艾灸镇痛下丘脑疼痛相关信号分子的PCR Array筛选研究

目的:明确下丘脑疼痛相关信号分子在艾灸镇痛作用中的表达情况,筛选与艾灸镇痛相关的分子靶点,为揭示艾灸镇痛原理提供新的实验依据。方法:选取健康、雄性C57BL/6J小鼠,随机分为空白组、模型组、模灸组。皮下注射20μL弗氏完全佐剂(Freund’s Complete Adjuvant,CFA)于小鼠右后足底,建立形成炎症疼痛实验小鼠模型。CFA足底注射后第4 d,给予“足三里”穴温和灸干预1次,持续时间为30 min。选取热刺激-缩腿潜伏期(Thermal-Withdraw Latency,TWL)作为判定疼痛指标,测定造模前、造模后、艾灸前、艾灸后即刻、30 min、60 min、90 min以及120 min痛阈值。运用PCR Array高通量分子筛选技术,筛选显著差异表达的下丘脑疼痛相关信号分子;用实时荧光定量PCR技术对差异表达基因进行重复验证。结果:(1)模型组、模灸组TWL较空白组均下降明显(P<0.01);模灸组在艾灸结束即刻、艾灸后30 min、60 min、90 min、120 min的痛阈较模型组均显著上升(P<0.01),艾灸后0 min即刻即出现痛阈上升,艾灸后60 min痛阈开始下降。(2)模型组7个疼痛相关基因差异表达显著,其中CCL12、CCR2、IL-1α、TAC1显著上调;NTRK1、SCN10a、SLC6a2显著下调;模灸组13个疼痛相关基因显著下调,即CCL12、CCR2、IL-1α、IL-1β、IL6、CX3CR1、CD4、EDNRA、TLR4、PTGS2、SCN10a、CNR1、TRPa1。模型组上调疼痛信号相关分子CCL12、CCR2、IL-1α,艾灸后下调。结论:艾灸“足三里”对小鼠炎性疼痛有显著镇痛效应;下丘脑内可能有多种潜在的疼痛相关信号分子参与艾灸镇痛,CCL12、CCR2、IL-1α等可能是参与艾灸镇痛新的关键分子靶点。

Effect of moxibustion on N-methyl-D-aspartate receptor subtype 2B expression in hippocampus of rheumatoid arthritis model rats

Objective:To observe the effect of moxibustion on the expression of N-methyl-D-aspartic acid(NMDA)receptor subtype 2B(NR2B)in the hippocampus of rheumatoid arthritis(RA)rats,and to explore the analgesic mechanisms of moxibustion in RA treatment.Methods:Sixty male Sprague-Dawley rats were randomly divided into a normal group,a model group,a moxibustion group,a moxibustion+NMDA receptor antagonist(AP-5)group,and a moxibustion+NMDA receptor agonist(NMDA)group,with 12 rats in each group.Except for the normal group,rats in the other four groups were treated with complete Freund's adjuvant in a windy,cold,and damp environment to replicate RA models.Rats in the moxibustion group received suspended moxibustion with moxa sticks at Shenshu(BL23)and Zusanli(ST36),and the two points were used alternately.After intraperitoneal injection of AP-5 or NMDA,rats in the moxibustion+AP-5 group and the moxibustion+NMDA group received the same moxibustion intervention as in the moxibustion group,once a day for 15 d.The thermal withdrawal latency(TWL)of rats in each group was detected before and after modeling and after the 15-day intervention.After the 15-day intervention,hematoxylin-eosin staining was performed to observe the pathological changes in knee joints.The real-time fluorescence quantitative polymerase chain reaction method was used to detect the mRNA expression of NR2B in the hippocampus;Western blotting assay was used to detect the protein and the phosphorylated protein expression of hippocampal NR2B.Results:The synovial tissue was proliferated,the synovial lining was significantly thickened,the pannus was formed,and the cartilage and bone tissues were significantly damaged in the model group.After intervention,the pathological morphology of the knee joints in the moxibustion group,the moxibustion+AP-5 group,and the moxibustion+NMDA group was significantly improved,and the improvement in the moxibustion+AP-5 group was more notable than that in the moxibustion+NMDA group.Compared with the normal group,the TWL was significantly decreased(P<0.01),and the mRNA,protein,and phosphorylated protein expression levels of hippocampal NR2B were significantly increased in the model group(P<0.01).Compared with the model group,the TWL of each intervention group was significantly increased(P<0.01 or P<0.05),and the mRNA,protein,and phosphorylated protein expression levels of hippocampal NR2B were significantly decreased(P<0.01).Compared with the moxibustion group,the TWL was significantly increased(P<0.01),and the mRNA,protein,and phosphorylated protein expression levels of hippocampal NR2B were significantly decreased in the moxibustion+AP-5 group(P<0.01);the TWL was significantly decreased(P<0.01),and the mRNA,protein,and phosphorylated protein expression levels of hippocampal NR2B were significantly increased in the moxibustion+NMDA group(P<0.01).Conclusion:Moxibustion reduces hyperalgesia in RA inflammatory rats.The analgesic effect may be related to the decrease in the expression and phosphorylation levels of NR2B in the hippocampus.