肠胶质细胞系TRPV4依赖性Ca^(2+)内流、ATP释放与炎症因子表达

Transient receptor potential vanilloid 4-dependent calcium influx,ATP release and expression of inflammatory factors in enteric glial cells

目的探讨瞬时感受器电位离子通道香草素受体4(transient receptor potential vanilloid 4,TRPV4)在调节肠胶质细胞外Ca^(2+)内流中的作用及其意义。方法收集大鼠肠胶质细胞系CRL-2690、大鼠肠上皮细胞系IEC6和人胚肾细胞系HEK293T样本,采用RT-qPCR、Western blot和免疫荧光方法检测TRPV4的mRNA、蛋白表达与定位;将CRL-2690细胞依次分为GSK组与GSK+HC组,低渗组与低渗+HC组,GdCl_(3)组与GdCl_(3)+HC组以及CaCl_(2)组与CaCl^(2+)HC组,采用Fura-2荧光探针和单细胞荧光Ca^(2+)检测仪评估CRL-2690细胞中TRPV4通道活性。将CRL-2690细胞分为对照组、GSK组与GSK+HC组,通过荧光素-荧光素酶实验测量细胞ATP的释放水平;采用RT-qPCR检测炎症相关基因GFAP、IL-1β、IL-6、IL-10的mRNA表达。结果在大鼠肠胶质细胞系CRL-2690中检测到TRPV4 mRNA和蛋白表达;TRPV4特异性激动剂GSK1016790A和低渗透压刺激明显增强了CRL-2690细胞内钙荧光强度,而TRPV4特异性抑制剂HC067047可抑制此作用(0.43038±0.06365 vs 0.00423±0.00578,P<0.0001);钙敏感受体(calcium-sensing receptor,CaSR)激活诱导的外Ca^(2+)内流也可被HC067047所抑制(P<0.05);功能上,激活TRPV4可促ATP释放、EGC反应性增生标志物GFAP和炎症因子IL-1β、IL-6的表达,并抑制抗炎因子IL-10的表达,该作用可被HC067047所抑制(P<0.05)。结论激活TRPV4可引起肠胶质细胞外Ca^(2+)内流,并促进ATP释放与炎症因子表达,可能参与肠道炎症的发生。

ObjectiveTo investigate the role and physiological significance of transient receptor potential vanilloid receptor 4(TRPV4)in regulation of calcium influx in rat intestinal glial cells.MethodsThe expression of TRPV4 at mRNA and protein levels and its localization were detected by RT-qPCR,Western blotting and immunofluorescence assay in rat enteric glial cell line CRL-2690,rat intestinal epithelial cell line IEC6 and human embryonic kidney cell line HEK293T.The CRL-2690 cells were divided into GSK1016790A(GSK,TRPV4-specific agonist)group and GSK+HC067047(HC,TRPV4-specific inhibitor)group,hypotonic group and hypotonic+HC group,GdCl_(3)group and GdCl_(3)+HC group,and CaCl_(2)group and CaCl^(2+)HC group,while the activity of TRPV4 channel was assessed by Fura-2 fluorescent probe and single-cell fluorescent calcium ion assay.CRL-2690 cells were also divided into control group,GSK group and GSK+HC group,and the level of cellular ATP release was measured by luciferin-luciferase assay,and the mRNA levels of inflammatory genes GFAP,IL-1β,IL-6 and IL-10 were detected by RT-qPCR.ResultsTRPV4 was expressed at mRNA and protein levels in rat intestinal glial cell line CRL-2690.GSK and low osmolarity stimulation significantly enhanced intracellular calcium fluorescence intensity in CRL-2690 cells,which was inhibited by HC(0.43038±0.06365 vs 0.00423±0.00578,P<0.0001).Calcium-sensing receptor(CaSR)activation-induced Ca^(2+)influx was also inhibited by HC(P<0.05).Functionally,activation of TRPV4 promoted ATP release,expression of EGC reactive proliferation marker,GFAP,and inflammatory factors IL-1βas well as IL-6,and inhibited the expression of anti-inflammatory factor IL-10 respectively.But all these effects were inhibited by HC treatment(P<0.05).ConclusionActivation of TRPV4 induces extracellular Ca^(2+)influx and promotes ATP release and expression of inflammatory cytokines in enteric glial cell,which may be closely involved in the underlying mechanisms of bowel inflammation.