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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 detect whether connective tissue growth factor (CTGF) induces Fractalkine production in mesangial cells. To determine whether lipoxin A4 (LXA4) regulates the effect of CTGF on the synthesis of Fractalkine and to explore its mechanism. Methods: The cultured rat glomerular mesangial cells were stimulated with CTGF. The expression of Fractalkine mRNA was detected by reverse transcription polymerase chain reaction (RT - PCR). The expression of Fractalkine in the supernatant was detected by enzyme - linked immunosorbent assay (ELISA). Chemotaxis assays were performed on the chemotaxis of monocytes (THP-1) using a chemotaxis assay. Western blot was used to detect the expression of p42 / 44mitogen-activatedprotein kinase (p42 / 44MAPK), phosphoinositide 3-kinase (PI3-K) and protein kinase B (PKB). The electrophoretic mobility shift assay was used to determine nuclear factor-κB (NF-κB). Results: CTGF stimulation increased the expression and secretion of Fractalkine mRNA in mesangial cells and increased the expressions of phosphorylated p42 / 44 MAPK, P-PI3-K, P-PKB and NF-κB. P-p42 / 44 MAPK inhibitor PD98059 inhibits CTGF-induced phosphorylation of p42 / p44 MAPK and secretion of Fractalkine. The PI3-K inhibitor LY294002 inhibits CTGF-induced PI3-K, PKB, NF-κB activation and Fractalkine secretion. NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) inhibits CTGF-induced NF-κB activation and Fractalkine secretion. LXA4 dose-dependently inhibited the above changes caused by CTGF. Conclusion: LXA4 can inhibit the secretion of Fractalkine induced by mesangial cells induced by CTGF. The mechanism may depend on the inhibition of the phosphorylation of p42 / p44 MAPK and PI3-K / PKB and the activation of NF-κB.