| Purpose:To observe the effect of electroacupuncture at “Quchi”(LI 11),“Xuehai”(SP10)and “Zusanli”(ST 36)on the degranulation of mast cells,the activation of Lyn and Syk,known as the rate-limiting proteins of the FcεRI cascade,and the expression of mitogen-activated protein kinase(MAPK)and nuclear factor-kappa B(NF-κB)pathway-associated proteins in urticaria rats,so as to reveal its mechanism urderlying improvement of urticaria and provide theoretical basis for clinical treatment.Material and method:1.MaterialThirty-seven SPF-grade male SD rats with a body mass of(180～220)g were provided by Liaoning Changsheng Biotechnology Co.,Ltd.(No: SCXK(liao)2015-0001)and acclimatized to their new housing for a week before beginning experimental protocols.Rats were bred and housed under specific pathogen-free(SPF)conditions in the animal laboratory of Liaoning University of Traditional Chinese Medicine.Experimental rat rooms were maintained at a temperature of 23±3℃,relative humidity of 40%-70%,light/dark cycle of 12 h.Five rats were used to obtain anti-OVA serum,and the thirty-two remaining rats were randomly divided into control,model,EA and medication groups(n=8 in each group).Experimental procedures were approved by the Institutional Ethics Committee of Liaoning University of Traditional Chinese Medicine concerning the Care and Use of Laboratory Animals.2.Method2.1 Animal modeling : The urticaria model was established by using passive cutaneous anaphylaxis(PCA)reaction method.Preparation for Anti-OVA Serum : 5 of 37 rats were randomly selected for the preparation of anti-OVA serum.0.25 mg of Bacillus Calmette-Guerin was suspended in 0.5ml of Sterile Water,and 1 ml of the BCG diluted solution was intraperitoneally injected into the SD rats 3 times at 1 day intervals.AT the same time,each side of the hindlimb was injected with 0.5 ml of 1% ovalbumin saline solution 3 times at 1 day intervals.Ten days after the 3th injection of OVA and BCG,whole blood was collected to obtain anti-OVA serum.After being anesthetized with chloral hydrate,the rats were shaved within 1.5 cm of the spine on both sides of their midline,and marked at the area of the back skin with a red penwhere the serum is to be injected.In the model,EA and medication groups,0.1 ml of the anti-OVA serum was intracutaneously injected into the both side of the back.In the control group,saline was intracutaneously injected instead of antiserum.After 48 h,1 mg/m L OVA and 0.5% Evans blue in 0.9% Na Cl was intravenously injected to induce the PCA reaction.Thirty minutes after antigen-challenge,mice were sacrificed and their tissues were collected.2.2Intervention methods:EA(2 Hz/15 Hz,1 m A)was applied to bilateral “Zusanli”(ST 36),“Quchi ”(LI 11),“Xuehai”(SP 10)for 20 min,once daily for 7 consecutive days before antigen attack.Rats of the medication group were treated by gavage of Loratadine(1mg×kg-1×d-1)for 7 days.Rats of the control and model group were only bound to the fixator without interventions.Corresponding modeling operations were performed 1 h after the intervention treatment was completed.2.3Indicator detection:2.3.1The diameter of cutaneous Evan’s blue spots were measured to evaluate the severity of PCA.The effusion of dye at allergized site was detected by spectrophotometer.The pathological changes of the skin tissue were detected by HE staining.The contents of Ig E and histamine in the serum were detected by ELISA.2.3.2The mast cells in the loose connective tissue were displayed by Toluidine blue staining.The expressions of Lyn and Syk in the mast cell of loose connective tissue were detected by immunohistochemistry.2.3.3The mast cells in the skin tissue were displayed by Toluidine blue staining.The expressions of TNF-α and IL-6 in the mast cell of skin tissue were detected by immunohistochemistry.The expression of extracellular signal-regulated kinase(ERK),phosphorylated(p)-ERK,c-Jun N-terminal kinase(JNK),p-JNK,P38 MAPK and p-P38 MAPK of the skin tissue was detected by Western blot.The expression of NF-κBp65and IκBα of the skin tissue was detected by Western blot.2.3.4Intraperitoneal fluid smears were prepared to observe the degranulation state of MCs.The contents of TNF-α and IL-6 in the intraperitoneal fluid were detected by ELISA.The expression of ERK,p-ERK,JNK,p-JNK,P38 MAPK,p-P38 MAPK of the acquired intraperitoneal MCs was detected by Western blot.The expression of NF-κBp65 and IκBα of the acquired intraperitoneal MCs was detected by Western blot.2.4Statistical methods:Statistical analyses were performed by repeated one-way analysis of variance(ANOVA)and the Dunnett’s multiple t-test calculated with SPSS 18.0 statisticalsoftware.All data are presented as the mean values ± standard error of the mean(SEM)with three times experimental replicates and a probability value(p<0.05)was considered to be statistically significant.Results:1.The skin Pathomorphological and serum biochemistry changes1.1The diameter of cutaneous Evan’s blue spots in rats of each groupThe diameter of cutaneous Evan’s blue spot was significantly increased in the model group than in the normal group(P<0.01),and considerably decreased in both EA and medication groups compared with the model group(P<0.01).In comparison with the EA group,no significant change was found about the diameter of cutaneous Evan’s blue spot in the medication group(P>0.05).1.2The skin dye exudation in rats of each groupCompared with the normal group,the extravasated dye in the skin was significantly increased in the model group(P<0.01).After the EA or medication intervention,the skin dye exudation was significantly decreased relevant to the model group(P<0.01).In comparison with the EA group,no significant changes were found about the skin dye exudation in the medication group(P>0.05).1.3The pathological changes of the skin tissue in rats of each groupIn the normal group,the cells of the epidermal layer were arranged regularly,and no inflammatory cell infiltration was observed in the dermis.Histopathological examination showed epidermal hyperplasia and a large number of inflammatory cells were infiltrated in the dermis of model group.While in EA group and the medication group,the epidermal became thinner,and the infiltration of inflammatory cells in dermis was significantly lower than that in the model group.1.4 The serum levels of Ig E and histamine in rats of each groupAfter modeling,the contents of Ig E and histamine in the serum were remarkably increased in the model group in comparion with the normal group(P<0.01).Whereas,after EA or medication intervention,the contents of Ig E and histamine in the serum were obviously decreased relevant to the model group(P<0.05,P<0.01).In comparison with the EA group,the contents of Ig E were obviously decreased(P<0.05),but no significant changes were found about the levels of histamine in the medication group(P>0.05).2 The results of mast cell-related indexes in the loose connective tissue2.1 The percentage of degranulated MCs in the loose connective tissue in rats of each groupCompared with the normal group,the percentage of degranulated MCs in the loose connective tissue were significantly increased in the model group(P<0.01).After the EA or medication intervention,the degranulation rate of mast cell were significantly decreased relevant to the model group(P<0.01).In comparison with the EA group,no significant changes were found about the mast cell degranulation rate in the medication group(P>0.05).2.2The expression of p-Lyn and Lyn in the mast cells of loose connective tissue in rats of each groupCompared with the normal group,the expression levels of p-Lyn and Lyn in the mast cells of loose connective tissue were significantly increased in the model group(P<0.01);After the EA or medication intervention,the expression levels of p-Lyn and Lyn were significantly decreased relevant to the model group(P<0.01);In comparison with the EA group,no significant changes were found about the expression levels of p-Lyn and Lyn in the medication group(P>0.05).2.3The expression of Lyn and Syk in the mast cells of loose connective tissue in rats of each groupCompared with the normal group,the expression levels of p-Syk and Syk in the mast cells of loose connective tissue were significantly increased in the model group(P<0.01);After the EA or medication intervention,the expression levels of p-Syk and Syk were significantly decreased relevant to the model group(P<0.01);In comparison with the EA group,the level of p-Syk was obviously decreased(P<0.01),but no significant change was found about the expression level of Syk in the medication group(P>0.05).3.The results of mast cell-related indexes in the skin tissue3.1 The percentage of degranulated MCs in the skin tissue in rats of each groupCompared with the normal group,the percentage of degranulated MCs in the skin tissue was significantly increased in the model group(P<0.01).After the EA or medication intervention,the percentage of degranulated MCs was significantly decreased relevant to the model group(P<0.01).In comparison with the EA group,no significant change was found about the percentage of degranulated MCs in the medication group(P>0.05).3.2 The expression levels of TNF and IL-6 in the mast cell of skin tissue in rats of each groupAfter modeling,the expression levels of TNF-α and IL-6 in the mast cell of skin tissue were remarkably increased in the model group in comparion with the control group(P<0.01).Whereas,after EA or medication intervention,the levels of TNF-α and IL-6 were obviously decreased relevant to the model group(P<0.01).In comparison with the EA group,the levels of TNF-α and IL-6 were obviously decreased(P<0.01).3.3 The ratios of p-ERK/ERK,p-JNK/JNK,p-P38MAPK/P38 MAPK in rats of each groupAfter modeling,the ratios of p-ERK/ERK,p-JNK/JNK,p-P38MAPK/P38 MAPK in the skin tissue were remarkably increased in the model group in comparion with the control group(P<0.01);Whereas,after EA or medication intervention,the ratios of p-ERK/ERK,p-JNK/JNK,p-P38MAPK/P38 MAPK were obviously decreased relevant to the model group(P<0.05,P<0.01);In comparison with the EA group,the ratios of p-ERK/ERK,p-JNK/JNK were obviously decreased(P<0.01),but no significant change was found about the ratio of p-P38MAPK/P38 MAPK in the medication group(P>0.05).3.4The expression of NF-κBp65 and IκBα in the mast cell of skin tissue in rats of each groupCompared with the normal group,the expression levels of NF-κBp65 in the skin tissue were significantly increased in the model group(P<0.05)and the levels of IκBα in the skin tissue were significantly decreased(P<0.01).After the EA or medication intervention,the expression levels of NF-κBp65 in the skin tissue were significantly decreased relevant to the model group(P<0.05),while the levels of IκBα in the skin tissue were significantly increased(P<0.01).In comparison with the EA group,no significant changes were found about the expression levels of NF-κBp65 and IκBα in the medication group(P>0.05).4.The results of Peritoneal mast cell-related indexes4.1 The percentage of degranulated MCs in the intraperitoneal fluid in rats of each groupThe Peritoneal mast cells in the normal group displayed a complete outline and the membrane of Peritoneal mast cells in the model group was incompletely ruptured with heterogeneous particles scattered around the cells.The cell membrane in the EA or medication group was intact and the cytoplasm was uniformly stained.After modeling,the percentage of mast cell degranulation was remarkably increased in the model group in comparion with the normal group(P<0.01).Whereas,after EA or medication intervention,the percentage of mast cell degranulation was obviously decreased relevant to the model group(P<0.01).In comparison with the EA group,no significant changes were found about the percentage ofmast cell degranulation in the medication group(P>0.05).4.2 The expression levels of TNF and IL-6 in the intraperitoneal fluid in rats of each groupAfter modeling,the contents of TNF-α and IL-6 in the intraperitoneal fluid were remarkably increased in the model group in comparion with the control group(P<0.01).Whereas,after EA or medication intervention,the contents of TNF-α and IL-6 were obviously decreased relevant to the model group(P<0.05,P<0.01).In comparison with the EA group,no significant changes were found about the contents of TNF-α and IL-6 in the medication group(P>0.05).4.3The expression of MAPKs in the Peritoneal mast cell in rats of each groupCompared with the normal goup,the expression levels of p-ERK,ERK,p-JNK,JNK,p-P38 MAPK and P38 MAPK in the Peritoneal mast cell were significantly increased in the model group(P<0.05,P<0.01).Following EA intervention and in comparison with the model group,the expression levels of p-ERK,p-JNK,JNK and p-P38 MAPK were down-regulated(P<0.05),but without significant changes in the expressions of ERK and P38MAPK(P>0.05).Following medication intervention and in comparison with the model group,the expression levels of p-ERK,p-JNK,JNK,p-P38 MAPK and P38 MAPK were down-regulated(P<0.05),but without significant changes in the expressions of ERK(P>0.05).In comparison with the EA group,no significant changes were found in all abovementioned indexes of medication group except the down-regulated P38MAPK(P>0.05).4.4The expression of NF-κBp65 and IκBα in the Peritoneal mast cell in rats of each groupCompared with the normal group,the expression levels of NF-κBp65 in the Peritoneal mast cell were significantly increased in the model group(P<0.01)and the levels of IκBα in the skin tissue were significantly decreased(P<0.01);After the EA or medication intervention,the expression levels of NF-κBp65 in the Peritoneal mast cell were significantly decreased relevant to the model group(P<0.05,P<0.01),while the levels of IκBα were significantly increased(P<0.01);In comparison with the EA group,the expression levels of NF-κBp65 in the medication group were significantly decreased(P<0.01)and the levels of IκBα were significantly increased(P<0.01).Conclusion:1.Electroacupuncture at“Quchi”(LI 11),“Xuehai”(SP 10)and “Zusanli”(ST 36)acupointscan effectively reduce serum Ig E and histamine levels,reduce the vascular permeability and plasma extravasation,improve the pathological changes of skin tissue in urticaria rats.2.Electroacupuncture at“Quchi”(LI 11),“Xuehai”(SP 10)and “Zusanli”(ST 36)acupoints can reduce allergic reaction by inhibiting the degranulation of mast cells in the loose connective tissue of urticaria rats,which may be related to its effects in regulating the activation of the FcεRI cascade response rate limiting proteins Lyn and Syk in mast cells.3.Electroacupuncture at“Quchi”(LI 11),“Xuehai”(SP 10)and “Zusanli”(ST 36)acupoints has the effect of reducing allergic reaction in rats with urticaria by inhibiting the degranulation of skin mast cells,which is closely associated with its effects in down-regulating the expression of MAPK/NF-κB signaling pathway-related proteins and pro-inflammatory factors TNF-α and IL-6 in mast cells.4.Electroacupuncture at“Quchi”(LI 11),“Xuehai”(SP 10)and “Zusanli”(ST 36)acupoints can reduce allergic reaction by inhibiting the degranulation of peritoneal mast cells in rats with urticaria,which may be related to its effects in down-regulating the expression of MAPK signaling pathway-related proteins,blocking the degradation of IκBα,inhibiting the translocation of NF-κBp65 into the nucleus,and decreasing the expression of pro-inflammatory factors TNF-α and IL-6 in mast cells. |
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