中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2009年
6期
430-436
,共7页
张云芳%阳晓%伍军%王雅宁%邹循亮%张锐%刘眉%郭群英%骆宁%董秀清%余学清
張雲芳%暘曉%伍軍%王雅寧%鄒循亮%張銳%劉眉%郭群英%駱寧%董秀清%餘學清
장운방%양효%오군%왕아저%추순량%장예%류미%곽군영%락저%동수청%여학청
NF-κB%脂多糖类%抗原,CD40%胞问黏附分子1%罗格列酮%腹膜间皮细胞
NF-κB%脂多糖類%抗原,CD40%胞問黏附分子1%囉格列酮%腹膜間皮細胞
NF-κB%지다당류%항원,CD40%포문점부분자1%라격렬동%복막간피세포
NF-kappa B%Lipopolysaccharides%Antigens,CD40%Intercellular adhesion molecule 1%Rosiglitazone%Peritoneal mesothelial cells
目的 探讨罗格列酮对脂多糖(LPS)诱导的体外培养大鼠腹膜间皮细胞CD40和胞间黏附分子1(ICAM-1)表达的影响以及调节机制.方法 分离及培养大鼠原代腹膜间皮细胞.将细胞随机分为正常对照组、LPS(5 mg/L)组、BAY11-7085(NF-κB抑制剂)组(5μmol/L预刺激3 h后加入LPS作用3 h)、不同浓度罗格列酮(过氧化物酶体增殖蛋白激活性受体γ配体)组(10、20 μmol/L分别预处理3 h再加入LPS 5 mg/L)、GW9662(过氧化物酶体增殖蛋白激活性受体γ拮抗剂)预处理组(预处理3 h后加入罗格列酮10 μmol/L,3 h后再加入LPS 5 mg/L)和溶媒对照组.加入LPS后1 h收集细胞检测核因子κB(NF-κB)p65水平;3 h收集细胞分别检测CD40和ICAM-1基因表达;24 h收集细胞分别检测CD40和ICAM-1蛋白表达.RT-PCR法检测基因表达;Western印迹和免疫荧光方法检测蛋白表达及核因子磷酸化.结果 (1)常规培养的腹膜间皮细胞表达基础量CD40和ICAM-1,LPS显著上调其表达(P<0.05);LPS作用1 h时腹膜间皮细胞磷酸化NF-κB p65活化水平显著增高,与对照组差异有统计学意义(1.10±0.17比0.55±0.06,P<0.05).(2)NF-κB抑制剂BAY11-7085预处理后LPS诱导的磷酸化NF-κB p65水平、CD40和ICAM-1表达显著低于LPS组(0.22±0.11比1.10±0.17,P<0.01;0.34±0.02比0.50±0.06,P<0.05;0.35±0.16比0.74±0.03,P<0.05).(3)罗格列酮预处理后,LPS诱导的磷酸化NF-κB p65水平、CD40以及ICAM-1蛋白表达亦显著低于LPS组(0.77±0.08比0.90±0.10,P<0.01;0.79±0.16比0.99±0.06,P<0.05;0.83±0.20比1.22±0.13,P<0.05).GW9662和罗格列酮联合预处理后,LPS诱导的磷酸化NF-κB p65水平与罗格列酮预处理组差异无统计学意义,但CD40和ICAM-1表达显著高于罗格列酮预处理组(0.95±0.19比0.79±0.16;1.04+0.24比0.83±0.20,均P<0.05).结论 NF-κB信号通路参与调节LPS诱导的腹膜间皮细胞表达CD40和ICAM-1.罗格列酮通过NF-κB途径下调CD40和ICAM-1表达,从而发挥抗炎作用.
目的 探討囉格列酮對脂多糖(LPS)誘導的體外培養大鼠腹膜間皮細胞CD40和胞間黏附分子1(ICAM-1)錶達的影響以及調節機製.方法 分離及培養大鼠原代腹膜間皮細胞.將細胞隨機分為正常對照組、LPS(5 mg/L)組、BAY11-7085(NF-κB抑製劑)組(5μmol/L預刺激3 h後加入LPS作用3 h)、不同濃度囉格列酮(過氧化物酶體增殖蛋白激活性受體γ配體)組(10、20 μmol/L分彆預處理3 h再加入LPS 5 mg/L)、GW9662(過氧化物酶體增殖蛋白激活性受體γ拮抗劑)預處理組(預處理3 h後加入囉格列酮10 μmol/L,3 h後再加入LPS 5 mg/L)和溶媒對照組.加入LPS後1 h收集細胞檢測覈因子κB(NF-κB)p65水平;3 h收集細胞分彆檢測CD40和ICAM-1基因錶達;24 h收集細胞分彆檢測CD40和ICAM-1蛋白錶達.RT-PCR法檢測基因錶達;Western印跡和免疫熒光方法檢測蛋白錶達及覈因子燐痠化.結果 (1)常規培養的腹膜間皮細胞錶達基礎量CD40和ICAM-1,LPS顯著上調其錶達(P<0.05);LPS作用1 h時腹膜間皮細胞燐痠化NF-κB p65活化水平顯著增高,與對照組差異有統計學意義(1.10±0.17比0.55±0.06,P<0.05).(2)NF-κB抑製劑BAY11-7085預處理後LPS誘導的燐痠化NF-κB p65水平、CD40和ICAM-1錶達顯著低于LPS組(0.22±0.11比1.10±0.17,P<0.01;0.34±0.02比0.50±0.06,P<0.05;0.35±0.16比0.74±0.03,P<0.05).(3)囉格列酮預處理後,LPS誘導的燐痠化NF-κB p65水平、CD40以及ICAM-1蛋白錶達亦顯著低于LPS組(0.77±0.08比0.90±0.10,P<0.01;0.79±0.16比0.99±0.06,P<0.05;0.83±0.20比1.22±0.13,P<0.05).GW9662和囉格列酮聯閤預處理後,LPS誘導的燐痠化NF-κB p65水平與囉格列酮預處理組差異無統計學意義,但CD40和ICAM-1錶達顯著高于囉格列酮預處理組(0.95±0.19比0.79±0.16;1.04+0.24比0.83±0.20,均P<0.05).結論 NF-κB信號通路參與調節LPS誘導的腹膜間皮細胞錶達CD40和ICAM-1.囉格列酮通過NF-κB途徑下調CD40和ICAM-1錶達,從而髮揮抗炎作用.
목적 탐토라격렬동대지다당(LPS)유도적체외배양대서복막간피세포CD40화포간점부분자1(ICAM-1)표체적영향이급조절궤제.방법 분리급배양대서원대복막간피세포.장세포수궤분위정상대조조、LPS(5 mg/L)조、BAY11-7085(NF-κB억제제)조(5μmol/L예자격3 h후가입LPS작용3 h)、불동농도라격렬동(과양화물매체증식단백격활성수체γ배체)조(10、20 μmol/L분별예처리3 h재가입LPS 5 mg/L)、GW9662(과양화물매체증식단백격활성수체γ길항제)예처리조(예처리3 h후가입라격렬동10 μmol/L,3 h후재가입LPS 5 mg/L)화용매대조조.가입LPS후1 h수집세포검측핵인자κB(NF-κB)p65수평;3 h수집세포분별검측CD40화ICAM-1기인표체;24 h수집세포분별검측CD40화ICAM-1단백표체.RT-PCR법검측기인표체;Western인적화면역형광방법검측단백표체급핵인자린산화.결과 (1)상규배양적복막간피세포표체기출량CD40화ICAM-1,LPS현저상조기표체(P<0.05);LPS작용1 h시복막간피세포린산화NF-κB p65활화수평현저증고,여대조조차이유통계학의의(1.10±0.17비0.55±0.06,P<0.05).(2)NF-κB억제제BAY11-7085예처리후LPS유도적린산화NF-κB p65수평、CD40화ICAM-1표체현저저우LPS조(0.22±0.11비1.10±0.17,P<0.01;0.34±0.02비0.50±0.06,P<0.05;0.35±0.16비0.74±0.03,P<0.05).(3)라격렬동예처리후,LPS유도적린산화NF-κB p65수평、CD40이급ICAM-1단백표체역현저저우LPS조(0.77±0.08비0.90±0.10,P<0.01;0.79±0.16비0.99±0.06,P<0.05;0.83±0.20비1.22±0.13,P<0.05).GW9662화라격렬동연합예처리후,LPS유도적린산화NF-κB p65수평여라격렬동예처리조차이무통계학의의,단CD40화ICAM-1표체현저고우라격렬동예처리조(0.95±0.19비0.79±0.16;1.04+0.24비0.83±0.20,균P<0.05).결론 NF-κB신호통로삼여조절LPS유도적복막간피세포표체CD40화ICAM-1.라격렬동통과NF-κB도경하조CD40화ICAM-1표체,종이발휘항염작용.
Objective To investigate the effect and mechanism by which PPARγ ligand, rosiglitasone, regulates the expression of CD40 and intercellular adhesion molecule 1 (ICAM-1) in the rat peritoneal mesothelial cells (RPMCs) induced by lipopolysaccharide (LPS). Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and maintained under defined in vitro conditions. The cells were randomly divided into groups as follows: medium, LPS (5 mg/L), LPS (5 mg/L)+BAY11-7085(5 μmol/L, NF-κB inhibitor), rosiglitazone (10 μmol/L or 20 μmol/L, peroxisome proliferator-activated receptor γ activator), LPS (5 mg/L)+rosiglitazone (10 μmol/L)+GW9662 (3 μmol/L, peroxisome proliferator-aetivatcd receptor γ antagonist), and LPS (5 mg/L)+vehicle (DMSO 0.2 ml/L). The expressions of CD40 and ICAM-1 RNA in RPMCs were examined by RT-PCR after 3 hour treatment, and the protein expressions of CD40, ICAM-1, p-NF-κB p65 and p-IκBα were examined by Western blot or immunofluorescence after 24 hour treatment. Results Following treatment with LPS, both the expressions of CD40 and ICAM-1 protein in RPMCs were up-regulated significantly (P<0.05), and the phosphoralation of p65 was increased greatly (1.10±0.17 vs 0.55±0.06, P<0.05). BAY11-7085 (5 μmol/L) significantly decreased the protein expression of p-p65 (0.22±0.11 vs 1.10±0.17, P<0.01), CD40 (0.34±0.02 vs 0.50±0.06, P<0.05) and ICAM-1 (0.35±0.16 vs 0.74±0.03, P<0.05). Pretreated with rosiglitazone for 3 h then added with LPS for 1 h, the levels of p-p65, CD40 and ICAM-1 in RPMCs were significantly decreased compared with those of LPS group (0.77±0.08 vs 0.90±0.10, P<O.01; 0.79±0.16 vs 0.99±0.06, P<0.05; 0.83±0.20 vs 1.22±0.13, P<0.05). However, for the group pretreated with rosiglitazone(10 μmol/L) and GW9662(3 μmol/L) for 3 h then added with LPS, the levels of p-p65 in RPMCs did not change significantly compared with those of rosiglitazone-pretreated group. The expressions of CD40 and ICAM-1 in RPMCs were significantly increased compared with those of rosiglitazone-pretreated group (0.95±0.19 vs 0.79±0.16, and 1.04±0.24 vs 0.83±0.20, P<0.05). Conclusion Rosiglitazone can decrease LPS-induced expression of CD40 and ICAM-1 in RPMCs by inhibition of NF-κB activation, which suggests that rosiglitazone may mediate its antiinflammatory effect through a NF-κB dependent mechanism.
