Pilot Study Of The Action Mechanism Of Acupuncture Meridian Based On The Properties Of Fibroblasts

Previous researches suggest that connective tissue is material basis of acupuncture medicine meridian and reactive oxygen species are closely related to meridian.Furthermore,function of connective tissue depends mainly on fibroblasts.Therefore,this research started from fibroblasts,especially focusing on its relationship with fibroblasts and reactive oxygen species,type I collagen in extracellular matrix,mast cells which are the crucial cell of connective tissue,and calcium ions with relative high concentration in acupuncture meridian to pilot study the action mechanism of acupuncture meridian.The experiment included two parts:in vivo and in vitro experiments.The details are as follows.(1)In vivo experiment:Rat linea alba,the connective tissue of anatomical overlapping with ren meridian,were selected for determination of reactive oxygen species level,compared with related biochemical indicators of reactive oxygen species in other groups.The rats were injected with hydrogen peroxide,glutathione and normal saline respectively through tail vein.We analysed the effect of different reactive oxygen species levels on ERK signaling pathway through Western Blot.(2)In vitro experiment:By constructing test system of reactive oxygen species in vitro,whether the main working part is in the intracellular or extracellular was determined.The interaction between fibroblasts,reactive oxygen species,type I collagen and mast cells was explored through single-cultured and co-cultured cell systems.The effect of Calcium ions with relative high concentration in acupuncture meridian on the level of reactive oxygen species inside fibroblasts and cell viability was also detected by DCFH-DA.The results are as follows.(1)In vivo experiment:Reactive oxygen species level in Linea alba is relatively high and the cells are mostly in the environment of high oxidative stress.The effect of hydrogen peroxide and antioxidant glutathione was very significant on the ERK signal pathway of connective tissue.(2)In vitro experiment:L929 cell survival rate is relatively stable when the reactive oxygen concentration is low.Type I collagen promotes proliferation of the connective tissue fibroblast L929,provide protection when they suffer from extracellular reactive oxygen species attack.Mast cells P815 can promote the secretion of type I collagen from L929 cell and change its cells form.Extracellular reactive oxygen species mainly damage type I collagen while intracellular reactive oxygen species can promote the secretion of type I collagen from L929 cell.Morphology change of L929 cell due to reactive oxygen species is reversible to a certain extent.Calcium ion in culture affects intracellular reactive oxygen species level.Low concentration of calcium ion in flow is beneficial to cell proliferation.Since it can also influence L929 cell indirectly through affecting the mast cell degranulation.Conclusion:High concentration of ROS was confirmed in linea alba,which overlapping the secretion of collagen by fibrolasts,co-existing collagen and mast cell helped to maintain the morphology and function of fibroblasts,so that ensuring the stability of the material basis of acupuncture meridian.The prompt and substantial effect of calcuim ion on fibroblasts may be one of the intend mechanism of the acupuncture.