艾灸镇痛下丘脑疼痛相关信号分子的PCR Array筛选研究  

Pain-Related Signaling Molecules Responded to Moxibustion-Induced Analgesia in Hypothalamus

作　　者：尹海燕[1] 罗钦[1] 侯帅 陈莎莎[1] 余曙光[1] Yin Haiyan;Luo Qin;Hou Shuai;Chen Shasha;Yu Shuguang(Acupuncture and Tuina School,Chengdu University of Traditional Chinese Medicine,Chengdu,610075,China)

机构地区：[1]成都中医药大学针灸推拿学院/第三附属医院,四川成都610075

出　　处：《成都中医药大学学报》2020年第3期8-13,共6页Journal of Chengdu University of Traditional Chinese Medicine

基　　金：国家重点研发计划项目(2019YFC1709001),国家自然科学基金项目(81904312,81774437)。

摘　　要：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。Objective:To determine moxibustion-triggered analgesic effect on inflammatory pain and explore the pain-related-gene involved mechanism in hypothalamus of moxibustion analgesia.Methods:Healthy male C57BL/6J mice were randomly divided into control,Complete Freund's Adjuvant injection(CFA),and CFA+Moxibustion groups.The inflammatory-pain model was established by injecting 20μl CFA into the right hind paw.At day 4 after the pain model established,mild moxibustion lasted for 30 minutes was administrated on the right Zusanli acupoints(ST36).The thermal-withdraw latency(TWL)was measured by thermal infrared radiation before and after CFA injection,before moxibustion,0,30,60,90,120 minutes after moxibustion.The hypothalamuses were cut off immediately after the determination of pain threshold,and the pain-related molecules expression in hypothalamus was screened by high throughput PCR Array which including 84 pain related genes.The result of PCR Array was verified by Q-PCR method.Results:Compare to control group,the TWL decreased obviously in CFA and CFA+Moxibustion group(P<0.01).The TWL increased sharply at 0,30,60,90,and 120 min after moxibustion(P<0.01).Compared with control group,7 genes(4 upregulated genes:CCL12,CCR2,IL-1α,TAC1;3 downregulated genes:Ntrk1,Scn10a,and Slc6a2)were changed significantly;Compared with CFA group,13 down-regulated genes(CCL12,CCR2,IL-1α,CX3cr,CD4,EDNRA,IL-1β,IL-6,TLR4,PTGS2,CNR1,SCN10a,TRPa1)were significantly expressed in moxibustion group.Conclusion:Moxibustion could trigger analgesia effect on inflammatory pain;CCL12,CCR2,and IL-1αin hypothalamus would be novel target to get involved in moxibustion-induced analgesia.

关 键 词：艾灸镇痛 下丘脑 炎性疼痛 疼痛信号分子 

分 类 号：R245.8[医药卫生—针灸推拿学] R-332[医药卫生—中医临床基础]