Objectives: This study aims to explore the analgesic mechanism of fire needle on peripheral sensitization in rats with neuropathic pain(NP) induced by oxaliplatin, so as to investigate its mechanism in improving peri-pheral sensitization.
Methods: Male SD rats aged 8 weeks were randomly divided into 4 groups：normal group(n=6), model group(n=6), fire needle group(n=6), and medication group(n=6). NP rat model was established by intraperitoneal injection of oxaliplatin(4 mg/kg) on days 1, 2, 8, 9, 15, 16, 22, and 23. For rats in the fire needle group, fire needle treatment was performed at the "Jiaji"(EX-B2) acupoints of the L4-L6 segments on days 24, 26, and 28, ie. 1 day, 3 and 5 days after modeling. The medication group received intraperitoneal injection of pregabalin(100 mg/kg). Mechanical pain thresholds of the rats were measured before modeling, after modeling and intervention. Serum contents of tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and chemokine ligand 12(CXCL12) were detected by ELISA. Skin histopathology changes in the acupoint area were observed using HE staining. The number of mast cells in the skin of the acupoints was observed using toluidine blue staining. Immunohistochemical staining was performed to detect the postive expressions of transient receptor potential vanilloid 1(TRPV1), protease-activated receptor 2(PAR2) and tryptase(TPS) in the skin of the acupoint area. Western blot was used to detect the protein expressions of TRPV1 and PAR2 in the dorsal root ganglia(DRG).
Results: Compared with the normal group, the model group had decreased paw withdrawal threshold(PWT) after modeling(P<0.05), increased serum contents of IL-6, TNF-α, and CXCL12(P<0.05), increased number of mast cells in the acupoint area(P<0.05), and increased positive protein expressions of TPS, TRPV1, and PAR2 in the skin of the acupoint area(P<0.05). Compared with the model group, the fire needle group and medication group had increased PWT after intervention(P<0.05), decreased serum contents of IL-6, TNF-α, and CXCL12, and postive protein expressions of TPS, TRPV1, and PAR2 in the skin of the acupoint area(P<0.05)；while the medication group had decreased protein expressions of TRPV1 and PAR2 in DRG(P<0.05). HE staining showed thickened epidermis, disordered cellular arrangement, significant intercellular edema, and inflammatory cell infiltration in the model group. In the medication and fire needle groups, the epidermis was thinner, cellular arrangement was clearer, and the extent of tissue edema and inflammatory cell infiltration was reduced compared to the model group.
Conclusions: Fire needle can improve mechanical pain threshold and reduce the contents of peripheral inflammatory factors in rats with oxaliplatin-induced NP. This effect may be related to the inhibition of mast cell activation and the inhibition of TPS, TRPV1 and PAR2 protein expressions, in the local areas of acupoints.
目的: 从外周敏化角度探讨火针治疗奥沙利铂诱导的神经病理性疼痛（NP）大鼠外周敏化的镇痛机制。方法: SD大鼠随机分为正常组（n=6）、模型组（n=6）、火针组（n=6）和药物组（n=6）。采用腹腔注射奥沙利铂（4 mg/kg）建立NP大鼠模型。造模后的第1、3、5天（即实验的第24、26、28天），火针组行火针L4—L6“夹脊”治疗，药物组行普瑞巴林（100 mg/kg）腹腔注射治疗。Von Frey丝检测大鼠缩足阈值（PWT）代表机械痛阈值，ELISA法检测大鼠血清肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、趋化因子12（CXCL12）含量，HE染色法观察大鼠穴区皮肤病理形态，甲苯胺蓝染色法观察各组大鼠穴区皮肤肥大细胞数量，免疫组织化学法检测大鼠穴区皮肤类胰蛋白酶（TPS）、瞬时受体电位香草酸通道1（TRPV1）、蛋白酶活化受体2（PAR2）阳性表达，Western blot法检测大鼠背根神经节（DRG）中TRPV1和PAR2蛋白的表达。结果: 与正常组相比，模型组大鼠造模后的PWT降低（P<0.05），血清IL-6、TNF-α、CXCL12含量升高（P<0.05），穴区皮肤肥大细胞数量增加（P<0.05），穴区皮肤TPS、TRPV1和PAR2蛋白阳性表达升高（P<0.05）。与模型组相比，火针组和药物组大鼠干预后的PWT升高（P<0.05），血清IL-6、TNF-α、CXCL12含量，穴区皮肤TPS、TRPV1、PAR2蛋白阳性表达降低（P<0.05）；药物组DRG中TRPV1和PAR2蛋白表达量降低（P<0.05）。HE染色结果显示，模型组大鼠穴区表皮层增厚，细胞排列层次紊乱，细胞间水肿明显，真皮层内可见大量炎性细胞浸润；药物组、火针组穴区表皮层较薄，细胞排列层次较清晰，组织水肿和炎性细胞浸润情况均较模型组减轻。结论: 火针可以改善奥沙利铂诱导的NP大鼠的机械痛阈、降低外周炎性因子的水平，该作用可能与抑制肥大细胞活化，抑制穴区局部TPS、TRPV1和PAR2蛋白的表达有关。.
Keywords: Fire needle; Mast cells; Neuroinflammation; Neuropathic pain; Oxaliplatin; Transient receptor potential vanilloid 1/protease-activated receptor 2.