2细胞,能显著缓解由LPS引起的BV2细胞活力和miR-124-3p表达降低。结论:LPS可以通过下调miR-124-3p表达,影响小胶质细胞存活。同时,自噬也可在一定程度上负反馈调节miR-124-3p表达,影响BV2细胞存活。
Objective : To investgate the effect of miR-124-3 p and autophagy in microglia cell death induced by lipopolysaccharides(LPS) and the interaction between mi R-124-3 p and autophagy. Methods : 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was used to detect the BV2 cell viability with 1 μg/mL LPS treatment in vitro. MiR-124-3 p expression was performed by real time PCR and LC3 B expression was detected by Western Blot.Mimic miR-124-3 p lentivirus was constructed to demonstrate the effect of miR-124-3 p in microglia cell death induced by LPS. Pre-treatment of BV-2 cells with 0.1 μmol/L Rapamycin illuminated the effect of autophagy in microglia cell death induced by LPS and the relationship between miR-124-3 p and autophagy. Results : LPS significantly inhibited BV2 microglia cell viability and miR-124-3 p expression. LPS significantly induced BV2 microglia autophagy.MiR-124-3 p inhibited the BV2 microglia cell death and the increase of autophagy induced by LPS. Rapamycin treatment in advanced rescued BV2 microglia cell death and decrease of miR-124-3 p. Conclusion : LPS inhibited BV2 microglia cell viability via miR-124-3 p. The negative feedback regulation of autophagy affected BV2 microglia cell viability and miR-124-3 p expression to some extent.
Objective : To investgate the effect of miR-124-3 p and autophagy in microglia cell death induced by lipopolysaccharides(LPS) and the interaction between mi R-124-3 p and autophagy. Methods : 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was used to detect the BV2 cell viability with 1 μg/mL LPS treatment in vitro. MiR-124-3 p expression was performed by real time PCR and LC3 B expression was detected by Western Blot.Mimic miR-124-3 p lentivirus was constructed to demonstrate the effect of miR-124-3 p in microglia cell death induced by LPS. Pre-treatment of BV-2 cells with 0.1 μmol/L Rapamycin illuminated the effect of autophagy in microglia cell death induced by LPS and the relationship between miR-124-3 p and autophagy. Results : LPS significantly inhibited BV2 microglia cell viability and miR-124-3 p expression. LPS significantly induced BV2 microglia autophagy.MiR-124-3 p inhibited the BV2 microglia cell death and the increase of autophagy induced by LPS. Rapamycin treatment in advanced rescued BV2 microglia cell death and decrease of miR-124-3 p. Conclusion : LPS inhibited BV2 microglia cell viability via miR-124-3 p. The negative feedback regulation of autophagy affected BV2 microglia cell viability and miR-124-3 p expression to some extent.
引文
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[16]LI H, JIN X, CHEN B, et al. Autophagy-regulating microRNAs:potential targets for improving radiotherapy[J]. J Cancer Res Clin Oncol, 2018, 144(9):1623-1634.
[2]任怡,丁新生,汪锡金,等. CD200调控小胶质细胞KATP通道在抑制帕金森病发病中的作用[J].江苏医药, 2015,41(8):876-879.
[3]ZHANG Y, SHEN K, BAI Y, et al. Mir143-BBC3 cascade reduces microglial survival via interplay between apoptosis and autophagy:implications for methamphetamine-mediated neurotoxicity[J]. Autophagy, 2016, 12(9):1538-1559.
[4]GARGOURI B, YOUSIF NM, BOUCHARD M, et al. Inflammatory and cytotoxic effects of bifenthrin in primary microglia and organotypic hippocampal slice cultures[J]. J Neuroinflammation, 2018, 15(1):159.
[5]WANG JY, ZHANG Q, WANG DD, et al. Mi R-29a:a potential therapeutic target and promising biomarker in tumors[J]. Biosci Rep, 2018, 38(1). pii:BSR20171265.
[6]ZHANG F, WANG B, LONG H, et al. Decreased miR-124-3p expression prompted breast cancer cell progression mainly by targeting beclin-1[J]. Clin Lab, 2016, 62(6):1139-1145.
[7]HUANG R, ZHANG Y, HAN B, et al. Circular RNA HIPK2regulates astrocyte activation via cooperation of autophagy and ER stress by targeting MIR124-2HG[J]. Autophagy, 2017,13(10):1722-1741.
[8]CHEN YZ, WANG F, WANG HJ, et al. Sphingosine 1 phosphate receptor-1(S1PR1)signaling protects cardiac function by inhibiting cardiomyocyte autophagy[J]. J Geriatr Cardiol,2018, 15(5):334-345.
[9]陶金,孙钰,王静成,等.细胞自噬与纤维化疾病的研究进展[J].江苏医药, 2018, 44(4):432-437.
[10]王栋,侯博儒,杨文桢,等.小胶质细胞自噬在缺血性脑卒中中作用的研究进展[J].中国康复理论与实践, 2016,22(12):1416-1419.
[11]TATEDA S, KANNO H, OZAWA H, et al. Rapamycin suppresses microglial activation and reduces the development of neuropathic pain after spinal cord injury[J]. J Orthop Res,2017, 35(1):93-103.
[12]LIVELY S, SCHLICHTER LC. Microglia responses to pro-inflammatory stimuli(LPS, IFNγ+TNFα)and reprogramming by resolving cytokines(IL-4, IL-10)[J]. Front Cell Neurosci, 2018, 12:215.
[13]CHERRY JD, OLSCHOWKA JA, O'BANION MK. Neuroinflammation and M2 microglia:the good, the bad, and the inflamed[J]. J Neuroinflammation, 2014, 11:98.
[14]HERDER V, ISKANDAR CD, KEGLER K, et al. Dynamic changes of Microglia/Macrophage M1 and M2 polarization in theiler's murine encephalomyelitis[J]. Brain Pathol, 2015,25(6):712-723.
[15]张博.瘦素对小胶质细胞炎症反应调节的研究[D].天津:天津医科大学, 2016.
[16]LI H, JIN X, CHEN B, et al. Autophagy-regulating microRNAs:potential targets for improving radiotherapy[J]. J Cancer Res Clin Oncol, 2018, 144(9):1623-1634.