| ObjectiveTo study the immunomodulation effects of polyporus polysaccharide with BCG on the microenvironment of bladder cancer,and explore the immunological mechanism of polyporus polysaccharide with BCG in treatment of bladder cancer,and to provide molecular basis for finding new treatment strategy and target for bladder cancer.Methods1.After the Fisher344 rat bladder cancer model was established by giving OH-BBN,the bladder cancer rat is randomly divided into Model,BCG,PPS,BPH,BPM and BPL group.The drugs were given once a week for 6 weeks by intravesical perfusion.2.The effect of drugs on bladder cancer rat was observed:HE staining of bladder tissue sections of rats was used for model evaluation.Morphological changes of bladder were observed.The indexes of bladder,spleen,thymus,kidney,liver and volume of bladder were calculated.3.The effect of drugs on immune cells of bladder,spleen,peripheral blood and related cytokines in bladder cancer rats was observed:CD3+,CD4+,CD8+and CD25+T cells,CD161+NK cells as well as CD11b+,CD68+and CD86+macrophage cells in peripheral blood and spleen were detected by FACS.The expressions of CD3,CD161 and CD68 in bladder tumor microenvironment were evaluated by IHC.The expressions of IL-6,TNF-α,IFN-y and IL-10 in serum and urine were analyzed by ELISA.4.The changes of TLR4/NF-κB and JAK2/STAT3 signal pathway molecules were evaluated by IHC.Investigated the therapeutic effects of PPS combined with BCG in bladder cancer rats and explore the possible mechanisms using UPLC-Q-TOF-MS/MS based untargeted serum metabolomic technique.Results1.In Model group,mamillary tumor protuberance were seen in the inner bladder wall,and the tumor protuberance in bladder wall of treatment groups were significantly decreased.Compared with the Model group,the volume of the bladder in treatment groups was significantly reduced(P<0.01),the bladder and spleen index of BCG,PPS and BPH group were significantly reduced(P<0.05 or P<0.01).2.FACS analysis showed that,compared with Model group,the expression of CD3+CD4+T cells in peripheral blood was promoted by BCG(P<0.05),and combination of two drugs could promote the expression of CD3+CD4+T cells,CD3+CD4+CD25+T cells(P<0.05 or P<0.01);BCG,PPS and BPH group promoted expression of CD3-CD16+NK cells(P<0.05 or P<0.01),and all drugs can promote expression of CD11b+CD68+CD86+macrophages(P<0.05 or P<0.01).Compared with Model group,BCG,PPS and BPH group promoted expression of CD3+CD4+T cells in spleen(P<0.01);BPH and BPM group promoted expression of CD3-CD161+NK cells(P<0.01).3.IHC showed that IOD SUM of CD3,CD161 and CD68 in BCG and PPS group were significantly higher than Model group(P<0.05 or P<0.01).Compared with Model group,all drugs could increase the level of IFN-6 in serum(P<0.05 or P<0.01)and lower the level of IL-10(P<0.05).The combination of drugs could increase the level of IL-6(P<0.05 or P<0.01),and PPS could increase the level of IL-6 in urine(P<0.05).All drugs could activate TLR4/NF-κB and JAK2/STAT3 signaling pathway.4.The results of discriminant analysis OPLS-DA indicated a clear separation among the control group and model group,while rats in BCG,PPS and BPH treated group fairly differed from the model group and closed to the control groupl.A total of 24 potential biomarkers were identified,including oleamide,LysoPC(20:5),linolenic acid,11-deoxycortisol,LysoPC(24:0),tryptophan,CPA(18:2),leucine,LysoPC(15:0),phytosphingosine,11,12-epoxyeicosatrienoic acid,2-phenylethanol glucuronide,glycocholic acid,LPI(18:0),cholic acid,tetrahydrocorticosterone,sphingosine 1-phosphate,docosahexaenoic acid,hydroxylinoleic acid,arachidonic acid,LPA(16:0),streptomycin,citric acid and oxymesterone.The potential mechanisms of BPH may be involved in the metabolic pathways of alpha-linolenic acid metabolism and arachidonic acid metabolism,which are the characteristics of inflammatory reaction.The therapeutical effect of BPH was confired by the serum metabolomics analysis,and the mechanism may be that PPS ameliorates the inflammatory side effects of BCG.ConclusionBCG,PPS and its combination could promote polarization of macrophages to Mlthrough TLR4/NF-κB and JAK2/STAT3 signaling pathway,and could effectively regulate the level of T cells,upregulated the levels of NK cells.Thereby promoting recovery of innate immunity and adaptive immune,and achieving the aim of treating Fisher344 bladder cancer rats induced by OH-BBN.Moreover,PPS could reduce the inflammatory reaction of BCG,which may be regulated by a-linolenic acid and arachidonic acid metabolism pathway. |
PDF Full Text Request