中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2015年
6期
440-450
,共11页
周敏%郭银凤%宋志霞%张晓良
週敏%郭銀鳳%宋誌霞%張曉良
주민%곽은봉%송지하%장효량
骨化三醇%PPARγ%巨噬细胞%表型转换%高糖%VDR-PPARγ通路
骨化三醇%PPARγ%巨噬細胞%錶型轉換%高糖%VDR-PPARγ通路
골화삼순%PPARγ%거서세포%표형전환%고당%VDR-PPARγ통로
Calcitriol%PPAR gama%Macrophages%Phenotype switch%High glucose%VDR-PPARγ pathway
目的 探讨1,25(OH)2D3对高糖环境下巨噬细胞活化状态的调节作用及其信号转导机制.方法 体外培养小鼠巨噬细胞系(RAW264.7),检测细胞内诱导型一氧化氮合酶(iNOS)活力.10-8 mol/L 1,25 (OH)2D3干预高糖预处理的巨噬细胞,分别检测巨噬细胞亚型M1标志物iNOS、肿瘤坏死因子(TNF)α、白细胞介素(IL)12和M2标志物甘露糖受体(MR)、精氨酸酶1(Arg-1)、IL-10的表达以及维生素D受体(VDR)和过氧化物酶体激活物增殖受体γ(PPARγ)的水平.然后用VDR siRNA和PPARγ拮抗剂分别沉默和阻断相应受体后,再次观察活性维生素D对上述M1和M2各标志物的影响.结果 细胞内iNOS活力呈葡萄糖浓度和时间依赖性表达增加,并且在25 mmol/L葡萄糖刺激细胞24h后iNOS活力达到最大.与对照组相比,25 mmol/L葡萄糖干预RAW 264.7细胞24 h后,不仅上清液中炎性细胞因子TNF-α、IL-12表达增多,实时荧光定量PCR和Western印迹结果显示M1标志物iNOS也表达上调(P＜0.05),而M2标志物MR和Arg-1的表达则显著下降(P＜0.05).1,25(OH)2D3干预后,上述趋势被逆转:M1标志物包括TNF-α、IL-12和iNOS的表达明显减少(P＜0.05),而M2标志物IL-10、Arg-1和MR则表达增加(P＜0.05),并且核受体VDR和PPARγ也显著增多(P＜0.05).进一步研究发现,用VDR siRNA和PPARγ拮抗剂阻断相应受体后,活性维生素D的上述作用均被阻断,而沉默VDR后PPARγ表达也相应减少(P＜0.05).结论 1,25(OH)2D3能促使高糖诱导的经典活化巨噬细胞(M1)向替代活化巨噬细胞(M2)转变,这一作用是通过VDR-PPARγ信号通路实现的.
目的 探討1,25(OH)2D3對高糖環境下巨噬細胞活化狀態的調節作用及其信號轉導機製.方法 體外培養小鼠巨噬細胞繫(RAW264.7),檢測細胞內誘導型一氧化氮閤酶(iNOS)活力.10-8 mol/L 1,25 (OH)2D3榦預高糖預處理的巨噬細胞,分彆檢測巨噬細胞亞型M1標誌物iNOS、腫瘤壞死因子(TNF)α、白細胞介素(IL)12和M2標誌物甘露糖受體(MR)、精氨痠酶1(Arg-1)、IL-10的錶達以及維生素D受體(VDR)和過氧化物酶體激活物增殖受體γ(PPARγ)的水平.然後用VDR siRNA和PPARγ拮抗劑分彆沉默和阻斷相應受體後,再次觀察活性維生素D對上述M1和M2各標誌物的影響.結果 細胞內iNOS活力呈葡萄糖濃度和時間依賴性錶達增加,併且在25 mmol/L葡萄糖刺激細胞24h後iNOS活力達到最大.與對照組相比,25 mmol/L葡萄糖榦預RAW 264.7細胞24 h後,不僅上清液中炎性細胞因子TNF-α、IL-12錶達增多,實時熒光定量PCR和Western印跡結果顯示M1標誌物iNOS也錶達上調(P＜0.05),而M2標誌物MR和Arg-1的錶達則顯著下降(P＜0.05).1,25(OH)2D3榦預後,上述趨勢被逆轉:M1標誌物包括TNF-α、IL-12和iNOS的錶達明顯減少(P＜0.05),而M2標誌物IL-10、Arg-1和MR則錶達增加(P＜0.05),併且覈受體VDR和PPARγ也顯著增多(P＜0.05).進一步研究髮現,用VDR siRNA和PPARγ拮抗劑阻斷相應受體後,活性維生素D的上述作用均被阻斷,而沉默VDR後PPARγ錶達也相應減少(P＜0.05).結論 1,25(OH)2D3能促使高糖誘導的經典活化巨噬細胞(M1)嚮替代活化巨噬細胞(M2)轉變,這一作用是通過VDR-PPARγ信號通路實現的.
목적 탐토1,25(OH)2D3대고당배경하거서세포활화상태적조절작용급기신호전도궤제.방법 체외배양소서거서세포계(RAW264.7),검측세포내유도형일양화담합매(iNOS)활력.10-8 mol/L 1,25 (OH)2D3간예고당예처리적거서세포,분별검측거서세포아형M1표지물iNOS、종류배사인자(TNF)α、백세포개소(IL)12화M2표지물감로당수체(MR)、정안산매1(Arg-1)、IL-10적표체이급유생소D수체(VDR)화과양화물매체격활물증식수체γ(PPARγ)적수평.연후용VDR siRNA화PPARγ길항제분별침묵화조단상응수체후,재차관찰활성유생소D대상술M1화M2각표지물적영향.결과 세포내iNOS활력정포도당농도화시간의뢰성표체증가,병차재25 mmol/L포도당자격세포24h후iNOS활력체도최대.여대조조상비,25 mmol/L포도당간예RAW 264.7세포24 h후,불부상청액중염성세포인자TNF-α、IL-12표체증다,실시형광정량PCR화Western인적결과현시M1표지물iNOS야표체상조(P＜0.05),이M2표지물MR화Arg-1적표체칙현저하강(P＜0.05).1,25(OH)2D3간예후,상술추세피역전:M1표지물포괄TNF-α、IL-12화iNOS적표체명현감소(P＜0.05),이M2표지물IL-10、Arg-1화MR칙표체증가(P＜0.05),병차핵수체VDR화PPARγ야현저증다(P＜0.05).진일보연구발현,용VDR siRNA화PPARγ길항제조단상응수체후,활성유생소D적상술작용균피조단,이침묵VDR후PPARγ표체야상응감소(P＜0.05).결론 1,25(OH)2D3능촉사고당유도적경전활화거서세포(M1)향체대활화거서세포(M2)전변,저일작용시통과VDR-PPARγ신호통로실현적.
Objective To investigate the effect of 1,25(OH)2D3 on high glucose induced macrophage activation and its underlying signal transduction mechanism.Methods RAW 264.7 cells were used to perform cell culture,the activity of intracellular iNOS was measured.VDR siRNA and PPARγ antagonist pre-treatment with macrophages were done before using 10-8 mol/L1,25(OH)2D3 to intervene high glucose pre-incubated macrophages.M1 markers including iNOS,TNF-α,IL-12,M2 markers including MR,Arg-1,IL-10 and nuclear receptors VDR and PPARγ were separately examined.Results The iNOS activity was increased in a glucose-dose and time dependent manner.Particularly,25 mmol/L glucose at 24 h gave the maximum response.After being treated with 25 mmol/L glucose for 24 h,not only inflammatory cytokines of TNF-α,IL-12 in the supernatant were increased,but quantitative real-time PCR and Western blotting analysis showed iNOS was also up-regulated (P ＜ 0.05).However,M2 markers,i.e.MR and Arg-l were significantly decreased (P ＜ 0.05).When in the presence of 1,25(OH),D3,the trends were reversed:the markers of M1,including TNF-α,IL-12 and iNOS were obviously reduced (P ＜ 0.05),while M2 markers,IL-10,Arg-1 and MR were increased (P ＜ 0.05).In addition,VDR and PPARγ were also increased (P ＜ 0.05).However,the above effects of 1,25 (OH)2D3 were abolished when further inhibited the expression of VDR and PPARγby VDR siRNA and PPARγ antagonist.Besides,accompanied by VDR,PPARγwas also decreased upon the treatment with VDR siRNA (P ＜ 0.05).Conclusion 1,25(OH)2D3 can promote high glucose induced classically activated macrophages (M1) converting to alternatively activated macrophages (M2) and this is achieved through VDR-PPARγ pathway.
