LIU Nannan, WANG Yu, ZHANG Xiaohong, HUANG Shiqi, ZHOU Minjie, JING Mingyi, CAI Dingjun

（Chengdu University of TCM, Chengdu 610032）

Abstract:  Objective:  The  excess  and deficiency  of  yingfen  and  weifen,  yin and  yang,  the  mutural restriction and reinforcement of yin and yang of zang-fu organs, as well as the circulation of qi and blood and body fluid inside human body change with time.Based on the  theory, acupuncture methods related  colosely  to  chronomedicine  such  as  acupuncture  based  on  midnight-noon  ebb-flow  were formed.These methods of puncturing certain acupoints at different time points can be more effective. Changes over time in acupoint area  may be important to the choices of puncture time. Fibroblasts, as the largest number of cells in loose connective tissue, are the cellular basis for mechanical stimulation of acupuncture. Acupuncture can cause fibroblast skeletal remodeling and has obvious time specificity. Previous studies have shown that acupuncture cause the fibroblast skeletal changes at the acupoint area is  most  significant  at  the  ZT12  time  point,  whereas  the  least  significant  at  the  ZT16  time point.Fibroblasts  can  sense  and  transmit  mechanical  stimulation,  they  can  also  transform  this mechanical stimulation into mechanical signals, resulting a series of biological effects in cells. This signal transformation of fibroblasts is due to the receptor effect of its cytoskeleton. But the molecular mechanism of how this stimulation transform into chemical signals is still not concluded. P38MAPK is a classical pathway in the MAPK signaling pathway, and the P38MAPK inhibits the MRP8/MRP14 complex  by  causing  MAP14(S100A9)  in  the  excess  calcium  phosphate  binding  protein  to  cause microtubule multimerization.  In  this  study,  we  selected  the  classical  P38MAPK protein pathway  to explore the molecular mechanism of how acupuncture mechanical stimulation change into chemical signals. Methods: Put clean graded male SD rats(180-220g) into animal domestication rhythm isolated units for 7 days, choose a total of 36 rats that have basic pain threshold within 3-10s. The selected 108 rats were randomly divided into two groups, including model group (n＝24) and blank group(n＝12). The method of modeling was to inject CFA 0.1 ml into plantar surface of the model rats' right hind paws by  intradermal  injection,  and  the  blank  group  was  injected  with  equal  amount  of  NS.  After  the succession of modeling, the rats were randomly divided into model group and acupuncture group, each group contains 12 rats. Then according to the different treatment time, each group was classified into 2 subgroups (ZT12、ZT16), each subgroup contains 6 rats. On the second day of successful modeling, the rats in acupuncture group received acupuncture treatment at the corresponding time point, while the blank group and the model group are only tied   up at corresponding time point without any treatment. Methods of operation: use 0.5 inch needles, puncture the rats’ “Zusanli” on the affected limb, with Twirling manipulation for a minute after every five minutes, the treatment lasts thirty minutes in total. After 7 days of treatments, take the skin and subcutaneous tissue of rats’ acupoint area of “Zusanli” on the  affected  limb,  and  the  expression  of   P38  /  P-P38  at  the  acupoint  area  was  detected  by immunohistochemistry.  Results:  In the present study, at the ZT12 time point, the expression levels of P-P38MAPK  and  P38MAPK  in  the  model  group  were  significantly  higher  than  those  in  the  blank group(P <0.05).While the expression of P-P38MAPK and P38MAPK in the acupuncture group was significantly lower than that of model group (P <0.05).At the time point of ZT16, the expression of PP38MAPK  and  P38MAPK  in  the  model  group  was  higher  than  that  in  the  blank  group  (P <0.05).Comparison at different time points between groups show that, the P-P38MAPK and P38MAPK expression at ZT16 time points in the blank and acupuncture group is   higher than the ZT12 time point(P  <0.05),and  the  expression  of  P-P38MAPK  and  P38MAPK  in  the  model  groupat  ZT16  is significantly higher that ZT12 time point(P <0.01).  Conclusion:  Inflammatory pain can significantly 

increase the expression of P38MAPK and phosphorylated P-P38MAPK in the acupoint area. The effect of acupuncture on the reconstruction of fibroblast skeleton at ZT12 was better than that of ZT16. This may be related to the decrease of P38MAPK expression level is more obvious at ZT12 than ZT16 time point and the reduction amplitude of P-P38MAPK by acupuncture was smaller than that of ZT16.

Key words: Acupuncture, Fibroblasts Cytoskeleton, Signal Channel Mechanism