Effects Of Electroacupuncture (ea) On The Expression Of Cb2 Recepter Protein In Skin And Subcutaneous Tissue Around Inflammatory Pain Focus Of Rats

Objective : To study the effects of electroacupuncture (EA) on the expression of CB2 receptors (cannabinoid receptor 2) protein and the distribution pattern of CB2 receptors on keratinocytes and immune cells recruited to the inflammatory skin tissues.Methods : A total of 54 adult male SD rats were randomly divided into normal control group, model group, 2Hz EA group, 15Hz EA group,100Hz EA group and sham EA group. Inflammatory pain model was established by hypodermic injection of complete Freund's adjuvant (CFA , 50μl) into the dorsal surface of left hindpaws using a 25-gauge hypodermic needle . EA (2, 15, 100Hz ; 1 mA) was applied to"Huantiao"(GB 30) and"Yanglingquan"(GB 34). On the 7th day after injection of CFA, the effects of different frequency of EA on the expression of CB2 receptors protein and the distribution of CB2 receptors on keratinocytes and immune cells recruited in the inflammatory skin tissues were studied by western blot and double-labeled fluorescent immunohistochemistry. Then, according to the relevance of the effect of electroacupuncture analgesia and the expression of CB2 receptors in local inflammatory skin tissue, we screened the cells in which the expression of CB2 receptors increased the most significantly.Results :①Compared with normal control group and sham EA group, the protein expression levels of CB2 receptors in local inflammatory skin tissue of 2Hz, 100Hz EA group had significantly increased (P <0.05), but the protein expression level of CB2 receptors of 15Hz EA group didn't. The CB2 receptors protein expression level of 2Hz EA group was significantly higher than that of 100Hz EA group (P <0.05).②The result of double-labeled fluorescent immunohistochemistry:A. As to sections for macrophages: The dermis and subcutaneous layers of the model group, EA group and sham EA group were thicker than that of normal group, with a large number of infiltrating macrophages.Double-labeled cells were mainly distributed in the dermis and shallow subcutaneous layers of inflammatory side of the skin tissues, each group have macrophages and CB2 receptors double-labeled cells. The percentage of positive area of macrophages that macrophage-CB2 receptors double-labeled cells acoounted for of 2Hz EA group significantly increased than that of normal group and sham EA group (P <0.05).B. As to sections for T lymphocytes: The dermis and subcutaneous layers of model group, EA group and sham EA group were thicker than that of normal group, with a large number of infiltrating T lymphocytes. Double-labeled cells were mainly distributed in the dermis and shallow subcutaneous layers of inflammatory side of the skin tissues, each group have T lymphocytes and CB2 receptors double-labeled cells. The percentage of positive area of T lymphocytes that T lymphocyte-CB2 receptors double-labeled cells acoounted for of 2Hz EA group had no significant differences with that of normal group , model group (P <0.05).C. As to sections for Keratinocytes: The epidermal layers of model group, EA group and sham EA group were thicker than that of normal group. Double-labeled cells are mainly distributed in the epidermis of inflammatory side of the skin tissues, each group have keratinocytes and CB2 receptors double-labeled cells. The percentage of positive area of keratinocytes that keratinocytes-CB2 receptors double-labeled cells acoounted for of 2Hz EA group significantly increased than that of normal group, model group and sham EA group (P <0.05).D. Comparing the difference between the percentage of positive area of CB2 receptors positive cells that keratinocytes, macrophages and T lymphocytes accounted for, we found that keratinocytes occupied significantly higher percentage of positive area of CB2 receptors positive cells than macrophages and T lymphocytes in normal group, model group, EA group and sham EA group, (P <0.05).Conclusions : It indicated that the analgesic effect of 2Hz and 100Hz EA may be related to its function of increasing the expression of CB2 receptors protein in the inflammatory skin tissues. After the treatment of electroacupuncture, the expression of CB2 receptors in keratinocytes'membrane increased the most significantly, so the keratinocytes are probably the main cells involved in electroacupuncture analgesia.