LPS诱导的A1型星形胶质细胞的能量代谢特征  

Characteristics of energy metabolism in lipopolysaccharide-induced A1-type astrocytes

作　　者：赵静 陈茹 沈桂萍 张慧丰 范彦英[1] ZHAO Jing;CHEN Ru;SHEN Gui-ping;ZHANG Hui-feng;FAN Yan-ying(Department of Pharmacology,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西医科大学基础医学院药理教研室,山西太原030001

出　　处：《中国病理生理杂志》2022年第6期970-977,共8页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81872854,No.81202520)。

摘　　要：目的:探讨在脂多糖(lipopolysaccharide,LPS)诱导下,小鼠皮层星形胶质细胞转化为A1毒性表型的同时,其能量代谢所发生的变化。方法:小鼠皮层星形胶质细胞培养8~9 d后,分为对照(control,CON)组和LPS组;采用CCK-8细胞增殖及毒性检测试剂盒检测不同LPS处理浓度及不同处理时间下的细胞活力;通过细胞免疫荧光染色技术检测胶质细胞原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达来鉴定星形胶质细胞的纯度;通过补体C3(complement component 3,C3)与GFAP细胞免疫荧光染色共定位,检测C3的表达水平;利用RT-qPCR技术检测C3、鸟苷酸结合蛋白2(guanylate-binding protein 2,GBP2)、S100钙结合蛋白A10(S100 calcium-binding pro⁃tein A10,S100A10)、转谷氨酰胺酶1(transglutaminase 1,TGM1)及白细胞介素1β(interleukin-1β,IL-1β)的mRNA表达变化;采用Western blot技术检测C3蛋白表达变化。利用Seahorse XFp活细胞生物能量检测技术检测细胞线粒体呼吸功能和糖酵解水平的变化。结果:免疫荧光染色结果显示,GFAP阳性细胞比例达98%以上。CCK-8结果显示,在不同浓度和不同处理时间下,LPS对星形胶质细胞的活力均无显著影响;RT-qPCR结果显示,在LPS剂量为100μg/L时,C3和GBP2的mRNA表达在24 h均显著升高(P<0.01),而S100A10和TGM1的mRNA表达则无显著变化。Western blot和免疫荧光染色结果显示,LPS处理后C3的蛋白表达显著升高(P<0.01);RT-qPCR结果显示,IL-1β的mRNA表达水平显著升高(P<0.01)。线粒体压力测定结果显示,LPS处理后的线粒体呼吸相关指标--氧消耗速率(oxygen consumption rate,OCR)与CON组相比无显著差异,而糖酵解速率相关指标--细胞外酸化速率(ex⁃tracellular acidification rate,ECAR)、基础糖酵解质子流出速率(glycolytic proton efflux rate,glycoPER)、代偿性糖酵解、glycoPER百分比及线粒体质子流出速率(mitochondial proton efflux rate,mitoPER)/glycoPER在LPS处理后均显著下降(P<0.01)。AIM:To investigate the change of energy metabolism during transformation of mouse cortical as⁃trocytes to the A1 toxic phenotype induced by lipopolysaccharide(LPS).METHODS:Primary mouse cortical astrocytes were divided into control(CON)group and LPS group after cultured for 8 to 9 d.Cell Counting Kit-8(CCK-8)was used to detect the cell viability.The expression of glial fibrillary acidic protein(GFAP)was detected by immunofluorescence.The expression of complement component 3(C3)was detected by costaining with GFAP.The mRNA levels of C3,gua⁃nylate-binding protein 2(GBP2),S100 calcium-binding protein A10(S100A10),transglutaminase 1(TGM1)and interleukin-1β(IL-1β)after LPS treatment were detected by RT-qPCR.The expression of C3 protein was assessed by Western blot.The levels of cellular mitochondrial respiratory function and glycolysis were detected by Seahorse XFp live-cell bio⁃energy detection technology.RESULTS:Immunofluorescence staining showed that the percentage of GFAP reached more than 98%.Treatment with LPS did not change the viability of astrocytes.The mRNA levels of C3 and GBP2 were significantly increased at 24 h after treatment with LPS at the concentration of 100μg/L(P<0.01),while the expression of S100A10 and TGM1 did not change.Both Western blot and immunofluorescence staining showed C3 was significantly in⁃creased after treated with LPS(P<0.01).The results of RT-qPCR showed that the mRNA level of IL-1βwas significantly increased(P<0.01).Mitochondrial pressure measurement showed that there was no significant difference in oxygen con⁃sumption rate(OCR),an indicator of mitochondrial respiration,between control group and LPS group.Glycolysis rate-related indicators such as extracellular acidification rate(ECAR),basal glycolytic proton efflux rate(glycoPER),compensatory glycolysis,the percentage of glycoPER,and mitochondial proton efflux rate(mitoPER)/glycoPER were decreased significantly after LPS treatment(P<0.01).CONCLUSION:LPS induces the transformation of astrocytes to A1 pheno⁃type an

关 键 词：星形胶质细胞 脂多糖 能量代谢 

分 类 号：R741.02[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]