Electroacupuncture Promotes The Intestinal Motility By Regulating BMP2 Secretion Of Muscularis Macrophages

Objective:Recent studies have shown that the mechanism of intestinal motility disorder in diabetes mellitus is related to the changes in the number and the function of intestinal muscularis macrophages.Bone morphogenetic protein 2(BMP2),as a member of TGF-? family,plays an important role in the progress of growth,development and differentiation of enteric neurons.BMP2 selectively acts on the surface of enteric neurons and influences intestinal motility.Previous studies have confirmed that electroacupuncture(EA)at Zusanli(ST36)can affect full-layer gastrointestinal macrophages and enteric neurons,but whether electroacupuncture has similar effect on muscularis macrophages and their secreted bone morphogenesis protein 2 is unclear.Based on the theories above,our study uses a long-term chronic electroacupuncture of diabetic mice as model to explore the role of phenotype and number of muscularis macrophages,bone morphogenetic protein 2,and enteric neurons in the intestinal dynamic disorder caused by diabetes.Methods: C57bl/6 healthy adult male mice were randomly divided into 5 groups:normal control group,diabetes mellitus group(DM),diabetes mellitus with sham electroacupuncture group(DM+SEA 30min/ day),diabetes mellitus with low frequency electroacupuncture group(DM+LEA,10 Hz 1mA 30min/ day),diabetes mellitus with high frequency electroacupuncture group(DM+HEA,100 Hz 1mA30min/ day).The stimulation time lasts for 8 weeks after formation of diabetic models.Flow cytometry,Western Blot,real-time PCR and Immunofluorescence were used to determine the total number of M2-type macrophages in the whole colonic layer,the relative number of M2-type macrophages in the muscular layer,the different expressions of molecules in BMP2/BMPR-Smad pathway and PGP9.5,nNOS of enteric neurons in the colon of each group.Results:(1)low frequency and high frequency electroacupuncture stimulation can improve theintestinal transmission time of diabetic mice;(2)colonic muscularis macrophages are dominated by M2 phenotype.Diabetes mellitus can lead to the decrease of the absolute content of M2-type macrophages in the whole colonic layer and the relative content of M2-type macrophages in the muscular layer.High-frequency electroacupuncture stimulation can improve these changes.(3)muscularis M2-type macrophages act through BMP2/BMPR-Smad pathway on enteric neurons.Down-regulations of BMP2,BMPR1 b,Smad1,PGP9.5 and nNOS can be observed in diabetic mice.All of these molecules can be up-regulated by High-frequency electroacupuncture stimulation,through which way it improves intestinal motility.Conclusions: The intestinal transmission time is prolonged in diabetic mice,and the decreasing relative amount of M2-type muscularis macrophages in diabetic can be observed,which also causes a down-regulation of BMP2 /BMPR-Smad signaling pathway,as well as the decreased expression of PGP9.5 and nNOS.High frequency electroacupuncture can restore all those down-regulations to improve intestinal dynamics.