CAJ | 학술논문

构建Toll样受体4(TLR4)shRNA腺病毒表达载体(pGSadeno-TLR4)并感染人主动脉血管平滑肌细胞(HA-VSMC),采用软脂酸和不同的信号通路阻断剂处理HA-VSMC,以实时定量PCR检测白细胞介素6(IL-6) mRNA的表达,酶联免疫吸附法(ELISA)检测NF-κB p65活性和IL-6的蛋白水平.结果显示,软脂酸增加HA-VSMC IL-6 mRNA和蛋白水平,且具有明显的剂量依赖关系,软脂酸(400 μmol/L)处理6h作用最明显,IL-6 mRNA的表达水平为对照组的10.43倍(P＜0.01).IL-6蛋白水平在软脂酸作用24h时达到峰值,约为对照组的2.18倍(P＜0.01).NF-κB阻断剂parthenolide下调软脂酸诱导的IL-6 mRNA和蛋白表达分别为65％和59％(均P＜0.01),蛋白激酶C(PKC)阻断剂白屈菜红碱下调软脂酸诱导的IL-6mRNA和蛋白表达分别为24％和28％(均P＜0.05).而细胞外信号调节激酶抑制剂PD98059和磷酸肌醇3激酶抑制剂wortmannin对软脂酸诱导的IL-6 mRNA和蛋白表达均无明显作用(均P＞0.05).pGSadeno-TLR4下调软脂酸诱导的IL-6 mRNA和蛋白表达分别为72％和75％(均P＜0.01),下调软脂酸刺激的NF-κB p65活性约62％ (P＜0.01).这些结果提示TLR-4/NF-κB和PKC信号通路介导了软脂酸诱导的人主动脉血管平滑肌细胞IL-6基因表达.
구건Toll양수체4(TLR4)shRNA선병독표체재체(pGSadeno-TLR4)병감염인주동맥혈관평활기세포(HA-VSMC),채용연지산화불동적신호통로조단제처리HA-VSMC,이실시정량PCR검측백세포개소6(IL-6) mRNA적표체,매련면역흡부법(ELISA)검측NF-κB p65활성화IL-6적단백수평.결과현시,연지산증가HA-VSMC IL-6 mRNA화단백수평,차구유명현적제량의뢰관계,연지산(400 μmol/L)처리6h작용최명현,IL-6 mRNA적표체수평위대조조적10.43배(P＜0.01).IL-6단백수평재연지산작용24h시체도봉치,약위대조조적2.18배(P＜0.01).NF-κB조단제parthenolide하조연지산유도적IL-6 mRNA화단백표체분별위65％화59％(균P＜0.01),단백격매C(PKC)조단제백굴채홍감하조연지산유도적IL-6mRNA화단백표체분별위24％화28％(균P＜0.05).이세포외신호조절격매억제제PD98059화린산기순3격매억제제wortmannin대연지산유도적IL-6 mRNA화단백표체균무명현작용(균P＞0.05).pGSadeno-TLR4하조연지산유도적IL-6 mRNA화단백표체분별위72％화75％(균P＜0.01),하조연지산자격적NF-κB p65활성약62％ (P＜0.01).저사결과제시TLR-4/NF-κB화PKC신호통로개도료연지산유도적인주동맥혈관평활기세포IL-6기인표체.
The recombinant adenovirus Toll like receptor 4 (TLR4) shRNA vector (pGSadeno-TLR4) was constructed and transfected into human aortic vascular smooth muscle cells (HA-VSMC).After HA-VSMC were treated with palmitate or different signaling pathway inhibitors,the mRNA and protein levels of interleukin-6 (IL-6)and NF-κB activity were tested with real-time PCR and ELISA,respectively.The results showed that palmitate increased mRNA and protein levels of IL-6 in HA-VSMC in a dose-dependent manner.The expression of IL-6 mRNA reached peak after treatment with 400 μmol/L of palmitate for 6 h,being 10.43 fold of control (P＜0.01).Treatment with 400 pmol/L of palmitate for 24 h maximally upregulated the protein level of IL-6,which was 2.18 fold of control (P＜0.01).NF-κB inhibitor parthenolide markedly inhibited palmitate-stimulated increased in IL-6 mRNA level by 65％ and protein level by 59％ (both P＜0.01).Protein kinase C (PKC) inhibitor chlerythrine suppressed palmitateinduced IL-6 mRNA expression by 24％ and IL-6 protein level by 28％.By contrast,extracellular signal-regulated protein kinase inhibitor PD98059 and phosphatidylinositol 3-kinase inhibitor wortmannin had no effect on the induction of IL-6 by palmitate.Blockade of TLR4 with pGSadeno-TLR4 significantly suppressed palmitate-induced IL-6 mRNA expression by 72％ and IL-6 protein expression by 75％ (both P＜0.01),along with decrease of NF-κB p65 activity decreased by 62％.These results suggest that TLR4/NF-κB and PKC pathways mediate palmitate-induced IL-6 expression in HA-VSMC.