Impact And Mechanism Studies Of Astragalus Polysaccharides On Macrophages’ Chemotaxis To Adipocytes

Background It was found that APS in rats could reduce ATMs(adipose tissue macrophages ATMs), improve IR(if insulin resistance IR) and lower BG. ATMs play an important role in the inflammatory response of adipose tissue. As found in IR appeared, the number of ATMs and pro-inflammatory macrophages increased. As macrophages migrate to adipose tissue, Adipose tissue’s release of IL-1β, IL-6, TNF-α, MCP-1 and other cytokines stimulates adipocytes’release of leptin and resistin, both of which can induce macrophages to migrate to adipose tissue, so that they release more TNF-a, IL-6 and IL-12. APS was used to impact on adipocytes and macrophages’ secretion of inflammatory cytokines so that the chemotaxis of macrophages to adipocytes could be observed. The mechanism by which reduced ATMs? Need further exploration.Objectives APS was used for inhibiting inflammatory cytokines of adipocytes and intervening in macrophages and adipocytes,This study aims to reveal the action site and mechanism of APS to reduce macrophages in adipose tissue, further improve the function of APS to prevent macrophages’ chemotaxis to adipocytes and assess the potential of APS and its preparations in clinical immunoregulation.Methods1.Cell culture:adipocytes used were 3T3-L1 cell strains of preadipocytes, and macrophages were ANA-1 cell strains. High glucose DMEM containing 10% fetal bovine serum was used to inoculate preadipocytes 3T3-L1 in culture plates which were cultured in the incubator at 37℃ with 5% CO2; after 80-90% of the plate was covered,2 days’cell fusion was carried out, and 10%fetal bovine serum high glucose DMEM was added with 0.5 mmol/L IBMX,1umol/L DEX and 10 ug/ml insulin (final concentration) to culture them for 48h; 10% fetal bovine serum high glucose DMEM with 10 ug/ml insulin (final concentration) was used for the culture for another 48h; 10% fetal bovine serum high glucose DMEM was used for the culture, changed after two days. After 8-12 days’induced differentiation,3T3-L1 preadipocytes were more than 90%, and complex lipid droplets were adipocytes. The experiment was carried out after the oil red O staining.2. In this study, Transwell technology was used. There were three groups.normal control group (NC), APS macrophages group (AM) and APS adipocytes group (AF), and each had three transwells. The macrophages were inoculated in the upper compartment of Transwell, and the adipocytes were inoculated in the lower compartment. The macrophages in the upper compartment can penetrate the 8 um polycarbonate membrane and enter the lower compartment. After preadipocytes 3T3-L1 were induced into adipocytes, the 0.1 g/L APS was added to the culture medium of adipocytes and macrophages as an intervention factor, except NC group; after 48 hours, the well was taken out, the culture medium was removed and the macrophages on the polycarbonate membrane were wiped. After fixed with 4% paraformaldehyde for 30 minutes at room temperature,30 minutes’crystal violet staining was carried out, and then the cells at the bottom of the upper compartment were counted through an inverted microscope to test the chemotaxis; the culture medium in upper and lower compartments in each group was collected for ELISA of the expression of cytokines IL-6 and TNF-a.Results1.3T3-L1 preadipocytes were induced into mature adipocytes by hormonal cocktail successfully.Oil red O staining was used to clarified matural adipocytes.2. According to Transwell count, the macrophages in AF group that entered the lower compartment due to chemotaxis were significantly less than NC group and AM group, and with P<0.05, the difference was statistically significant3. According to ELISA results of IL-6, for NF group and AF group under the lower chamber, the IL-6 expreeesion was reduced, P<0.05, so the difference was statistically significant; Comparison of AM group and NC group on the upper chamber room, for group AM and group NC on the upper chamber room, for AF room and the room under the NC group, P>0.05, the difference was not statistically significant.According to ELISA results of TNF-a, for NC group and AF group under the lower chamber, the TNF-a expreeesion was reduced, P<0.05, so the difference was statistically significant; for NM group and AM group, P>0.05, so the difference was not statistically significant. Comparison of AM group and NC group on the upper chamber room, for group AM and group NC on the upper chamber room, for AF room and the room under the NC group, P>0.05,the difference was not statistically significant.Conclusions The APS function of target cells was adipocytes to reduce macrophages’ chemotaxis to adipose tissue. APS inhibit adipocytes’release of inflammatory cytokines TNF-a and IL-6 so as to reduce ATMs.