巨噬细胞雄激素受体通过调控白细胞介素1β基因表达影响高磷诱发的血管平滑肌细胞钙化  

Interleukin-1βmediates the effect of macrophage androgen receptor on hyperphosphate-induced vascular smooth muscle cell calcification

作　　者：庞海燕[1] 卢志[1] 肖龙飞 陈海燕[1] 尚芝群[2] 蒋宁[2] 王晓娟 魏芳[1] 姜埃利[1] 王林[3] 牛远杰[2] Pang Haiyan;Lu Zhi;Xiao Longfei;Chen Haiyan;Shang Zhiqun;Jiang Ning;Wang Xiaojuan;Wei Fang;Jiang Aili;Wang Lin;Niu Yuanjie(Kidney Diseases and Blood Purification Center,the Second Hospital of Tianjin Medical University,Tianjin 300211,China;Department of Urology,the Second Hospital of Tianjin Medical University,Tianjin 300211,China;Department of Geriatrics,the Second Hospital of Tianjin Medical University,Tianjin 300211,China)

机构地区：[1]天津医科大学第二医院肾脏病血液净化科,天津300211 [2]天津医科大学第二医院泌尿外科,天津300211 [3]天津医科大学第二医院老年病科,天津300211

出　　处：《中华肾脏病杂志》2022年第5期420-427,共8页Chinese Journal of Nephrology

基　　金：天津市自然科学基金(19JCYBJC24900)

摘　　要：目的探讨巨噬细胞雄激素受体(androgen receptor,AR)是否通过调控白细胞介素(interleukin,IL)-1β基因表达影响高磷诱发的血管平滑肌细胞钙化。方法采用人单核细胞系THP-1细胞,利用染色质免疫沉淀实验检测AR是否与IL-1β启动子的雄激素受体元件(androgen receptor element,ARE)序列结合,通过荧光素酶分析实验检测AR是否调控IL-1β基因表达。基因沉默THP-1细胞的AR,用携带载体或shRNA的慢病毒转染THP-1细胞,流式细胞术分选出带荧光标记的阳性转染细胞THP-1ARsc(对照组)和THP-1ARsi(沉默单核细胞AR),Western印迹法检测THP-1ARsc、THP-1ARsi细胞的AR表达水平,通过佛波酯(50 ng/ml)诱导获得巨噬细胞MФARsc(对照组)或MФARsi(沉默巨噬细胞AR),酶联免疫吸附试验检测MФARsc或MФARsi条件培养基的IL-1β表达水平。用MФARsc或MФARsi的条件培养基加入磷酸盐(磷酸二氢钠,终浓度2.5 mmol/L)后对人主动脉平滑肌细胞(human aortic smooth muscle cells,HASMC)进行培养,茜素红S染色分析HASMC的钙化情况,Western印迹法检测成骨细胞标志物RUNX2和HASMC标志物SM22α的表达水平;中和实验分析IL-1β介导巨噬细胞AR对HASMC钙化的影响。结果AR与IL-1β启动子的ARE序列结合并调控IL-1β基因表达。与MФARsc细胞比较,MФARsi细胞条件培养基的IL-1β蛋白表达水平显著下降(P<0.001)。与MФARsc条件培养基比较,MФARsi条件培养基HASMC钙沉积显著减少,RUNX2蛋白表达下降而SM22α蛋白表达增多(均P<0.05);MФARsi条件培养基+IgG抗体组较MФARsc条件培养基+IgG抗体组HASMC钙化显著抑制,MФARsc条件培养基+IL-1β抗体组较MФARsc条件培养基+IgG抗体组HASMC钙化显著抑制,而MФARsi条件培养基+IgG抗体组和MФARsi条件培养基+IL-1β抗体组均较MФARsc条件培养基+IL-1β抗体组HASMC钙化抑制程度更大(均P<0.05)。结论巨噬细胞AR通过与IL-1β启动子内的ARE序列结合调控IL-1β的表达,IL-1β介导巨噬细胞AR对�Objective To investigate whether it is by regulating interleukin 1β(IL-1β)gene expression that androgen receptor(AR)in macrophages affects hyperphosphate-induced vascular smooth muscle cell calcification.Methods The chromatin immunoprecipitation(ChIP)experiment was used to determine whether AR was bound to the androgen receptor element(ARE)sequence of IL-1βpromoter in THP-1 cells.Whether the AR regulated IL-1βgene expression was detected by luciferase assay experiments.AR of THP-1 cells was silenced and transfected by lentivirus with vector or shRNA.Flow cytometry was used to select positive transfected cells THP-1ARsc(control)and THP-1ARsi(AR silencing)with fluorescent markers.Western blotting was used to detect AR protein levels of THP-1ARsc(control)and THP-1ARsi cells(AR silencing in monocytes).Macrophages MФARsc(control)or MФARsi(AR silencing)were induced by 50 ng/ml phorbol ester.Enzyme-linked immunosorbent assay was used to detect IL-1βexpression levels of MФARsc or MФARsi conditioned medium.The human aortic smooth muscle cells(HASMC)were cultured in MФARsc or MФARsi conditioned medium with phosphate(2.5 mmol/L final concentration of sodium dihydrogen phosphate),and Alizarin red S staining was used to analyze HASMC calcification degree.Western blotting was used to detect the expression levels of RUNX2(osteoblast marker)and SM22α(HASMC marker),and neutralization assay was performed to test IL-1β-mediating effect of macrophages AR on HASMC calcification.Results AR was bound to ARE sequence of IL-1βpromoter and regulated IL-1βgene expression.The expression level of IL-1βprotein in conditioned medium of MФARsi cells decreased significantly compared to MФARsc cells(P<0.001).Compared with MФARsc conditioned medium group,HASMC calcium deposition in MФARsi conditioned medium group decreased significantly,RUNX2 protein decreased and SM22αprotein increased(all P<0.05).The degree of HASMC calcification in the MФARsi conditioned medium+IgG antibody group decreased than that in the MФARsc conditio

关 键 词：受体 雄激素 巨噬细胞 肌细胞 平滑肌 白细胞介素1Β 炎症 

分 类 号：R692[医药卫生—泌尿科学]