脂氧素A_4抑制结缔组织生长因子诱导的系膜细胞产生Fractalkine

Lipoxin A_4 inhibits CTGF-induced production of fractalkine in mesangial cells

目的:检测结缔组织生长因子(connectivetissuegrowthfactor,CTGF)是否诱导肾小球系膜细胞产生Fractalkine;检测脂氧素A4(lipoxinA4,LXA4)是否调节CTGF对合成Fractalkine的作用,并探讨其作用机制。方法:应用CTGF刺激培养的大鼠肾小球系膜细胞,应用逆转录多聚酶链反应测定FractalkinemRNA表达,应用酶联免疫吸附试验测定上清液中Fractalkine蛋白表达。应用趋化试验测定上清液对单核细胞(THP-1)的趋化作用。应用Westernblot测定分裂原激活的蛋白激酶(p42/44mitogen-activatedproteinkinase,p42/44MAPK)、磷脂酰肌醇3-激酶(phosphoinositide3-kinase,PI3-K)、蛋白激酶B(proteinkinaseB,PKB)。应用凝胶电泳迁移率试验测定核因子-κB(nuclearfactor-κB,NF-κB)。结果:CTGF刺激使系膜细胞FractalkinemRNA表达与分泌量增加,增加磷酸化p42/44MAPK、P-PI3-K、P-PKB及NF-κB表达。P-p42/44MAPK抑制剂PD98059抑制CTGF诱导的p42/p44MAPK磷酸化与Fractalkine分泌。PI3-K抑制剂LY294002抑制CTGF诱导的PI3-K、PKB、NF-κB活化与Fractalkine分泌。NF-κB抑制剂吡咯烷二硫氨基甲酸酯(pyrrolidinedithio-carbamate,PDTC)抑制CTGF诱导的NF-κB活化与Fractalkine分泌。LXA4呈剂量依赖性地抑制CTGF所致的上述变化。结论:LXA4可抑制CTGF引起的系膜细胞分泌Fractalkine,其机制依赖于抑制p42/p44MAPK、PI3-K/PKB的磷酸化与NF-κB活化。

Objective： To determine the regulatory role of connective tissue growth factor （CTGF） on production of fraetalkine in rat glomerular mesangial cells, and the modulating effect of lipoxin A4 （LXA4） on action of CTGF, and to explore the mechanisms of action of CTGF and LXA4. Methodology： Cultured rat mesangial cells were treated with CTGF, with or without pre-incubation with LXA4, The expression of mRNA was analyzed by reverse transcription polymerase chain reaction （RT-PCR）, The protein levels of fractalkine in the supernatants were determined by enzyme-linked immunosorbent assay （ELISA）. The monocyte transmigration was assessed by in vitro ehemotaxis assay. The expression of p42/44 mitogen-activated protein kinase （MAPK）, phosphoinositide 3-kinase （PI3-K） and protein kinase B （PKB） was assessed by Western blotting, The activity of nuclear factor-κB （NF-κB） was determined by electrophroretic mobility shift assay, Results： CTGF enhanced the mRNA expression and protein production of fractalkine, the expression of phospho （P）-p42/44 MAPK, P-PI3-K, P-PKB and NF-κB. P-p42/44 MAPK blockade inhibited the CTGF-induced expression of P-p42/44 MAPK and partially decreased the level of fractalkine in supernatants, P-PI3-K blockade down-regulated the CTGF-stimulated expression of P-PI3-K, P-PKB and NF-κB, and partially decreased the release of fractalkine. NF-κB blockade abrogated the CTGF-activated NF-κB and partially decreased the secretion of fractalkine. LXA4 dose-dependently inhibited the CTGF-stimulated above action, Conclusion： LXA4 inhibits CTGF-induced production of fractalkine via p42/44 MAPK, PI3-K/PKB and NF-κB-dependent signal pathway.