氧化石墨烯片层对小胶质细胞神经免疫效应的调控

Effects of graphene oxide slice on neuro-immunity modulation of microglia cells

[背景]氧化石墨烯(GO)广泛应用于神经生物学领域。近年来研究表明,GO能够参与神经系统的调节过程,但关于其神经免疫效应调控具体机制尚无相关报道。[目的]探究GO在脂多糖(LPS)和白细胞介素4(IL-4)诱导小胶质细胞M1经典活化表型与M2选择活化表型转化中的神经调控作用。[方法]在GO基底培养基和对照培养基上对BV2细胞进行培养,分别加入20μg·L^-1 LPS或IL-4诱导BV2细胞表型转化,分组为空白对照组、GO对照组、空白-LPS组、空白-IL-4组、GO-LPS组和GO-IL-4组。比较24 h后培养上清中的细胞数量。利用实时荧光定量PCR(RT-qPCR)和流式细胞术分别检测BV2细胞表面M1表型标志物[如肿瘤坏死因子α(TNF-α)、诱导型一氧化氮合酶(i NOS)、细胞因子CD86]以及M2表型标志物[如重组人精氨酸酶1(Arg1)、白细胞介素-10(IL-10)、细胞因子CD206]表达量的变化,同时使用分光光度法检测培养上清中一氧化氮(NO)的含量。[结果]与空白对照组相比,GO处理后的BV2细胞细胞计数降低(218.13±8.77和175.63±8.24,P <0.01)。相比于空白-LPS组,GO-LPS组BV2细胞上M1表型标志物TNF-α、iNOS的mRNA相对表达量(8.16±0.26和5.35±0.42;45.64±2.03和30.39±1.84)以及CD86阳性细胞率[(14.65±0.85)%和(10.29±0.46)%]降低,并且培养上清中NO的含量[(19.62±1.07)μmol·L^-1和(8.37±1.25)μmol·L^-1]降低,M2表型标志物IL-10的mRNA相对表达量升高(1.63±0.09和2.09±0.15)(P <0.05)。与空白-IL-4组相比,GO-IL-4组M1表型标志物iNOS的mRNA相对表达量增加(0.41±0.03和1.05±0.07),M2表型标志物Arg1以及IL-10的mRNA相对表达量下降(201.63±12.31和4.74±0.38;2.63±0.14和0.71±0.13),CD206的阳性细胞率和平均荧光强度下降[(17.01±1.03)%和(3.66±0.41)%;8 893.2±347.62和1 299.9±159.67](P <0.01)。[结论]GO抑制BV2小胶质细胞生长,并对LPS和IL-4诱导下的神经免疫效应所引起的BV2小胶质细胞向M1和M2表型的转化起到抑制作用。

[Background]Graphene oxide(GO)is widely applied to neuroscience.Recent studies have shown that GO can regulate the nervous system,but there are limited reports about the mechanism underlying its neuro-immunity modulation.[Objective]This experiment is designed to investigate the regulation of GO towards M1/M2 polarization of microglia induced by lipopolysaccharide(LPS)and interleukin-4(IL-4).[Methods]BV2 cells cultured on GO substrate and control medium were added with 20μg·L-1 LPS and IL-4 respectively to induce M1/M2 phenotype of BV2 cells,and were divided into blank control group,GO control group,LPS control group,IL-4 control group,GO-LPS group,and GO-IL-4 group.After 24 h of incubation,the number of cells was counted.The expression levels of M1 markers[tumor necrosis factorα(TNF-α),inducible nitric oxide synthase(iNOS),and cytokine CD86]and M2 markers[recombinant human arginase-1(Arg1),interleukin-10(IL-10),and cytokine CD206)]were detected by real-time fluorescence quantitative PCR(RT-qPCR)and flow cytometry,and the NO concentration in supernatant was measured by spectrophotometry.[Results]After the designed GO treatment,the number of BV2 cells was decreased(218.13±8.77 and 175.63±8.24)compared with the blank control group(P<0.05).The mRNA expression levels of M1 markers TNF-α(8.16±0.26 and 5.35±0.42),iNOS(45.64±2.03 and 30.39±1.84),CD86 positive rate[(14.65±0.85)%and(10.29±0.46)%,and NO production[(19.62±1.07)μmol·L-1 and(8.37±1.25)μmol·L-1]were all decreased in the GO-LPS group,as well as the mRNA expression levels of M2 marker IL-10(1.63±0.09 and 2.09±0.15)increased,compared with the LPS control group(P<0.05).Under the IL-4 inducement,the mRNA expression level of M1 marker iNOS was upregulated(0.41±0.03 and 1.05±0.07),the levels of Arg1(201.63±12.31 and 4.74±0.38)and IL-10(2.63±0.14 and 0.71±0.13)were downregulated,and the positive rate[(17.01±1.03)%and(3.66±0.41)%]and average fluorescence intensity(8893.2±347.62 and 1299.9±159.67)in the GO-IL-4 group(P<0.01).[Conclusion]GO inhibits the growth of BV2 cells and regulates the activation of microglia to resist the effects of LPS/IL-4 induced microglial polarization.