基于MrgC受体的慢性炎症疼痛新型调节机制及电针干预的研究

Chronic inflammatory pain exists in the process of various diseases. There is little effect when applied with Morphine and dramatic side effects with anti-inflammatory drugs(such as nonsteroidal antiinflammatory drugs, NSAID) . So it is an important content to elucidate the function of peripheral cell and molecular concerning conduction and regulation of chronic inflammatory pain. In the present study，a chronic inflammatory pain animal models will be induced by the right hind paw hypodermic injection of complete freund's adjuvant(CFA). After blocking peripheral opiate receptor with naloxone , the expression of MrgC in L4-L6 dorsal root ganglion(DRG) and the transport to inflamed paw and lumbar dorsal hom(LDH), the expression of μ、δ和κopioid receptor in L4-L6 DRG and inflamed paw will be observed to expound the regulatory effect of MrgC on chronic inflammatory pain by BAM22 and different frequency of electroacupuncture(EA) , with the Bilateral thermal pain threshold and paw-swelling as an index of therapeutic effect. Simultaneously the best frequency of EA wil be uncovered. Then a MrgC knockdown CFA model with small interfering RNA (siRNA) will be built to block MrgC receptor. With a control of MrgC agonist, the influence of MrgC on chronic inflammatory pain, as well as the specific molecule mechanism of MrgC on NMDA-NO-PKCγ pathway and the role of EA on it will be observed. Thus revealing a new peripheral regulatory mechanism of opioid's non- typical opioid receptor-MrgC receptor mechanism in chronic inflammatory pain. Meanwhile a new peripheral mechanism of electroacupuncture analgesia will be uncovered.

慢性炎症疼痛广泛地存在于各种疾病的进程中，应用阿片受体激动剂吗啡与单应用炎性介质的抑制剂临床疗效都不佳，因此慢性疼痛传导与调制有关的外周细胞与分子功能研究成为镇痛研究的重要方向。本项目拟建立完全弗氏佐剂(CFA)诱导的大鼠慢性炎症疼痛模型，给与纳络酮拮抗外周阿片受体，观察L4-L6背根神经节MrgC受体的表达及至炎症足垫与脊髓背角的转运，与外周三类阿片受体表达的情况，阐明阿片类多肽BAM22与不同频率电针经MrgC受体对慢性炎症疼痛的干预效应，确立电针调节MrgC受体的最佳频率；采用 siRNA封闭MrgC受体表达，以其特异性激活剂作对照，给予电针干预，观察MrgC受体途径在慢性炎症疼痛中的作用及其调控NMDA-NO-PKCγ通路的具体分子机制，揭示阿片类物质的非阿片受体-MrgC受体机制这一新型的慢性炎症疼痛调节机制；明确电针对这一机制的调控作用，从而阐明电针治疗慢性炎症疼痛的新机理。

本项目在建立完全福氏佐剂（complete freund’s adjuvant，CFA）慢性炎性痛大鼠模型基础上，从行为学到形态学等不同方面，采用免疫组化及免疫荧光、免疫印迹和荧光定量PCR、siRNA（small interference ＲNA）干扰等分子生物学技术，观察阿片类多肽牛肾上腺髓质22肽(Bovine adrenal medulla 22, BAM22)与电针经MrgC（Mas -related G protein-coupled receptor C）对慢性炎症疼痛的干预效应，确立电针调节MrgC受体的最佳频率，观察MrgC途径在慢性炎性疼痛中及电针镇痛的作用及其调控N-甲基-D-天门冬氨酸受体-蛋白酶Cγ(NMDA -PKCγ)通路的具体分子机制，以揭示阿片类物质的非阿片受体-MrgC受体机制这一新型的慢性炎症疼痛调节机制；明确电针对这一机制的调控作用，从而阐明电针治疗慢性炎症疼痛的新机理。研究结果如下： .1．明确2/100Hz电针既能有效地干预CFA诱导的慢性炎性疼痛，又能高效地诱导MrgC基因表达，从而确立其为最佳的电针镇痛频率；.2. 鞘内注射BAM22多肽通过MrgC及阿片肽受体双重途径参与了抗慢性炎性痛，但是在炎症局部BAM22多肽及其MrgC途径没有发挥抗慢性炎性痛作用。.3. 慢性炎症除能导致炎性痛外，也能诱导背根神经节MrgC及BAM22多肽的表达，电针可通过调节MrgC途径发挥抗慢性炎性痛作用，其作用包括上调背根神经节MrgC的表达，促进背根神经节与脊髓背角BAM22多肽含量上升。.4． MrgC参与对慢性炎性痛的抑制，其作用通过下调脊髓背角NMDA受体亚基NR1相应ser890位点的磷酸化，而不包括对NR1、PKCγ的表达及其膜转位的调控。.5. 电针可通过调节MrgC发挥干预慢性炎性疼痛的作用，其MrgC途径包括下调脊髓背角NR1相应ser890位点的磷酸化，而不包括对NR1和PKCγ的表达及膜转位的调控。.