重组人神经生长因子通过抑制小胶质细胞炎症反应发挥神经保护作用

Neuroprotective effect of recombinant human nerve growth factors by inhibiting microglia inflammation

目的探讨重组人神经生长因子(rhNGF)的神经保护作用及其机制。方法BV2小胶质细胞分为细胞对照组、脂多糖(LPS)组及LPS+rhNGF 50和500μg·L^(-1)组。除细胞对照组外,其余3组用LPS 1 mg·L^(-1)刺激BV2细胞24 h诱导BV2细胞炎症反应,随后LPS+rhNGF组加rhNGF继续孵育48 h。孵育结束后收集BV2细胞,并收集各组培养上清即条件培养基(MCM),各组BV2细胞和MCM分别与小鼠脑神经瘤细胞Neuro-2a(N2a)共培养12或24 h。CCK-8法检测BV2细胞存活率,实时荧光定量PCR检测BV2细胞中促炎因子白细胞介素(IL-6)、肿瘤坏死因子α(TNF-α)和IL-1β及抗炎因子IL-4和IL-10 mRNA表达水平,CBA法检测BV2细胞培养上清中IL-6,TNF-α,IL-4和IL-10浓度,免疫荧光法检测BV2细胞M1表型标志物CD16和M2表型标志物CD206表达,NeuN/TUNEL共染法和AnnexinⅤ/7-AAD法检测N2a细胞凋亡率。结果与细胞对照组相比,LPS组BV2细胞存活率显著下降(P<0.01),细胞变圆且胞体变大;促炎因子IL-1β,IL-6和TNF-α及抗炎因子IL-10 mRNA水平显著升高(P<0.01),抗炎因子IL-4 mRNA水平下降(P<0.01);IL-6和TNF-α蛋白水平显著升高(P<0.01),IL-4蛋白水平无显著差异;CD16和CD206阳性细胞百分比明显升高(P<0.01),CD16阳性细胞升高更加明显;与BV2细胞或其MCM共培养的N2a细胞存活率显著降低且凋亡率显著升高(P<0.05,P<0.01)。与LPS组相比,LPS+rhNGF 50和500μg·L^(-1)组BV2细胞存活率显著提高(P<0.01);IL-1β,IL-6和TNF-αmRNA水平显著降低(P<0.05),IL-4和IL-10 mRNA水平显著提高(P<0.05,P<0.01);IL-6和TNF-α蛋白水平显著降低(P<0.05,P<0.01),IL-4蛋白水平无显著差异,IL-10浓度低于检测限;CD206阳性细胞百分比显著升高(P<0.01),CD16阳性细胞百分比显著降低(P<0.05)。与LPS处理的BV2细胞或MCM-LPS组相比,与rhNGF 50和500μg·L^(-1)处理的BV2细胞共培养组及与MCM-rhNGF 50和500μg·L^(-1)共培养组N2a细胞存活率显著升高且凋亡率显著降低(P<0.05,P<0.01)。结论rhNGF可通过抑制BV2细胞炎症反应诱导BV2细胞向M2表型转化,抑制N2a细胞凋亡,从而发挥神经保护作用。

OBJECTIVE To evaluate the neuroprotective effect of recombinant human nerve growth factor(rhNG)at the molecular level and explore the mechanism.METHODS BV2 microglia were divided into the cell control group,lipopolysaccharide(LPS)group,and LPS+rhNGF 50 and 500μg·L^(-1) groups.Except the cell control group,all the groups of BV2 cells were stimulated by LPS 1 mg·L^(-1) for 24 h to induce inflammatory reaction before LPS+rhNGF groups were cultured with rhNGF for another 48 h.After incubation,BV2 cells and microglial conditioned medium(MCM)were collected.The BV2 cells and MCM in each group were respectively co-cultured with mouse neuroblastoma cell Neuro-2a(N2a cells)for 12 or 24 h.The cell survival rate was detected by CCK-8 kit,the mRNA expression levels of pro-inflammatory cytokines interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)and IL-1β,and anti-inflammatory cytokines IL-4 and IL-10 in BV2 cells were detected by real-time fluorescence quantitative PCR(RTqPCR),and the concentrations of IL-6,TNF-α,IL-4 and IL-10 in the BV2 cell culture supernatant were detected by CBA kit.The expressions of M1 phenotypic marker CD16 and M2 phenotypic marker CD206 in BV2 cells were detected using the immunofluorescence method.The apoptosis rate of N2a cells was detected with NeuN/TUNEL co-staining and Annexin V/7-AAD methods.RESULTS Compared with the cell control group,the survival rate of BV2 cells in the LPS group decreased significantly(P<0.01),the cells became round and the cell body became larger,the mRNA levels of pro-inflammatory factors IL-1β,IL-6 and TNF-α,and anti-inflammatory factors IL-10 increased significantly(P<0.01),the mRNA level of anti-inflammatory factor IL-4 decreased(P<0.01),the protein levels of IL-6 and TNF-αincreased significantly(P<0.01),but there was no significant difference in the protein level of IL-4.The percentages of CD16+and CD206+cells were significantly up-regulated(P<0.01),particularly the former.The survival rate of N2a cells co-cultured with BV2 cells or their MCM decreased significantly while the apoptosis rate increased significantly(P<0.05,P<0.01).Compared with the LPS group,the survival rate of BV2 cells in LPS+rhNGF 50 or 500μg·L^(-1) groups increased significantly(P<0.01),the mRNA levels of IL-1β,IL-6 and TNF-αdecreased significantly(P<0.05),the mRNA levels of IL-4 and IL-10 increased significantly(P<0.05,P<0.01),the protein levels of IL-6 and TNF-αdecreased significantly(P<0.05,P<0.01),but the protein level of IL-4 hardly changed and the concentration of IL-10 was lower than the detection limit.The percentage of CD206+cells increased significantly(P<0.01),and that of CD16+cells decreased significantly(P<0.05).Compared with LPS-treated BV2 cell co-culture group or MCM-LPS-treated group,the survival rate of N2a cells co-cultured with BV2 cells treated with rhNGF 50 or 500μg·L^(-1) and with MCMrhNGF 50 or 500μg·L^(-1) increased significantly while the apoptosis rate decreased significantly(P<0.05,P<0.01).CONCLUSION rhNGF can induce the transformation of BV2 microglia into M2 phenotype and inhibit the apoptosis of N2a cells by inhibiting the inflammatory reaction of BV2 microglia.