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目的:观察中电导钙激活钾离子通道(intermediate-conductance Ca~(~(2+) ) -activated K+channel,K_(Ca)3.1)在软脂酸(palmitic acid,PA)诱导的单核细胞跨内皮迁移中的作用及其调控机制。方法:分离2型糖尿病(type 2 diabetes mellitus,T2DM)患者外周血单核细胞(peripheral blood mononuclear cells,PBMCs)并培养人单核细胞株(THP-1 cells),以PA刺激,通过Western blotting、RT-PCR、ELISA及细胞迁移实验观察PA对PBMCs及THP-1细胞跨内皮迁移的影响及其与K_(Ca)3.1的关系、K_(Ca)3.1与MCP-1之间的关系并探讨其信号转导通路。结果:100μmol/L PA上调体重指数(body mass index,BMI)位于20~27.9 kg/m~2的T2DM患者PBMCs K_(Ca)3.1的表达并促进其跨内皮迁移,对BMI≥28 kg/m~2的T2DM患者PBMCs无影响;K_(Ca)3.1特异性阻滞剂TRAM-34、NF-κB阻滞剂PDTC(100μmol/L)和Bay11-7082(10μmol/L)抑制PA诱导的BMI位于20~27.9 kg/m~2的T2DM患者PBMCs跨内皮迁移;TRAM-34和K_(Ca)3.1特异性si RNA显著减少PA(200μmol/L)诱导的THP-1细胞跨内皮迁移及THP-1细胞中MCP-1的分泌和表达,anti-TLR2/4(4 mg/L)、p38MAPK抑制剂SB203580(10μmol/L)及SB202190(10μmol/L)、PDTC(100μmol/L)和Bay11-7082(10μmol/L)显著减少PA诱导的THP-1细胞中K_(Ca)3.1和MCP-1的表达。结论:PA通过TLR2/4-p38MAPK-NF-κB通路上调K_(Ca)3.1促进MCP-1的表达,进而诱导单核细胞的跨内皮迁移。
OBJECTIVE: To investigate the effect of Ca2 + -activated K + channel (K-channel) on the activation of monocytes induced by palmitic acid (PA) Trans-endothelial migration and its regulatory mechanism. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with type 2 diabetes mellitus (T2DM) and cultured in vitro. PA was stimulated with THP-1 cells, The effect of PA on the trans-endothelial migration of PBMCs and THP-1 cells and the relationship with K (Ca) 3.1, the relationship between K (Ca) 3.1 and MCP-1 were investigated by RT-PCR, ELISA and cell migration assay Signal transduction pathway. Results: 100 μmol / L PA up-regulated body mass index (BMI) expression of K (Ca) 3.1 in PBMCs from 20 to 27.9 kg / m 2 and promoted trans-endothelial migration. ~ 2 in PBMCs of T2DM patients. Inhibition of PA-induced BMI by K (Ca) 3.1 specific blockers TRAM-34, NF-κB blockers PDTC (100μmol / L) and Bay11-7082 (10μmol / L) PBMCs from 20 to 27.9 kg / m ~ 2 migrated across the endothelium. TRAM-34 and K (Ca) 3.1-specific si RNA significantly reduced the migration of THP-1 cells induced by PA (200 μmol / L) The secretion and expression of MCP-1 in cells were detected by flow cytometry. Anti-TLR2 / 4 (4 mg / L), p38MAPK inhibitor SB203580 (10μmol / L) and SB202190 (10μmol / L) 10μmol / L) significantly reduced the expression of K_ (Ca) 3.1 and MCP-1 in PA-induced THP-1 cells. CONCLUSION: PA can up-regulate the expression of MCP-1 by up-regulating K (Ca) 3.1 through TLR2 / 4-p38MAPK-NF-κB pathway and induce the trans-endothelial migration of monocytes.