凝集素样氧化型低密度脂蛋白受体1通过β-连环蛋白/激活转录因子6α通路对高糖诱导的小鼠小胶质细胞吞噬功能影响的研究  

LOX-1 promotes hyperglycemia-induced phagocytosis dysfunction of BV2 microglia through the β-catenin/ATF6α pathway

作　　者：张雅娟 刘静[2] 田利民[2] 张娜 张延燕 刘亚倩 司惠玲 赵文淑 仇菊梅 张琦[2] ZHANG Yajuan;LIU Jing;TIAN Limin(The First School of Clinical Medical of Gansu University of Chinese Medicine,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学第一临床医学院,兰州730000 [2]甘肃省人民医院老年医学科

出　　处：《中国糖尿病杂志》2024年第6期450-457,共8页Chinese Journal of Diabetes

基　　金：国家自然科学基金(82160166、81960173);甘肃省重点研发计划(22YF7FA096);甘肃省人民医院院内科研基金(22GSSYA-1);兰州市人才创新创业项目(2021-RC-136);兰州市卫生健康科技发展项目(2021005);甜蜜医生培育项目(2021SD01)。

摘　　要：目的 探讨凝集素样氧化型低密度脂蛋白受体1(LOX-1)通过β-连环蛋白(β-catenin)/激活转录因子6α(ATF6α)通路,参与调控高糖(HG)诱导的小鼠小胶质(BV2)细胞吞噬功能障碍的分子机制。方法 体外培养BV2细胞,构建慢病毒LOX-1RNAi载体(LV-LOX-1)及慢病毒空载体(LV-Con),分为正常对照(NC)组、HG组、LV-LOX-1组和LV-Con组。LV-LOX-1及LV-Con感染BV2细胞后,用HG(25 mmol/L葡萄糖)培养24 h后,记为HG+LV-LOX-1组和HG+LV-Con组,分别使用15μmol/L的β-catenin抑制剂(FH535)和ATF6α抑制剂(AEBSF)处理HG+LV-LOX-1及HG+LV-Con感染的BV2细胞24 h后,记为HG+LV-LOX-1+FH535组、HG+LV-Con+FH535组、HG+LV-LOX-1+AEBSF组、HG+LV-Con+AEBSF组。荧光显微镜、RT-PCR和Western blot判断转染效率。CCK-8检测细胞活力,RT-PCR和Western blot检测各组LOX-1、β-catenin、ATF6α、乳脂肪球表面生长因子Ⅷ(MFG-E8)m RNA及蛋白表达。结果 LV-LOX-1重组体感染细胞72 h后,LV-LOX-1、LV-Con组细胞出现大量绿色荧光,NC组细胞无绿色荧光。与NC组比较,HG组LOX-1、ATF6α mRNA和蛋白表达升高(P<0.05),MFG-E8、β-catenin mRNA和蛋白表达降低(P<0.05)。与HG+LV-Con组比较,HG+LV-LOX-1组MFG-E8、β-catenin mRNA和蛋白表达升高(P<0.05),LOX-1、ATF6α mRNA和蛋白表达降低(P<0.05)。与HG+LV-LOX-1组比较,HG+LV-LOX-1+FH535组ATF6α mRNA和蛋白及p-β-catenin、p-ATF6α蛋白表达升高(P<0.05),MFG-E8、β-catenin mRNA和蛋白表达降低(P<0.05)。与HG+LV-LOX-1组比较,HG+LV-LOX-1+AEBSF组MFG-E8m RNA和蛋白表达升高(P<0.05),ATF6α mRNA和蛋白及p-ATF6α蛋白表达降低(P<0.05)。结论 LOX-1、MFG-E8、β-catenin、ATF6α参与调控BV2细胞吞噬功能,LOX-1通过β-catenin/ATF6α信号通路促进HG诱导的BV2细胞吞噬功能障碍。Objective To investigate the molecular mechanism of lectin-like oxidized low-density lipoprotein receptor 1(LOX-1) in the regulation of high glucose induced phagocytosis dysfunction of mouse microglia(BV2 microglia).Methods BV2 cells were cultured in vitro,lentivirus LOX-1RNAi vector(LV-LOX-1) and lentivirusempty vector(LV-Con) were constructed and divided into normal control(NC) group,HG group,LV-LOX-1 group and LV-Con group.After infecting BV2 cells with LV-LOX-1 and LV-Con,the cells were cultured with 25 mmol/L glucose for 24 h,and then divided into HG+LV-LOX-1group and HG+LV-Con group.After treatment of HG+LV-LOX-1 and HG+LV-Con infected BV2microglia with 15 μmol/L FH535(β-catenin inhibitor) and AEBSF(ATF6α inhibitor) for 24 h,respectively,they were denoted as HG+LV-LOX-1+FH535 group,HG+LV-Con+FH535 group,HG+LV-LOX-1+AEBSF group,and HG+LV-Con+AEBSF group.Transfection efficiency was determined by fluorescence microscopy,RT-PCR and Western blot.Cell viability was detected b CCK-8.RT-PCR and Western blot were used to detect the m RNA and protein expression of LOX-1,β-catenin,ATF6α and milk fat globular-surface growth factor Ⅷ (MFG-E8) in each group.Results After 72 h of LV-LOX-1 infection,the cells in LV-LOX-1 and LV-Con groups showed a lot of green fluorescence,but not in NC group.Compared with NC group,the m RNA and protein expression of LOX-1 and ATF6α were increased(P<0.05),while the mRNA and protein expression of MFG-E8 and β-catenin decreased in HG group(P<0.05).Compared with HG+LV-Con group,the mRNA and protein expression of LOX-1 and ATF6α were decreased(P<0.05),while the m RNA and protein expression of MFG-E8 and β-catenin increasedin HG+LV-LOX-1 group(P<0.05).Compared with HG+LV-LOX-1 group,the mRNA and protein expressions of MFG-E8 and β-catenin were decreased(P<0.05),and the m RNA and protein expressions of ATF6α and p-β-catenin and p-ATF6α were increased in HG+LV-LOX-1+FH535 group(P<0.05).Compared with HG+LV-LOX-1 group,the m RNA and protein expression were increased(P<0.05),ATF6α m

关 键 词：凝集素样氧化型低密度脂蛋白受体1 乳脂肪球表皮生长因子Ⅷ 吞噬功能 小鼠小胶质细胞 

分 类 号：R587.2[医药卫生—内分泌] R749.2[医药卫生—内科学]