Deng Shi-Yu, Tang Xue-Chun, Chang Yue-Chen, Xu Zhen-Zhen, Chen Qin-Yi, Cao Nan, Kong Liang-Jing-Yuan, Wang Yang, Ma Ke-Tao, Li Li, Si Jun-Qiang
The Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Department of Physiology, Shihezi University Medical College, Shihezi, China.
Department of Anesthesia, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Front Cell Neurosci. 2021 May 25;15:665596. doi: 10.3389/fncel.2021.665596. eCollection 2021.
Our aim was to investigate the effects of the protein expression and the function of sodium, potassium, and chloride co-transporter (NKCC1) in the dorsal root ganglion (DRG) after activation of transient receptor potential vanilloid 1 receptor (TRPV1) in capsaicin-induced acute inflammatory pain and the possible mechanism of action. Male Sprague-Dawley rats were randomly divided into control, capsaicin, and inhibitor groups. The expression and distribution of TRPV1 and NKCC1 in rat DRG were observed by immunofluorescence. Thermal radiation and acetone test were used to detect the pain threshold of heat and cold noxious stimulation in each group. The expressions of NKCC1 mRNA, NKCC1 protein, and p-NKCC1 in the DRG were detected by PCR and western blotting (WB). Patch clamp and chloride fluorescent probe were used to observe the changes of GABA activation current and intracellular chloride concentration. After intrathecal injection of protein kinase C (PKC) inhibitor (GF109203X) or MEK/extracellular signal-regulated kinase (ERK) inhibitor (U0126), the behavioral changes and the expression of NKCC1 and p-ERK protein in L DRG were observed. TRPV1 and NKCC1 were co-expressed in the DRG. Compared with the control group, the immunofluorescence intensity of NKCC1 and p-NKCC1 in the capsaicin group was significantly higher, and the expression of NKCC1 in the nuclear membrane was significantly higher than that in the control group. The expression of NKCC1 mRNA and protein of NKCC1 and p-NKCC1 in the capsaicin group were higher than those in the control group. After capsaicin injection, GF109203X inhibited the protein expression of NKCC1 and p-ERK, while U0126 inhibited the protein expression of NKCC1. In the capsaicin group, paw withdrawal thermal latency (WTL) was decreased, while cold withdrawal latency (CWL) was prolonged. Bumetanide, GF109203X, or U0126 could reverse the effect. GABA activation current significantly increased in the DRG cells of the capsaicin group, which could be reversed by bumetanide. The concentration of chloride in the DRG cells of the capsaicin group increased, but decreased after bumetanide, GF109203X, and U0126 were administered. Activation of TRPV1 by exogenous agonists can increase the expression and function of NKCC1 protein in DRG, which is mediated by activation of PKC/p-ERK signaling pathway. These results suggest that DRG NKCC1 may participate in the inflammatory pain induced by TRPV1.
我们的目的是研究辣椒素诱导的急性炎性疼痛中瞬时受体电位香草酸亚型1受体（TRPV1）激活后，背根神经节（DRG）中钠、钾、氯共转运体（NKCC1）的蛋白表达及功能变化，以及可能的作用机制。将雄性Sprague-Dawley大鼠随机分为对照组、辣椒素组和抑制剂组。采用免疫荧光法观察大鼠DRG中TRPV1和NKCC1的表达及分布。利用热辐射和丙酮试验检测各组热和冷有害刺激的痛阈。通过PCR和蛋白质印迹法（WB）检测DRG中NKCC1 mRNA、NKCC1蛋白和p-NKCC1的表达。采用膜片钳和氯离子荧光探针观察γ-氨基丁酸（GABA）激活电流和细胞内氯离子浓度的变化。鞘内注射蛋白激酶C（PKC）抑制剂（GF109203X）或丝裂原活化蛋白激酶/细胞外信号调节激酶（MEK/ERK）抑制剂（U0126）后，观察大鼠的行为变化以及L4 DRG中NKCC1和p-ERK蛋白的表达。TRPV1和NKCC1在DRG中共表达。与对照组相比，辣椒素组中NKCC1和p-NKCC1的免疫荧光强度显著更高，且核膜中NKCC1的表达显著高于对照组。辣椒素组中NKCC1 mRNA、NKCC1蛋白以及p-NKCC1的表达均高于对照组。注射辣椒素后，GF109203X抑制了NKCC1和p-ERK的蛋白表达，而U0126抑制了NKCC1的蛋白表达。在辣椒素组中，爪部热缩潜伏期（WTL）缩短，而冷缩潜伏期（CWL）延长。布美他尼、GF109203X或U0126可逆转此效应。辣椒素组DRG细胞中GABA激活电流显著增加，布美他尼可逆转此效应。辣椒素组DRG细胞中氯离子浓度升高，但给予布美他尼、GF109203X和U0126后降低。外源性激动剂激活TRPV1可增加DRG中NKCC1蛋白的表达及功能，这是由PKC/p-ERK信号通路的激活介导的。这些结果表明，DRG中的NKCC1可能参与TRPV1诱导的炎性疼痛。
