| Objective1.A homogeneous polysaccharide was extracted from polyporus,and its primary structure and physicochemical properties were analysed.2.To explore histopathological structure and relevant protein expression of the BBN bladder cancer rats treated with plyporus.3.To explore the effect on macrophage polarization treated with polyporus in the co-culture microenvironment and to uncover its possible molecular mechanisms at the level of gene and protein.4.To explore the polarization effect on IFN-?-stimulated macrophage treated with plyporus in the co-culture microenvironment,and to investigate its molecular mechanism at the level of phenotype,gene and protein.Methods1.The crude extract of Polyporus umbellatus(Pers.)Fries polysaccharide was obtained by the method of hot water.The non-sugar impurities and crude polysaccharide protein was removed through ethanol precipitation and sevag method.DEAE-52 assay was used to remove the depigmentation of the crude polysaccharide.A homogeneous polyporus polysaccharide,here named HPP,was purified and isolated through the mothed of Sephadex G-100 gel column chromatography.UV spectrum was used to investigate the physicochemical properties of homogeneous polysaccharide.The primary structure analysis of homogeneous polysaccharide was determinated by IR spectrum and NMR analysis.2.The control group,model group,water decoction of polyporus(WDP)group,and polyporus polysaccharide(PPS)group were set up by establishing the BBN bladder cancer model of rats,and then measured the body weight every day.HE stain and immunohistochemistry assay were used to observe the pathological changes and related proteins in rat bladder.3.The cells were incubated with fresh medium or T24 cell supernatant and the control group,the co-culture control group,the IFN-? group,the co-culture IFN-y group,the administration group and the co-culture administration group were set up.MTT assay was used to detect the viability of RAW264.7 macrophages treated with WDP and PPS.Griess method was used to measure the secretion of NO on RAW264.7 macrophage treated by WDP and PPS.PCR method was used to detect the expression of gene INOS,IL-6,TNF-? and IL-1? on RAW264.7 macrophage,which is treated by WDP and PPS.The expression of gene INOS,IL-6,TNF-? and IL-1? on IFN-?-stimulated macrophage RAW264.7 treated with PPS was detected too.ProcartaPlexTM Multiplex Immunoassay was used to detect the secretion of IL-6,IL-23,RANTES,MCP-1,MIP-?,MIP-1?,TNF-a and IL-1? in RAW264.7 macrophages treated with WDP and PPS.In addition,the secretion of IL-6,IL-23,RANTES and TNF-? of IFN-?-stimulated RAW264.7 macrophages treated with PPS were measured.The protein expression of P65-NF-KB,INOS,IKB-?,Cox2,STAT3,P38 and JNK on RAW264.7 macrophage treated with WDP and PPS were carried out using western blot assay.In addition,the members of NF-KB,including P65-NF-KB,INOS,I KB-? and Cox2 in IFN-y-stimulated RAW264.7 macrophage treated with PPS was detected.4.MTT method was used to detect the viability of RAW264.7 macrophage treated with HPP.The expression of INOS,IL-6,TNF-? and IL-1? mRNA was investigated through PCR assay after HPP treatment.Griess assay was used to determine the secretion of NO on macrophages treated with HPP.ProcartaPlexTM Multiplex Immunoassay observed the secretion of IL-6,IL-23,RANTES,MCP-1,MIP-1?,MIP-1?,TNF-? and IL-1? on RAW264.7 macrophages after treated with HPP.The membrane molecule CD16/32,CD40,CD11b,CD282,CD40 and CD284 of RAW264.7 macrophage treated with HPP were analysed by flow cytometry.In addition,the expression of CD282 and CD80 in IFN-y-stimulated RAW264.7 macrophage treated with HPP were carried out.The protein expression of Cox2,INOS,P50,P65-NF-KB,NLRP3,Caspase-1,P38,JAK2,stst3,IKB-? and JNK in RAW264.7 and THP-1 macrophages treated with HPP was investigated by western blotting.And the protein expression of Cox2,INOS,P50,P65-NF-KB and IKB-?in IFN-y-stimulated RAW264.7 macrophages treated with HPP were detected too.SC75741 and T2418 were used to inhibited P65-NF-KB and stat3 protein respectively,and then detected the protein expression of Cox2,INOS,P50,IK B-?,P65-NF-K B,NLRP3,Caspase-1,P38,JAK2,stst3,and JNK on RAW264.7 and THP-1 macrophages treated with HPP.After incubating with T2418,the secretion of IL-23,MCP-1 and TNF-? on RAW264.7 macrophages treated with HPP were measured by ProcartaPlexTM Multiplex Immunoassay.Results1.We obtained a homogeneous polysaccharide from polyporus through the method of hot water,alcohol precipitation,protein removal preparation,DEAE-52 assay and Sephadex G-100 gel column chromatography.2.Compared with the control group,the weight of BBN rats were decreased,while the weight of PPS and WDP group was increased.Compared with the control group,the expression of CD163 of PPS and WDP group was decreased,while the protein expression of IL-6 and INOS were increased.3.In the medium and the co-culture microenvironment,after treating with WDP and PPS,the secretion of NO on RAW246.7 macrophage were increased in comparison to the control group.The secretion of inflammatory cytokines including IL-6,IL-23,RANTES,MCP-1,MIP-1?,MIP-1?,TNF-? and IL-1?,and the expression of INOS,IL-6,TNF-? and IL-1? mRNA were enhanced on RAW246.7 macrophage treated with WDP and PPS,Compared to the control group,the proteins expression of P65-NF-KB,IKB-?,Cox2 and INOS of NF-KB pathway were increased after treatment with WDP and PPS.And the expression of P38,JNK and STAT3 protein of STAT3 pathway were increased too.In the medium and the co-culture microenvironment,WDP and PPS promoted macrophages to M1 subtype probably through NF-KB and STAT3 pathways.In addition,after treating with PPS,the phagocytic rate of IFN-?-induced RAW246.7 macrophage was increased;the expression of IL-6 and TNF-? mRNA were increased;the secretion of INOS,IL-6,TNF-? and RANTES were increased;the expression of macrophage membrane molecules CD284,CD86 and CD40 were increased;the protein expression of NF-KB members including Cox2,INOS and P65-NF-KB were increased after PPS treatment.4.In the co-culture microenvironment,after treated with HPP,the secretion of NO was gradually increased with the increased dose.After treated with HPP,the expression of INOS,IL-6,TNF-? and IL-1? mRNA on RAW246.7 macrophage were upregulated with the increased dose.After treated with HPP,the secretion of RANTES,MIP-1 ?,IL-23,MIP-1?,INOS,IL-6,MCP-1,TNF-? and IL-1? in RAW246.7 macrophage were gradually increased with the increased dose.After treated with HPP,the expression of macrophage membrane molecules CD14,CD284,CD282,CD86,CDllb and CD80 on RAW246.7 macrophage were upregulated with the increased dose.After treated with HPP,the protein expression of NF-KB/NLRP3 pathway including I KB-?,IKK ?/?,Tak1,Cox2,INOS,P50,P65-NF-KB,NLRP3 and Caspase-1 in RAW246.7 and THP-1 macrophage were upregulated with the increased dose.After treated with HPP,the protein expression of STAT3/P38MAPK pathway including JAK2?STAT3?P38 and JNK in RAW246.7 and THP-1 macrophage were upregulated with the increased dose.To provide further evidence that NF-KB pathways participate in macrophage activation induced by HPP,HPP treated macrophages were exposed to a specific P65-NF-K B inhibitor,SC75741 and a specific STAT3 inhibitor,T2418.Compared with the HPP group,the protein expression of IKB-?,IKK?/?,TAK1,Cox2,INOS,50,P65-NF-KB,NLRP3 and Caspase-1 of the NF-KB/NLRP3 pathway on RAW246.7 and THP-1 macrophage were inhibited after treated with SC75741.After treating with T2418,the protein expression of STAT3,P38 and JNK of the STAT3 pathway in RAW246.7 and THP-1 macrophage,and the secretion of IL-23,MCP-1 and IL-6 in RAW246.7 macrophage were decreased compared to the HPP group.After treating with HPP,the expression of IFN-?-induced RAW264.7 macrophage membrane molecules CD282 and CD80 was increased.The protein expression of NF-KB members including Cox2,INOS,IKB-? and P65-NF-KB in IFN-?-induced RAW246.7 macrophage were increased after HPP treatment.Conclusions1.Chemical structure characterizeation showed that HPP is a new polysaccharide.2.The mechanism of anticancer of polyporus treatment in bladder cancer rat may be associated with the polarization of the macrophages.WDP and PPS made macrophage polarize to M1 subtype may be through the NF-KB and P38MAPK pathway.And PPS can further activate the IFN-?-induced macrophages in tumor microenvironment.3.HPP polarizated macrophages to M1 subtype through the NF-KB/NLRP3 and P38MAPK/STAT3 pathway,and can further polarize the IFN-?-induced macrophages in tumor microenvironment. |
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