抑制核因子-κB活性对TNF-α诱导的肾小球系膜细胞血管紧张素原表达的影响

Effect of inhibiting nuclear factor-kappa B on TNF-α-induced expression of angiotensinogen and production of angiotensinⅡ in glomerular mesangial cells

目的:研究抑制核因子-κB(NF-κB)活性对肿瘤坏死因子-α(TNF-α)诱导的SD大鼠肾小球系膜细胞血管紧张素原表达及血管紧张素Ⅱ产生的影响。方法:分离SD大鼠肾小球系膜细胞分为下列3组:对照组,TNF-α处理组,TNF-α+NF-κB抑制剂吡咯烷二硫氨基甲酸酯(PDTC)处理组(TNF-α+PDTC处理组)。以电泳迁移率变动分析法(electrophoretic mobility shift assay,EMSA)检测NF-κB活性,放射免疫分析法检测培养液血管紧张素Ⅱ水平,RT-PCR方法检测血管紧张素原mRNA表达,蛋白质印迹法(Western blotting)检测血管紧张素原蛋白表达。结果:TNF-α处理组NF-κB活性[(20.67±9.14)×102μg/cell]显著高于对照组[(8.25±4.35)×102μg/cell,P<0.01]与TNF-α+PDTC处理组[(7.20±4.57)×102μg/cell,P<0.01],TNF-α+PDTC处理组与对照组比较无显著差异。血管紧张素原mRNA表达TNF-α处理组(0.27±0.05)显著高于对照组(0.20±0.05,P<0.05),与TNF-α+PDTC处理组(0.22±0.06,P>0.05)比较无显著差异;蛋白表达TNF-α处理组[(0.60±0.19)μg/cell]显著高于对照组[(0.37±0.15)μg/cell,P<0.05]及TNF-α+PDTC处理组[(0.37±0.17)μg/cell,P<0.05],TNF-α+PDTC处理组与对照组比较无显著差异(P>0.05)。培养液血管紧张素Ⅱ水平在TNF-α处理组[(9.73±2.38)×10-5ng.L-1/cell]显著高于对照组[(7.50±1.51)×10-5ng.L-1/cell,P<0.05]及TNF-α+PDTC处理组[(6.94±1.46)×10-5ng.L-1/cell,P<0.05],TNF-α+PDTC处理组与对照组比较无显著差异(P>0.05)。结论:TNF-α可激活SD大鼠肾小球系膜细胞NF-κB,并诱导血管紧张素原表达及血管紧张素Ⅱ产生增加;抑制NF-κB活性可降低血管紧张素原表达及血管紧张素Ⅱ产生。结果提示NF-κB介导TNF-α诱导的肾小球系膜细胞血管紧张素原表达及血管紧张素Ⅱ产生。

AIM ： To study the effect of inhibiting nuclear factor - kappa B （ NF -κB） activity on the expression of angiotensinogen （AGT） and the production of angiotensin Ⅱ （ Ang Ⅱ） induced by tumor necrosis factor - α （ TNF -α） in glomerular mesangial ceils （MCs） of SD rats. METHODS： The MCs of SD rats were isolated and divided into three groups as follows ： control ; MCs treated with TNF - α, and the MCs treated with TNF - α ＋ pyrrolidinedithiocarbam- ate （PDTC）. The activity of nuclear factor - kappa B was measured by electrophoretic mobility shift assay. The expression of AGT was determined by RT - PCR for mRNA and Western blotting for protein. The concentration of angiotensin Ⅱ in supernatant was measured by RIA. RESULTS. The NF - κB activity in the MCs treated with TNF - α （20.67± 9. 14） ×10^2 μg/cell was significantly higher than that in control cells [ （8. 25 ±4. 35） × 10^2 μg/cell, P 〈0. 01 ] and the MCs treated with TNF - α ＋ PDTC [ （ 7. 20 ± 4. 57 ）× 10^2 μg/cell, P 〈 0. 01 ], and no significant difference was found between control and the MCs treated with TNF - α ＋ PDTC （ P 〉 0. 05 ）. The AGT mRNA level in the MCs treated with TNF - α （0. 27 ± 0. 05 ） was higher than that in the control cells （0. 20±0. 05, P 〈 0. 05 ）, and no significant difference was observed when compared with that in the MCs treated with TNF - α ＋ PDTC （0. 22 ± 0. 06, P 〉 0. 05 ）. The expression of AGT protein in the MCs treated with TNF -α （0. 60 ±0. 19） μg/cell was higher than that in the control [ （0. 37 ±0. 15）μg/cell, P 〈 0. 05 ] and the MCs treated with TNF - α＋ PDTC [ （0. 37 ± 0. 17） μg/cell, P 〈 0. 05 ], and no significance was found between the MCs treated TNF - α ＋ PDTC and the control （P 〉 0. 05）. The Ang Ⅱ level in supernatant of cultured MCs treated with TNF - α [ （9.73 ±2. 38） × 10^-5 ng. L^-1/cell] was significantly higher than that in the control [ （7.50 ± 1.51 ） × 10^-5 ng. L^-1/cell, P 〈 0. 05 ] and in the MCs treated with TNF - α ＋ PDTC [ （6. 94 ±1.46） ×10^-5 ng . L^-1/cell, P 〈0. 05], however, the difference between the MCs treated with TNF-α ＋ PDTC and the control was of no significance （P 〉 0. 05）. CONCLUSION ： TNF - α activates the NF - κB in glomerular MCs, induces the AGT expression and the production of Ang Ⅱ. Inhibition of NF - κB decreases the AGT expression and the production of Ang Ⅱ. Therefore, the effects of TNF - α on AGT and Ang Ⅱ may be mediated by NF - κB.