Dendrobium Polysaccharides Promoted The Proliferation And Enhanced Anti-inflammatory Activity Of RAW264.7 Macrophages By Inhibiting STAT3 Pathway

Objective:The aim of this study was to investigate the regulatory effect of Huoshanense Dendrobium polysaccharides（DOPs）on cell proliferation,apoptosis and cycle of RAW264.7 macrophages.And we explored the function and mechanisms of the DOPs about the release of inflammation factors on RAW264.7 macrophages to enhance anti-inflammatory activity and its mechanism.Methods:We collected the fresh Dendrobium stems in Anhui province,Huoshan county.After washing,grinding,drying at 60℃to constant weight,we used hot water extraction to extract DOPs.The crude DOPs was extracted by petroleum ether and ethanol repeatedly,and then deproteinized,precipitated by ethanol and washed by organic reagent.The polysaccharide content of DOPs was calculated by phenol-sulfuric acid method and UV-spectrophotometer,and we employed the high-performance gel permeation chromatography（HPGPC）to detect the molecular weight of DOPs.High-performance ion exchange chromatography（HPIEC）was used to determine the monosaccharide composition of DOPs,and fourier transform infrared spectroscopy（FT-IR）was used to analyze the functional group composition of DOPs.Furthermore,we detected the relative m RNA level of signal transduction and activator of transcription（STAT3）in RAW264.7macrophages by q RT-PCR,as well as the inflammation factors like interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）and inducible nitric oxide synthase（i NOS）for anti-inflammatory activity studies.Moreover,after RAW264.7macrophages were steadily transfected with lentivirus and screened by puromycin,we obtained the stable RAW264.7 macrophage models of sh STAT3 and oe STAT3.We selected cell counting kit（CCK-8）assay to test the cell multiplication rate of RAW264.7macrophages.For the apoptotic cell and cell cycle of RAW264.7 macrophages,we chose the flow cytometry.In the meantime,the protein levels of Cysteinyl aspartate specific proteinase-3（Caspase-3）and Cyclin-dependent kinase-4（CDK-4）were detected by Western Blot.The results showed that DOPs were the treatment group and lentinan were deemed as positiye control group.Results:The content of polysaccharides from DOPs was 21.27%by phenolsulfuric acid method and UV-spectrophotometer.The molecular weight of DOPs determined by HPGPC was 3.74×105 Da.HPIEC determined that the monosaccharides composition of DOPs was primarily mannose,glucose.Moreover,the amount of substance ratio of mannose was 77.1%and glucose was 20.0%.FT-IR infrared spectrum identified DOPs in 3600-3200 cm^-1 is the stretching vibration absorption peak-OH,and this is characteristic of sugar absorption peak.And 2933cm-1 absorption peak is polysaccharide C-H stretching vibration.In the macrophage model of DOPs,the results showed that DOPs significantly promoted the proliferation of RAW264.7 macrophages,decreased the expression level of STAT3 in RAW264.7 macrophhages,inhibited the apoptosis,increased the number of cells in the S phase of cell cycle and increased the secretion levels of cytokines TNF-α,IL-1β,i NOS and IL-6.After the stable transfection of lentivirus,the sh STAT3 promoted the proliferation of RAW264.7 macrophages,inhibited the apoptosis and increased the S phase of cell cycle.Oe STAT3 showed the opposite effect.After treatment with DOPs,cell proliferation inhibition,apotosis increase and S phase cell number decrease caused by oe STAT3 were effectively reversed,and the effect was more obvious than the results of positive control group treated with lentinan.Conclusion:After extraction,purification and indentification of DOPs,we found the DOPs could significantly improve the proliferation of RAW264.7 macrophages,reduce the incidence of apoptosis,promote S phage of process of cell cycle,increase the secration of TNF-α,IL-1β,i NOS and IL-6 cytokines levels to exert anti-inflammatory effects.We concluded that the mechanism of action may be the STAT3 signaling pathway was inhibited.