| Objective:The rat model of Acute Gouty Arthritis(AGA)was established by injecting Monosodium Urate(MSU)suspension into the ankle joint cavity of the right hind limb.AGA rats were treated with bloodletting therapy,Zhuang medicinal thread moxibustion therapy,colchicine,and bloodletting therapy combined with Zhuang medicinal thread moxibustion therapy,the therapeutic effects of Zhuang medicinal thread moxibustion,bloodletting therapy,colchicine and bloodletting therapy combined with Zhuang medicinal thread moxibustion were compared,and the effect of bloodletting therapy combined with Zhuang medicinal thread moxibustion on TLRs/MyD88 signaling pathway in the treatment of gouty arthritis was explored.Methods:Sixty SPF male Sprague-Dawley rats,weighing about 200±20g,were kept in SPF animal room with room temperature of(22±1)℃ and room relative humidity of(50±10)%.Sixty rats were divided into blank group,model group,colchicine group,bloodletting group,drug thread group and bloodletting combined with drug thread group,with 10 rats in each group.The modeling site was the ankle joint of the right hind limb of the rat.0.2 ml sodium urate suspension was injected into the ankle joint lumen of the rats except for the blank group to replicate the AGA model.The blank group rats were injected with 0.2 ml physiological saline into the ankle joint lumen.The rats were given intervention treatment at 24h,48h and 72h after modelling,that is,colchicine group was given colchicine suspension with 5%concentration at the rate of 1mL per 100g of rat body weight.In the pricking blood group,the most swollen part of the ankle joint was punctured and bloodletting was performed,with the blood volume of about 0.1ml.In the medicine line group,moxibustion was carried out evenly around the most swollen part of the ankle joint.Acupuncture and blood combined with medicine line group was first treated with acupuncture and bloodletting and then moxibustion with medicine line.Except for the colchicine group,rats in all groups were given normal saline by gavage,that is,1 mL of normal saline per 100 g of rat body weight as the standard.The cross-sectional diameters of the ankle joints were observed at 6h,12h,24h,48h and 72h after modeling.HE staining was used to observe the pathological changes of ankle synovium.The contents of inflammatory factors IL-2,COX-2,IL-8,IL-37 and IL-4 were determined by ELISA kit.The expressions of MyD88,IKK-β and IκB-α in synovium were detected by Western blot.The expression of MyD88,IKK-βand IκB-αmRNA in synovium of each group was determined by RT-PCR.Results:1.During the intervention period,the rats in colchicine group developed diarrhea,while the stool and stool were normal in the other treatment groups.2.Cross-sectional diameter results of the inner and outer ankle joints in rats:The cross section diameter of ankle joint in all experimental groups reached the maximum 12h after modeling.Compared with blank group,the cross-sectional diameter of ankle joint was increased in experimental groups at 6h,12h and 24h after modeling(P<0.05),but there was no statistical significance between treatment groups and model group(P>0.05).After intervention,compared with model group,the cross-section diameter of ankle joint was decreased in all treatment groups to varying degrees(P<0.05).48h after modeling,the cross-section diameter of ankle joint in treatment groups was lower than that in model group(P<0.05),and the cross-section diameter of ankle joint in pricking blood group and drug line group was higher than that in colchicine group and pricking blood combined drug line group(P<0.05).There was no significant difference between the colchicine group and the pricking blood combination group(P>0.05).72h after modeling,the cross section diameter of ankle in treatment groups was lower than that in model group(P<0.05),the cross section diameter of ankle in pricking blood group and drug line group was higher than that in colchicine group and drug line of pricking blood group(P<0.05),and the cross section diameter of ankle in colchicine group was higher than that in drug line of pricking blood group(P<0.05).3.The results of HE staining of synovial tissue:the synovial tissue and cell structure were intact in the blank group,and a large number of inflammatory cell infiltration was observed in the synovial tissue of the model group.Compared with the model group,the inflammatory cell infiltration was improved in the blood prick group,drug line group,colchicine group and blood prick combined drug line group,and the inflammatory cell infiltration was improved in the colchicine group and blood prick combined drug line group than the blood prick group and drug line group.4.The results of ELISA:compared with the model group,the serum levels of IL-2,COX-2 and IL-8 in the blood prick group,drug line group,colchicine group and blood prick combined with drug line group were decreased,and the serum levels of IL-37 and IL-4 were increased(P<0.05).Compared with the blood prick group and the drug line group,the serum levels of IL-2,COX-2 and IL-8 in the colchicine group and the blood prick combined with drug line group were decreased,and the serum levels of IL-37 and IL-4 were increased(P<0.05).There was no significant difference in the content of each factor between the colchicine group and the acupuncture combined with blood line group(P>0.05).5.Western blot results:Compared with the model group,the expression levels of MyD88,IKK-β and IκB-αproteins in the blood prick group,drug line group,colchicine group and blood prick combined with drug line group were decreased(P<0.05).6.RT-PCR results:Compared with the model group,the expression of MyD88,IKK-βand IκB-αmRNA in the blood prick group,drug line group,colchicine group and blood prick combined with drug line group were decreased(P<0.05).The expression levels of MyD88,IKK-β and IκB-αmRNA in the colchicine group and the blood-prick combined with drug line group were lower than those in the blood-prick group and the drug line group(P<0.05).The expression of MyD88,IKK-β and IκB-αmRNA in the combination group was slightly higher than that in the colchicine group,but there was no statistical significance(P>0.05).Conclusions:1.Bloodneedling therapy combined with Zhuang medicinal thread moxibustion can significantly improve the degree of joint swelling in AGA rats,and there is no obvious adverse reaction during the treatment.It is better than colchicine in reducing the degree of joint swelling and adverse reactions of AGA rats.2.In terms of anti-inflammatory effect,the curative effect of bloodletting therapy combined with Zhuang medicinal thread moxibustion and colchicine is equivalent,and the anti-inflammatory effect is stronger than that of single bloodletting therapy and Zhuang medicinal thread moxibustion.Acupuncture therapy combined with Zhuang medicinal thread moxibustion can reduce the levels of pro-inflammatory factors IL-2,COX-2 and IL-8 in the serum of AGA rats,and increase the levels of anti-inflammatory factors IL-4 and IL-37,so as to regulate the inflammatory response,so as to play a therapeutic effect on AGA.3.The effect of bloodspicing therapy combined with Zhuang medicinal thread moxibustion on AGA may be related to the down-regulation of MyD88,IKK-β,IκB-α protein and gene expression,and the inhibition of TLRs/MyD88 inflammatory signaling pathway may be one of the anti-inflammatory mechanisms of bloodspicing therapy combined with Zhuang medicinal thread moxibustion in the treatment of AGA. |
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