短链脂肪酸对小胶质细胞活化及组蛋白乙酰化水平的影响

Effects of short chain fatty acids on activation and histone acetylation of microglia

目的:研究短链脂肪酸(SCFAs)对小胶质细胞活化及组蛋白乙酰化水平的作用。方法:将对数生长期BV2永生化小胶质细胞分为对照组(空白对照)、不同浓度曲古霉素(TSA)组(阳性对照)、不同浓度SCFAs组;采用斑点印迹法(Dot blot)筛选各组组蛋白乙酰化常见位点,寻找与TREM2关联最大的组蛋白乙酰化位点、组别,Western blot验证上述浓度组别;根据筛选结果,采用CCK8检测BV2细胞活性;免疫荧光染色检测BV2细胞Iba1表达;ELISA检测BV2细胞IL-4、IL-13、IL-10、TNF-α、iNOS表达;qRT-PCR检测BV2细胞目的基因TREM2、TNF-α、iNOS、Mincle、IL-10、Arg1 mRNA表达。结果:Dot blot显示H3、H4乙酰化位点中H3K27和H4K8与TREM2关联性较好,根据距离TSA各浓度最近的组别筛选SCFAs,得到10 mmol/L丙酸、1 mmol/L戊酸、10 mmol/L戊酸、1 mmol/L异戊酸、10 mmol/L异戊酸、1 mmol/L SCFAs、10 mmol/L SCFAs、100 mmol/L SCFAs组;Western blot显示H3K27、H4K8与TREM2聚类结果较好,验证了筛选结果,并在干预1 h后,高浓度SCFAs与高浓度TSA作用类似,干预24 h后,低浓度SCFAs与低浓度TSA作用类似;CCK8显示药物干预24 h后,高浓度SCFAs与TSA组相对增殖率均低于对照组(P<0.05);免疫荧光染色显示,各浓度SCFAs组对Iba1的调节作用与TSA基本一致,均可抑制BV2细胞活化,且随着时间延长作用增强(P<0.05),且高浓度SCFAs比低浓度SCFAs抑制作用更强;ELISA显示除1 mmol/L SCFAs外其余各组均可加快IL-4、IL-13释放(P<0.05),高浓度SCFAs可减缓IL-10、TNF-α、iNOS释放(P<0.05);qRT-PCR显示TNF-α、iNOS mRNA表达在各组药物干预1h后有一定下降(P>0.05),但24h后其mRNA表达多数从下降转为上升(P<0.05),TREM2、IL-10、Arg1、Mincle表达在干预1 h后略有上升(P>0.05),24 h后大范围明显下降(P<0.05),其中Arg1表达尤为显著,表现为高浓度SCFAs或TSA组表达明显下降。结论:SCFAs可促进组蛋白乙酰化,且高浓度SCFAs可抑制小胶质细胞增殖并促进其活化,干预早期(24 h内)可促进小胶质细胞M2a型活化,干预晚期(24 h后)可促进小胶质细胞M1型活化。

Objective:To study effects of short-chain fatty acids(SCFAs)on microglial activation and histone acetylation.Methods:BV2 immortalized microglia with logarithmic growth were divided into control group(blank control),different concentrations of trichomycin(TSA)groups(positive control)and different concentrations of SCFAs groups.Common sites of histone acetylation in each group were screened by Dot blot to find histone acetylation sites and groups most associated with TREM2,and the above concentration groups were verified by Western blot.According to screening results,CCK8 was used to detect activity of BV2 cells,and immunofluorescence staining to detect expression of Iba1 in BV2 cells.Expressions of IL-4,IL-13,IL-10,TNF-αand iNOS in BV2 cells were detected by ELISA,and mRNA expressions of TREM2,TNF-α,iNOS,Mincle,IL-10 and Arg1 in BV2 cells were detected by qRT-PCR.Results:Dot blot showed that among acetylation sites of H3 and H4,H3K27 and H4K8 had a good correlation with TREM2.According to groups closest to each concentration of TSA to screen SCFAs,10 mmol/L propionic acid,1 mmol/L valerate,10 mmol/L valerate,1 mmol/L isovalerate,10 mmol/L isovalerate,1 mmol/L SCFAs,10 mmol/L SCFAs,100 mmol/L SCFAs groups were obtained.Western blot showed that clustering results of H3K27,H4K8 and TREM2 were good,which verified screening results.After 1 h of intervention,effect of high concentration of SCFAs was similar to that of high concentration of TSA,and after 24 h of intervention,effect of low concentration of SCFAs was similar to that of low concentration of TSA.CCK8 showed that relative proliferation rates in high concentration of SCFAs and TSA groups were lower than those of control group after 24 h of drug intervention(P<0.05).Immunofluorescence staining showed that regulatory effect of SCFAs on Iba1 was basically same as that of TSA,and could inhibit activation of BV2 cells(P<0.05),and inhibitory effect of high concentration of SCFAs was stronger than that of low concentration of SCFAs.ELISA showed that all groups except 1 mmol/L SCFAs could accelerate release of IL-4 and IL-13(P<0.05),and high concentration of SCFAs could slow down release of IL-10,TNF-αand iNOS(P<0.05).qRT-PCR showed that mRNA expressions of TNF-αand iNOS were decreased after 1 h of drug intervention(P>0.05),but most of their mRNA expressions were changed from decrease to increase after 24 h(P<0.05).Expressions of TREM2,IL-10,Arg1 and Mincle were increased slightly after 1 h intervention(P>0.05),and were decreased significantly after 24 h(P<0.05),especially expression of Arg1 was significantly decreased in high concentration SCFAs or TSA groups.Conclusion:SCFAs can promote histone acetylation,and high concentration of SCFAs can inhibit proliferation of microglia and promote its activation,and promote activation of M2a type of microglia in early stage(within 24 h),and promote activation of M1 type of microglia in late stage(after 24 h).