The Role Of P2X₇ Receptor In Fibroblast Cytoskeleton Remodeling In Acupoint Area By Chrono-Acupuncture

Objective:To clarify the regulatory effect of P2X₇ receptor on the fibroblast cytoskeleton in the acupoint area in the specific time.Methods:The selected 18 wild-type C57BL/6J mice and 18 P2X₇^-/-mice were divided into groups by the pain threshold,according to the principle of complete randomness.Wild type mice were divided into blank group（6 mice）and model group（12 mice）.Then the model group was made into an inflammatory pain model,injecting CFA 0.1ml into plantar surface of the model mice’right hind paw by intradermal injection.After successful modeling,the mice were random divided into model group and acupuncture group according to the painthreshold,and each group contains 6mice.The grouping and modeling methods of P2X₇^-/-mice were the same as the wild type mice.According to the results of previous studies,the specific time point ZT8was selected.The acupuncture group was treated with acupuncture at Zusanli at ZT8,and the needles were twisted for 1 minute with every 5 minutes,with a frequency of120 times/min,totally 0.5h.Using acupuncture once a day for 7 days.The control groups were bound in the same way for 0.5h without acupuncture.Fluorescence double-standard method combined with laser confocal imaging method was used to detect the cytoskeleton morphology of fibroblasts in the acupoint area,the expression level of F-actin andβ-tubulin,and the expression level of P2X₇ receptor on the fibroblasts in the wild-type mice.The expression of p38MAPKmRNA in wild-type mice was detected by RT-PCR.Result:1.In acupuncture group,the optical density of P2X₇ receptor in the point area of wild type mice was significantly increased:compared with the blank group,P=0.006,P<0.01;Compared with the model group,P=0.044,P<0.05.2.The expression of p38MAPKmRNA in the mice point area in the model group was higher than that in the blank group,P=0.014,P<0.05.The p38MAPKmRNA expression in acupoint area of the acupuncture group was lower than that of the model group,P=0.034,P<0.05.3.Changes in the arrangement of P2X₇^-/-mouse fibroblasts were observed under LSCM,with only a few cytoskeleton distribution.After acupuncture,it was observed that the cytoskeleton cross-sectional area was slightly increased and arranged in a filamentous,parallel to the nucleus.Compared with C57BL/6J mice,the number of cytoskeletons in each group of P2X₇^-/-mice was very small,,and the staining was not strong enough.The expression ofβ-tubulin and F-actin in the P2X₇^-/-mice acupuncture group were significantly higher than that in the blank group,P=0.000,P<0.01;Compared with model group,P=0.000,P<0.01.Conclusion:Timing acupuncture has a clear regulatory effect on fibroblast cytoskeleton remodeling in the mouse acupoint area.P2X₇ receptor of fibroblasts is involved in this process,and this regulatory effect may be related to the p38MAPK signaling pathway.