中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2008年
10期
711-717
,共7页
伍军%阳晓%张云芳%张锐%董秀清%范瑾瑾%刘眉%余学清
伍軍%暘曉%張雲芳%張銳%董秀清%範瑾瑾%劉眉%餘學清
오군%양효%장운방%장예%동수청%범근근%류미%여학청
血管紧张素Ⅱ%核转录因子κB%炎症%脂多糖类%Toll样受体4%CD40%腹膜间皮细胞
血管緊張素Ⅱ%覈轉錄因子κB%炎癥%脂多糖類%Toll樣受體4%CD40%腹膜間皮細胞
혈관긴장소Ⅱ%핵전록인자κB%염증%지다당류%Toll양수체4%CD40%복막간피세포
Angiotensin Ⅱ%NF-kappa B%Inflammation%Lipopelysaceharides%Toll-like receptor 4%CD40%Peritoneal mesothelial cells
目的 观察血管紧张素Ⅱ(AngⅡ)对原代培养大鼠腹膜间皮细胞(RPMC)Toll样受体4(TLR4)表达的影响及其在脂多糖(LPS)诱导的核转录因子κB(NF-κB)活化及CD40表达中的作用.方法 分离及培养RPMC.用不同浓度AngⅡ(10-9、10-8、10-7、10-6mol/L)刺激细胞及用10-7mol/L AngⅡ刺激细胞不同时间(mRNA为1、2、4、8、12、24、48 h和蛋白为6、12、24、36、48 h),观察血管紧张素1型受体(ATIR)阻滞剂洛沙坦(10-5mol/L)和血管紧张素2型受体(AT2R)阻滞剂PDl23177(10-5mol/L)对AngⅡ诱导TLR4表达的影响.将细胞随机分为下列4组:对照组、Ang Ⅱ(10-7mol/L)组、LPS(1 mg/L)组、Ang Ⅱ(10-7mol/L)+LPS(1 mg/L)组.观察Ang Ⅱ对LPS诱导的NF-κB激活和CD40表达的影响.RT-PCR检测TLR4、CD40mRNA表达;Western印迹检测TLR4、IκBα、磷酸化IκBα(P-IκBα)、NF-κB p65、磷酸化NF-κB(p-p65)蛋白表达;免疫荧光检测细胞NF-κB p65亚单位的表达及分布.结果 (1)10-9、10-8、10-7、10-6mol/L Ang Ⅱ作用RPMC 12 h,TLR4 mRNA表达分别增加70.5%、89.5%、102.9%和121.9%;作用24 h TLR4蛋白表达分别增加12.1%、27.7%、51.2%和41.6%.AngⅡ(10-7mol/L)作用RPMC不同时问,TLR4 mRNA表达高峰为8 h和12 h(P(0.01),蛋白表达高峰为12 h和24 h(P<0.01).(2)洛沙坦阻断后,AngⅡ诱导的TLR4表达与未阻断组比较,下调33.5%(P<0.05).PD123177对Ang Ⅱ诱导的TLR4表达无显著影响(P0.05).(3)与正常对照组比较,LPS作用60 min p-IκBα/IκBα、p-p65/p65表达分别上调362.6%(P<0.01)和67.4%(P<0.05);作用4 h CD40 mRNA表达上调299.9%(P<0.01);与LPS组比较,Ang Ⅱ预刺激24h加LPS作用60 min,p-IκBα/IκBα、p-p65/p65表达分别上调49.1%(P<0.01)和29.3%(P<0.05);作用4 h CD40 mRNA表达上调56.8%(P<0.01).(4)免疫荧光结果显示正常对照组与AngⅡ组细胞中,p65信号定位于细胞胞质;LPS作用60 min,p65信号从胞质进入胞核;AngⅡ+LPS组NF-κB p65胞核信号显著增强.结论 Ang Ⅱ呈浓度、时间依赖性诱导RPMC TLR4表达,并显著增强LPS诱导NF-κB激活及CD40的表达.提示腹膜组织局部产生的AngⅡ可能对LPS诱导的腹膜组织炎性反应具有放大作用.
目的 觀察血管緊張素Ⅱ(AngⅡ)對原代培養大鼠腹膜間皮細胞(RPMC)Toll樣受體4(TLR4)錶達的影響及其在脂多糖(LPS)誘導的覈轉錄因子κB(NF-κB)活化及CD40錶達中的作用.方法 分離及培養RPMC.用不同濃度AngⅡ(10-9、10-8、10-7、10-6mol/L)刺激細胞及用10-7mol/L AngⅡ刺激細胞不同時間(mRNA為1、2、4、8、12、24、48 h和蛋白為6、12、24、36、48 h),觀察血管緊張素1型受體(ATIR)阻滯劑洛沙坦(10-5mol/L)和血管緊張素2型受體(AT2R)阻滯劑PDl23177(10-5mol/L)對AngⅡ誘導TLR4錶達的影響.將細胞隨機分為下列4組:對照組、Ang Ⅱ(10-7mol/L)組、LPS(1 mg/L)組、Ang Ⅱ(10-7mol/L)+LPS(1 mg/L)組.觀察Ang Ⅱ對LPS誘導的NF-κB激活和CD40錶達的影響.RT-PCR檢測TLR4、CD40mRNA錶達;Western印跡檢測TLR4、IκBα、燐痠化IκBα(P-IκBα)、NF-κB p65、燐痠化NF-κB(p-p65)蛋白錶達;免疫熒光檢測細胞NF-κB p65亞單位的錶達及分佈.結果 (1)10-9、10-8、10-7、10-6mol/L Ang Ⅱ作用RPMC 12 h,TLR4 mRNA錶達分彆增加70.5%、89.5%、102.9%和121.9%;作用24 h TLR4蛋白錶達分彆增加12.1%、27.7%、51.2%和41.6%.AngⅡ(10-7mol/L)作用RPMC不同時問,TLR4 mRNA錶達高峰為8 h和12 h(P(0.01),蛋白錶達高峰為12 h和24 h(P<0.01).(2)洛沙坦阻斷後,AngⅡ誘導的TLR4錶達與未阻斷組比較,下調33.5%(P<0.05).PD123177對Ang Ⅱ誘導的TLR4錶達無顯著影響(P0.05).(3)與正常對照組比較,LPS作用60 min p-IκBα/IκBα、p-p65/p65錶達分彆上調362.6%(P<0.01)和67.4%(P<0.05);作用4 h CD40 mRNA錶達上調299.9%(P<0.01);與LPS組比較,Ang Ⅱ預刺激24h加LPS作用60 min,p-IκBα/IκBα、p-p65/p65錶達分彆上調49.1%(P<0.01)和29.3%(P<0.05);作用4 h CD40 mRNA錶達上調56.8%(P<0.01).(4)免疫熒光結果顯示正常對照組與AngⅡ組細胞中,p65信號定位于細胞胞質;LPS作用60 min,p65信號從胞質進入胞覈;AngⅡ+LPS組NF-κB p65胞覈信號顯著增彊.結論 Ang Ⅱ呈濃度、時間依賴性誘導RPMC TLR4錶達,併顯著增彊LPS誘導NF-κB激活及CD40的錶達.提示腹膜組織跼部產生的AngⅡ可能對LPS誘導的腹膜組織炎性反應具有放大作用.
목적 관찰혈관긴장소Ⅱ(AngⅡ)대원대배양대서복막간피세포(RPMC)Toll양수체4(TLR4)표체적영향급기재지다당(LPS)유도적핵전록인자κB(NF-κB)활화급CD40표체중적작용.방법 분리급배양RPMC.용불동농도AngⅡ(10-9、10-8、10-7、10-6mol/L)자격세포급용10-7mol/L AngⅡ자격세포불동시간(mRNA위1、2、4、8、12、24、48 h화단백위6、12、24、36、48 h),관찰혈관긴장소1형수체(ATIR)조체제락사탄(10-5mol/L)화혈관긴장소2형수체(AT2R)조체제PDl23177(10-5mol/L)대AngⅡ유도TLR4표체적영향.장세포수궤분위하렬4조:대조조、Ang Ⅱ(10-7mol/L)조、LPS(1 mg/L)조、Ang Ⅱ(10-7mol/L)+LPS(1 mg/L)조.관찰Ang Ⅱ대LPS유도적NF-κB격활화CD40표체적영향.RT-PCR검측TLR4、CD40mRNA표체;Western인적검측TLR4、IκBα、린산화IκBα(P-IκBα)、NF-κB p65、린산화NF-κB(p-p65)단백표체;면역형광검측세포NF-κB p65아단위적표체급분포.결과 (1)10-9、10-8、10-7、10-6mol/L Ang Ⅱ작용RPMC 12 h,TLR4 mRNA표체분별증가70.5%、89.5%、102.9%화121.9%;작용24 h TLR4단백표체분별증가12.1%、27.7%、51.2%화41.6%.AngⅡ(10-7mol/L)작용RPMC불동시문,TLR4 mRNA표체고봉위8 h화12 h(P(0.01),단백표체고봉위12 h화24 h(P<0.01).(2)락사탄조단후,AngⅡ유도적TLR4표체여미조단조비교,하조33.5%(P<0.05).PD123177대Ang Ⅱ유도적TLR4표체무현저영향(P0.05).(3)여정상대조조비교,LPS작용60 min p-IκBα/IκBα、p-p65/p65표체분별상조362.6%(P<0.01)화67.4%(P<0.05);작용4 h CD40 mRNA표체상조299.9%(P<0.01);여LPS조비교,Ang Ⅱ예자격24h가LPS작용60 min,p-IκBα/IκBα、p-p65/p65표체분별상조49.1%(P<0.01)화29.3%(P<0.05);작용4 h CD40 mRNA표체상조56.8%(P<0.01).(4)면역형광결과현시정상대조조여AngⅡ조세포중,p65신호정위우세포포질;LPS작용60 min,p65신호종포질진입포핵;AngⅡ+LPS조NF-κB p65포핵신호현저증강.결론 Ang Ⅱ정농도、시간의뢰성유도RPMC TLR4표체,병현저증강LPS유도NF-κB격활급CD40적표체.제시복막조직국부산생적AngⅡ가능대LPS유도적복막조직염성반응구유방대작용.
Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) on the expression of TLR4 and its role in lipopolysaccharide (LPS)-induced NF-κB activation and CD40 expression in rat peritoneal mesothelial cells (RPMCs). Methods RPMCs were harvested from Spragne-Dawley rat peritoneal cavity and maintained under defined in vitro condition. The cells were treated with Ang Ⅱ at different concentrations (10-9, 10-8, 10-7, 10-6 mol/L) and exposed to Ang Ⅱ (10-7 mol/L) for different times (1, 2, 4, 8, 12, 24, 48 h for mRNA and 6, 12, 24, 36, 48 h for protein, respectively). Meanwhile, the influence of AT1 receptor antagonist (AT1R, losartan, 10-5 mol/L) and AT2 receptor blocker (AT2R, PD123177, 10-5 mol/L) on the TLR4 induced by Ang Ⅱ was observed. After synchronization for 24 hours, the cells were randomly assigned to four groups: the control group, the Ang Ⅱ (10-7 tool/L) group, the LPS (1 mg/L) group, the Ang Ⅱ (10-7 mol/L) plus LPS (1 mg/L) group, which were used to investigate the effects of Ang Ⅱ on the NF-κB activation and CD40 expression induced by LPS. The mRNA expression of TLR4 and CD40 was measured by RT-PCR and the protein abundance of TLR4, NF-κB p65, phospho-p65, IKBα and phospho-IκBα were analyzed by Western blot. Immunofluorescence was performed to determine the subcellular localization of p65 subunit of NF-κB. Results (1) Treatment of RPMCs with Ang Ⅱ resulted in a concentration-dependent increase in the expression of TLR4. Ang Ⅱ at 10-9, 10-8, 10-7 and 10-6 mol/L increased TLR4 mRNA expression by 70.5%, 89.5%, 102.9%, and 121.9%, respectively and protein expression by 12.1%, 27.7%, 51.2%, and 41.6%, respectively (P<0.01). Treatment of RPMCs with 10-7 mol/L Ang Ⅱ resulted in a time-dependent increase in the expression of TLR4, with the peak of mRNA expression at 8 and 12 h (P<0.01) and the protein expression at 12 and 24 h (P<0.01). (2) Losartan antagonized Ang Ⅱ-stimulated expression of TLR4 by 33.5% (P<0.05), PD123177 had no such effect (P0.05). (3) Treatment of RPMCs with LPS (1 mg/L) for 60 rain significantly increased the ratio of phospho-IκBα to IκBα by 362.6% (P< 0.01) , phospho-p65 to p65 by 67.4% (P<0.05), and LPS (1 mg/L) for 4 h significantly increased the expression of CD40 mRNA by 299.9% (P<0.01) compared to the control group. In comparison to the LPS (1 mg/L) group, preincubation of RPMCs with AngⅡ (10-7 mol/L) for 24 h then treated with LPS (1 mg/L) for 60 rain significantly increased the ratio of phospho-IκBα to IκBα by 49.1% (P<0.01), phospho-p65 to p65 by 29.3%(P<0.05), and LPS (1 mg/L) for 4 h significantly increased the expression of CD40 mRNA by 56.8%(P<0.01). (4) The p65 subunit of NF-κB was dominantly distributed in the cytoplasm in the control and Ang Ⅱ group. Following exposure to LPS for 60 min, p65 subunit labeling was upregulated and translocated into the nuclei. A significantly increased nuclear staining of p65 in ceils treated with Ang Ⅱ plus LPS were observed. Conclusions Ang Ⅱ induces the expression of TLR4 in dose- and time-dependent manner in RPMCs, resulting in enhanced NF-κB signaling and induction of CD40 expression, Locally produced Ang Ⅱ in the peritoneum may play an amplified role in LPS-induced peritoneal inflammation.
