预电针调控瞬时受体电位通道相关蛋白防治荨麻疹的机制研究

Electroacupuncture preconditioning relieves cutaneous passive anaphylaxis by down-regulating IP3 mediated ROS/TRPM2 signaling pathway and inhibiting mast cell degranulation in rats with urticaria

目的:观察预电针“曲池”“血海”对荨麻疹大鼠的防治作用,以及对瞬时受体电位(TRP)离子通道相关蛋白三磷酸肌醇(IP3)、活性氧(ROS)、TRPM2、钙调蛋白(CaM)介导肥大细胞(MCs)脱颗粒的影响,探讨其分子机制。方法:SD大鼠随机分为空白对照组、模型对照组、预电针组、阳性药物组,每组8只。造模前10 d,预电针组给予“曲池”“血海”预电针干预,20 min/次,1次/d,连续10 d;阳性药物组每天给予氯雷他定片稀释液灌胃(1 mg/kg),1次/d,连续10 d。采用卵白蛋白与免疫佐剂稀释液联合注射法制备大鼠抗卵白蛋白血清,皮肤被动过敏反应法制备荨麻疹大鼠模型。计数大鼠背部致敏皮肤搔抓次数,测量大鼠皮肤致敏蓝斑的直径,甲苯胺蓝染色法观察并计数大鼠皮肤MCs脱颗粒率,免疫组织化学法检测大鼠皮肤组织IP3、ROS表达,Western blot法检测大鼠皮肤组织TRPM2、CaM蛋白表达。结果:与空白对照组比较,模型对照组搔抓次数、皮肤致敏蓝斑直径均增加(P<0.01),MCs脱颗粒率升高(P<0.01),皮肤组织中IP3、ROS阳性表达及TRPM2、CaM蛋白表达水平均升高(P<0.01)。与模型对照组比较,预电针组、阳性药物组搔抓次数减少、皮肤致敏蓝斑直径减小(P<0.01),MCs脱颗粒率降低(P<0.01),皮肤组织中IP3、ROS阳性表达及TRPM2、CaM蛋白表达水平均降低(P<0.05,P<0.01)。结论:预电针“曲池”“血海”可减少荨麻疹模型大鼠背部致敏区域搔抓次数,缩短致敏蓝斑直径,从而抑制MCs脱颗粒,该效应可能与电针调控TRP通道相关蛋白IP3、ROS、TRPM2、CaM表达有关。

Objective To observe the effect of electroacupuncture(EA)preconditioning of“Quchi”(LI11)and“Xuehai”(SP10)on mast cell(MC)degranulation,and expressions of inositol triphosphate(IP3),reactive oxygen species(ROS),transient receptor potential(TRP)M2,calmodulin(CaM)in rats with urticaria,so as to reveal its molecular mechanism under-lying improving urticaria.Methods Thirty-two male SD rats were randomly divided into blank control,model,preconditioning of EA(Pre-EA)and medication groups(n=8 rats/group).The urticaria model was established by intradermal injection of dilute allogeneic antioalbumin serum at the spots of the bilateral symmetry of the spine on the back,and followed by tail venous injection of mixture solution of egg albumin diluent,plus 0.5%Evans blue and normal saline.Ten days before the end of modeling,rats of the pre-EA group received EA stimulation of LI11 and SP10 for 20 min,once a day for 10 consecutive days,and those of the medication group received gavage of loratadine tablets diluted solution(1 mg/kg)once a day for 10 days.The times of rat’s scratching the sensitized skin were recorded,the diameter of the sensitized blue spots was measured and the degranulation rate of skin MCs was counted under microscope after toluidine blue staining.The expression levels of IP3,ROS,TRPM2 and CaM in the skin tissue were measured by immunohistochemistry and western blot,respectively.Results Compared with the blank control group,the scratching times,diameter of the sensitized blue spots,degranulation rate of MCs,and the expression levels of ion channel related proteins(IP3,ROS,TRPM2 and CaM)were significantly increased(P<0.01)in the model group.In comparison with the model group,the scratching times,diameter of sensitized blue spot,degranulation rate of MCs,and the expression levels of IP3,ROS,TRPM2 and CaM in both pre-EA and medication groups were significantly down-regulated(P<0.01,P<0.05).No significant differences were found between Pre-EA and medication groups in down-regulating the levels of the above-mentioned 7 indexes.Conclusion EA-LI11 and SP10 preconditioning can reduce the cutaneous anaphylaxis in urticaria rats,which may be related to its effects in inhibiting the degranulation of MCs,and the expression of TRP channel related proteins.