| Background:More than one huandred kinds of polysaccharides have been reported to date,whose pharmacologic action including immunological regulation, anti-tumor,anti-trauma, anti-thromb and diabetes therapeutical effect. More than 90%polysaccharides show immunological regulation,which may be related to theregulation of macrophage(Mφ). Many kinds of polysaccharides can stimulateMφto release certain cytokines. So it is supposed that possible mechanisms mayrelated with the binding with pathogen recognition (PRR) on Mφand thefollowing activation of certain signal pathway. Manose recrptor (MR), a kind ofPPR, is expressed on Mφ. It specifically recognizes and binds to ligands, whichinclude terminal D-mannose, fucose, N-acetylglucosamine, mediate thephagocytose of antigen, regulate the secretion of cytokine and start the immuneresponse.Ourstudies indicatethat Rheum tanguticum polysaccharide (RTP)canincrease the proliferation of CD4+T cell and decrease the secretion of IFN-γ. While it shows the potential capacity of binding with MR. RTP hasimmunological regulation, but the immunological regulation is unclear. In the present study,we aims to explore the features of Rheum tanguticumpolysaccharide(RTP)getting mixed with macrophage mannose receptor(MMR)and the influence on macrophage's immunological function.Use Mannose-FITC-BSA as the model(which can bind with mannose receptor specially,)mannose aspositive control and galactose as negative control.Use fluorescence microscopeand multifunctional enzyme-marked equipment to detect the uptake ofMan-FITC-BSA after incubated with Mφfor 1h.Through competitive inhibitionexperiment,we incubate RTP,APS or Man with Man-FITC-BSA for 60min toobserve whether RTP,APS can get mixed with MMR.Then we use the ELISA kitto detect the secretion of Mφ's TNF-αand IL-4 after the stimulation of Mannose,RTP and APS.To investigate the mechanism of immunoregulation of plantpolysaccharides initially.Methods1.The express and disposition of MMR of rat:Peritoneal macrophage wereacquired through irrigation of peritoneal cavity.After purification andidentification,use immunohistochemistry to detect the express and disposition onmacrophage.2.The binding between Peritoneal MMR and its ligands:Peritonealmacrophage were acquired through irrigation of peritoneal cavity.Afterpurification,macrophage were incubated with fluorescent isothiocyanate(FITC)-labeled mannosylated albumin(Man-FITC-BSA)(10-6,10-5,10-4,10-3,10-2g/L)for 60 minutes,the affinity of MR and its lignads were measured bymultifunctional enzyme-marked equipment and fluorescene microscope.3.The binding between RTP and Peritoneal MMR of different condition:The ulcerative colitis model of rats was produced by coloclysis with TNBS/40%alcohol.Six days later,put rats to death and obtained peritoneal macrophage,then macrophages were divided into control guoup,the model group,D-mannosegroup,RTP-1,RTP-2,APS-1,APS-2,APS-3(10-4,10-3,10-2,10-1,1.0 g/L).After 1h's incubation with Man-FITC-BSA(10-2g/L),fluorescence intensity weremeasured by multifunctional enzyme-marked equipment.4.The influence on Mφ's function between RTP and Peritoneal MMR ofdifferent condition:Mφwere divided into control guoup,the model group,D-mannose group(1.0 g/L),RTP-2 group(10-2,10-1,1.0 g/L),APS-3 group(10-2,10-1,1.0 g/L),RTP-2(1.0 g/L)+Man(1.0 g/L)group,APS-3(1.0g/L)+Man(1.0 g/L)group.After 24h's incubation,TNF-α,IL-4 were measured byELISA kit.Result:1.The express and disposition of MMR:There are MR expressed on macrophage through immunohistochemistry.2.The binding between MMR receptor(MR)of rat and its ligands:The binding of MR and its ligands is calcium dependent,which can be inhibitedby D-mannose and EDTA,but not by D-galactose.3.The binding between RTPand MMR of different condition:The binding of MR between RTP is different.RTP-2 is more powerful than theothers,RTP-2>APS-3>RTP-1>APS-2>APS-1.On different condition the binding of MR is different,while under inflammationcondition the binding of MR is decreased compared with the nomormal.After thetreatment of RTP,the situation ban be improved.4.The influence of RTP and MMR of different condition:After stimulation Mφwith RTP and APS in vitro for 36h,RTP can induce thesecretion of TNF-α,while mannose can toltally inhibit the secretion of TNF-αinduced by RTP,while partly inhibit the function of APS.Conclusions 1.Mannose,RTP APS,can inhibit the Mφto phagocytose Man-FITC-BSA.Theintensity is Mannose>RTP-2>APS-3>RTP-1>APS-2>APS-1.Galactoseexhibits no inhibition.The difference may be related to the construction oftheir monosaccharide of the polysaccharides.2.After stimulated with APS and RTP in vitro for 36h,the secretion of TNF-αissignificant increased in normal situation compared with control groups,andhaving a character of dose dependent.At the meanwhile.No significantdifference was found in the secretion of TNF-αin Mφstimulated by mannosecompared to control groups.And No statistics difference was found in thesecretion of IL-4.RTP can induce the secretion of TNF-α,while mannose cantoltally inhibit the secretion of TNF-αinduced by RTP,while partly inhibit thefunction of APS.RTP can bind with MMR and induced the secretion ofTNF-α,which take part in Th1 cellular immunity.3.The difference between the two polysaccharide maybe related with theconstruction of their monosaccharide.
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