芒柄花素保护BV2小胶质细胞糖氧剥夺再灌注损伤及其机制研究

Protective effects of formononetin against oxygen-glucose deprivation/ reperfusion injury in BV2 microglia and associated mechanism

摘要

目的 探讨芒柄花素（formononetin，FN）对糖氧剥夺再灌注（glucose and oxygen deprivation/reperfusion，OGD/R）刺激的BV2小胶质细胞多聚ADP核糖聚合酶1［poly（ADP-ribose） polymerase 1，PARP1］/聚（ADP-核糖）糖水解酶［poly（ADP-Ribose） glycohydrolase，PARG］信号通路及神经炎症的影响。 方法 建立OGD/R BV2细胞模型，分为空白对照组、OGD/R组、10 μm FN组、OGD/R+10 μm FN处理组、OGD/R+抑制剂PJ34（10 μm）处理组、OGD/R+抑制剂Ethacridine lactate（7.5 μm）处理组。采用免疫荧光法（immunofluorescence method，IF）检测BV2细胞核因子-κB p65（Nuclear factor-kappa B p65，NF-κB p65）蛋白核转移及p53、凋亡诱导因子（apoptosis-inducing factor，AIF）、Toll样受体４（Toll-like receptor 4，TLR4）蛋白表达；免疫印迹法检测（Western blot，WB）PARP1/PARG通路相关蛋白表达；酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）检测血清γ干扰素（interferon-γ，IFN-γ）、白细胞介素-1β（interleukin-1β，IL-1β）以及肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）含量。 结果 WB结果显示，与对照组相比，进行糖氧剥夺6 h后再进行复糖复氧培养1 h，细胞中PARP1、PARG表达明显升高（P=0.000）；与OGD/R组相比，在OGD/R后加入FN处理，BV2细胞中PARP1（P=0.000）和PARG（P=0.000）蛋白表达量明显下降，Iduna蛋白表达量明显上升（P=0.000）；在OGD/R后加入PARP抑制剂PJ34处理后，PARP1蛋白表达量明显下降（P=0.000），但PARG和Iduna蛋白表达量无明显变化（P=0.061）；在OGD/R后加入PARG抑制剂Ethacridine lactate处理后，BV2细胞中PARG蛋白表达量明显下降（P=0.000），但PARP1和Iduna蛋白表达量无明显变化（P=0.072）。IF结果显示，与OGD/R组相比，OGD/R后加入FN，细胞中p53、AIF、TLR4、NF-κB p65表达均明显下降，且NF-κB p65核转移明显减少；在OGD/R后加入PARP抑制剂PJ34或PARG抑制剂Ethacridine lactate，p53、AIF、TLR4表达同样下降，NF-κB p65核转移也明显减少。ELISA结果显示，与OGD/R组BV2细胞相比，在OGD/R后加入FN处理，细胞中IL-1β（P=0.004）和TNF-α（P=0.040）表达明显降低，但INF-γ水平无明显变化（P=0.056）；在OGD/R后加入PARP抑制剂PJ34处理后，BV2细胞中促炎因子INF-γ（P=0.000）、IL-1β（P=0.021）和TNF-α（P=0.003）表达水平明显降低，或PARG抑制剂Ethacridine lactate处理后，BV2细胞中促炎因子INF-γ、IL-1β和TNF-α表达水平亦明显降低（P=0.000）。 结论 FN可抑制糖氧剥夺BV2小胶质细胞中神经炎症，其机制涉及PARP1/PARG信号通路调控。

Abstract

Objective To investigate the effects of formononetin on the PARP1/PARG signaling pathway and neuroinflammation in BV2 microglia activated by oxygen-glucose deprivation/reperfusion（OGD/R）. Methods An OGD/R model was established with BV2 cells. These microglia were divided into blank control group，OGD/R group，formononetin（10 μm） group，OGD/R+formononetin（10 μm） group，OGD/R+PJ34（PARP1 inhibitor，10 μm） group，and OGD/R+ethacridine lactate（PARG inhibitor，7.5 μm） group. We measured the nuclear translocation of nuclear factor-kappa B（NF-κB） p65 and the expression of p53，apoptosis-inducing factor （AIF），and Toll-like receptor 4（TLR4） proteins in BV2 cells by immunofluorescence assay；measured the expression of PARP1/PARG pathway-related proteins by Western blot；and determined the content of interferon（INF）-γ，interleukin（IL）-1β，and tumor necrosis factor-α（TNF-α） by enzyme-linked immunosorbent assay（ELISA）. Results Western blot results showed that compared with the control group，the expression of PARP1 and PARG in cells was significantly increased after 6-hour oxygen-glucose deprivation followed by 1-hour reperfusion（P<0.05）； compared with the OGD/R group，the OGD/R+formononetin group had significantly decreased expression of PARP1 and PARG proteins（P<0.05） and significantly increased expression of Iduna protein（P<0.05）；the OGD/R+PJ34 group had significantly decreased expression of PARP1 protein（P<0.05），but the expression of PARG and Iduna proteins had no significant changes（P>0.05）；the OGD/R+ethacridine lactate group had significantly decreased expression of PARG protein（P<0.05），but had no significant changes in the expression of PARP1 and Iduna proteins（P>0.05）. Immunofluorescence assay results showed that compared with the OGD/R group，formononetin significantly reduced the expression of p53，AIF，TLR4，and NF-κB p65 and the nuclear translocation of NF-κB p65 in OGD/R cells； and both PJ34 and ethacridine lactate significantly reduced the expression of p53，AIF，and TLR4 and the nuclear translocation of NF-κB p65 in OGD/R cells. ELISA results showed that compared with the OGD/R group，formononetin significantly decreased the expression of IL-1β and TNF-α（P<0.05），but did not significantly affect INF-γ levels in OGD/R cells （P>0.05）； and both PJ34 and ethacridine lactate significantly decreased the expression levels of the proinflammatory factors INF-γ，IL-1β，and TNF-α in OGD/R cells （P<0.05）. Conclusion Formononetin can inhibit neuroinflammation in BV2 microglia deprived of glucose and oxygen，through regulating the PARP1/PARG signaling pathway.

关键词

芒柄花素 / BV2小胶质细胞 / 糖氧剥夺再灌注 / 多聚ADP核糖聚合酶1抑制剂 / 聚（ADP-核糖）糖水解酶抑制剂

Key words

formononetin / BV2 microglia / oxygen-glucose deprivation/reperfusion / PARP1 inhibitor / PARG inhibitor

基金资助

国家自然科学基金资助项目(82160850)

贵州省科技基金资助项目(黔科合基础[2020]1Z071)