TRPV1激活对钠-钾-氯共转运体在DRG细胞上表达和功能的影响

EFFECT OF TRPV1 ACTIVATION ON THE EXPRESSION AND FUNCTION OF SODIUM-POTASSIUM-CHLORINE COTRANSPORTER ON DRG

摘要:

目的：探讨在急性神经源性炎性痛模型中，用辣椒素激活瞬时感受器电位香草酸受体（transient receptor potential vanilloid 1, TRPV1）后对背根神经节（dorsal root ganglion, DRG）细胞钠钾氯共转运体（sodium-potassium-chloride co-transorter 1, NKCC1）及其磷酸化后蛋白表达变化及其功能的影响。方法：雄性SD大鼠随机分成正常组（n=12），辣椒素组（n=30）和布美他尼组（n=6）。急性分离DRG，运用免疫荧光和Western blotting技术检测NKCC1和p-NKCC1的蛋白分布及其变化；运用膜片钳技术检测GABA激活电流大小；运用氯离子荧光探针检测DRG细胞内氯离子浓度变化。结果：免疫荧光结果显示，TRPV1和NKCC1在正常大鼠DRG细胞共表达，辣椒素组NKCC1和p-NKCC1的免疫荧光染色强度高于正常组，且核膜上的NKCC1较正常组表达明显增多；行为学结果显示辣椒素组热缩足潜伏期减小、冷缩足潜伏期延长，给予NKCC1的抑制剂布美他尼预处理后，则能逆转该效果。Western blotting结果显示辣椒素组NKCC1和p-NKCC1的蛋白表达升高，与行为学变化结果一致；膜片钳结果显示辣椒素组DRG细胞的GABA激活电流明显增加，布美他尼可以逆转此作用；荧光探针结果显示辣椒素组DRG细胞内氯离子浓度增加，给予布美他尼后DRG细胞中Cl-浓度降低。结论：外源性的激动剂激活TRPV1受体后促进了NKCC1的表达增加和功能变化，提示NKCC1参与了激活TRPV1引起的疼痛。

Abstract:

Objective: To investigate the changes of sodium and potassium chloride co-transporter (NKCC1) and its phosphorylated protein expression in dorsal root ganglia after activation of transient receptor potential vanilloid receptor (TRPV1) in capsaicin-induced acute neuropathic inflammatory pain model. Method: Male SD rats were randomly divided into control group (n = 12), capsaicin group (n = 30) and bumetanide group(n = 6). The DRG was isolated and the protein distribution and changes of NKCC1 and p-NKCC1 were detected by immunofluorescence and Western blotting. The patch-clamp technique was used to detect the activation current of GABA. The chloride ion fluorescence probe was used to detect the change of DRG intracellular chloride concentration. Results:The results of immunofluorescence showed that TRPV1 and NKCC1 were co-expressed in normal rat DRG cells. The immunofluorescence staining intensity of NKCC1 and p-NKCC1 in capsaicin group was higher than that in normal group, and the NKCC1 nuclear membrane on nuclear membrane was significantly increased compared with normal group. The results showed that the capsaicin group had a reduced latency of heat-shrinking and a prolonged incubation period of cold-shrinking. After pretreatment with NKCC1 inhibitor bumetanide, the effect could be reversed. Western blotting results showed that the protein expression of NKCC1 and p-NKCC1 in capsaicin group was increased, which was consistent with the behavioral changes. Patch clamp results showed that the GABA-activated current of capsaicin group increased significantly, and bumetanide could reverse this effect. Fluorescence probe results showed that the concentration of chloride ions in the capsaicin group increased in the DRG cells, and the concentration of Cl- in the DRG cells decreased after administration of bumetanide. Conclusion:Activation of the TRPV1 receptor by an exogenous agonist promotes increased expression and functional changes in NKCC1, suggesting that NKCC1 is involved in the activation of TRPV1-induced pain.