Immunoregulation and anti-tumor effects of Polyporusus Bellatus:a review of recent research

Miao-Miao Lin,Mei-Yu Cui,Hai-Yan Cao,Xue-Song Wu,Li-Jing Cai,Li-Hui Zhang,Guo-Wei Zhang*

1College of Chinese Medicine,Hebei University,Baoding,Hebei province,China.2Laiyuan hospital of Chinese Medicine,Laiyuan,Hebeiprovince,China.

Background

ZhuLing(Polyporus)is a common Chinese herbal medicine which is widely used in China for more than 2000 years.It is traditionally used as a diuretic agent to treat urinary tract infections[1].In recent years,the medicinal value of Zhuling has received more attention.Many medical scientists have conducted extensive researches on its active ingredients,pharmacological effects and mechanism.Polyporusus Bellatus(PPS)is the major component of Zhuling and has anti-tumor and immunoregulation efficacy.The PPS has particular effects in inhibiting tumor growth[2,3].

Polysaccharide composition known in Zhuling

Zhuling contains PPS.Five polysaccharides are currently isolated from Zhuling,whose names,types and composition of monosaccharides respectively are:(1)Polyporusus Bellatus, water-soluble polysaccharide 6-branched beta(1→3)-Dextran[4];(2)Dextran,formed by condensation of β-(1→3), β-(1→4)and β(1→6)glucosidic bonds[5];(3)Polyporusus Bellatus,composed of glucose and galactose[6];(4)Polyporusus Bellatus(1→6) β-D-glucopyranose skeleton,branched from β-D-Glcp, (1→3)β-DGlcp, (1→3) β-D-GlcpA,(1→4)β-D-Glcp and(1→4)β-D-GlcpA composition[7];(5)Polyporusus Bellatus(1→6,1→4)β-D-glucopyranose skeleton having a substituent of (1→6) β D-glucopyranose branched from (1→3)β-dglucopyranosyl[8].

The main pharmacological effectsof PPS

PPShas immunomodulatory effect

PPSis a bioactive polysaccharide.Li,XQ,et al.reported that PPS could induce the differentiation and maturation of murine bone marrow dendritic cells(BMDCs).PPS stimulates BMDCs production of Interleukin-12(IL-12)p40,up-regulates the expression of co-stimulatory molecules and enhances the stimulation of na?ve T cells via Toll-like Receptor 4(TLR4)[9].Jiang,Z.et al.concluded that M1 macrophages can be induced from RAW264.7 cells by stimulating Interferon-γ (IFN-γ)alone.In addition,PPS promoted mRNA expression of Interleukin-1β (IL-1β),Inducible Nitric Oxide Synthase(INOS),Interleukin-10(IL-10),Transforming Growth Factor-β (TGF-β)and Tumor Necrosis Factor(TNF-α)in M1 macrophages[10].Li,XQ.et al.observed the phenotype and function of PPSby 3H-TdRincorporation.Compared with the negative group,they found that PPS could increase the co-expression of CD11c and CD86 molecules on Dendritic Cells(DC)surface and the production of IL-12 and IL-10 in a dose-dependent manner.PPSalso enhanced matured BMDCs capacity of T cell initial activation and decreased phagocytosis of BMDCs.Anti-TLR4,but not anti-Toll-like Receptor 2(TLR2)or complement receptor 3(CR3)monoclonal antibodiesinhibited PPS-induced production of IL-12 p40 and blocked the combination between fluorescence-labeled PPS(f-PPS)and BMDCs.Their data demonstrate that PPS could promote the activation of murine BMDC via TLR4 and maturation of immunomodulationy activity [11].Li,JF,et al.investigated the actions of PPS, mycobacterium polysaccharide (MPS) and lentinan (LEN) on lymphokine-activated killer(LAK)cell activity in vitro in this study.Human peripheral blood mononuclear cells(PBMC)were cultured for 96 hours with medium containing different concentrations of the above-mentioned drugs in combination with Recombinant Interleukin-2(rIL-2).Then cell-mediated lysis was determined by 1H-TdR release assay including natural killer cell(NK)sensitive and natural killer cell resistant target cells.They concluded that when combined with rIL-2 in a certain concentration,all three kinds of polysaccharides could enhance the LAK activity by 42%-56.9%,and reduce the dose of rIL-2 by 50%.It suggested that the PPS,mycobacterium polysaccharide and lentinan could be used as bioactivity regulators in LAK cell therapy in tumor treatment[12].Nie Hong,et al.confirmed that the single Rprescription of PPS showed a rebound in lymphocyte transformation ability,killing activity of NK cells,total number of T cells and IgG antibody production ability in mice with cyclophosphamide[13].Zhang Juncai,et al.confirmed that PPS can enhance humoral and cellular immune responses in Cavia porcellus,and PPS is a potent immunopotentiator[14].Hou Gan,et al.found that PPS can increase INOS activity in mouse peritoneal macrophages,promote Nitric Oxide(NO)synthesis,and consume intracellular Glutathione(GSH).It suggested that intracellular GSH may play a role in regulating NO production in macrophages and protecting host cells from NO-mediated cytotoxicity[15].Zhang,et al.showed in vivo studies that the synergistic effect of PPS combined with Bacille Calmette-Guerin(BCG)could significantly increase the expression of co-stimulatory molecules CD86,CD40 and TLR4/CD14.Immunohistochemical analysis showed that CD86 and CD40 staining were more pronounced[16].Jiang Zb,et al.studied the effect of PPS on the regulation of lipopolysaccharide(LPS)-induced cytokines in the J774 macrophage inflammation model,and studied its anti-inflammatory mechanism which may be through the mitogen-activated protein kinase(MAPK)signaling pathway to reduce inflammatory damage[17].Jiang Zb et al.observed the effect of PPS on INF-γ inducing M1 subtype macrophage membrane surface protein and its effects on related cytokines IL-1β,Interleukin-6(IL-6),and TNF-αmRNA.In macrophage M1 type,increasing the expression of M1 inflammatory cytokines induced by INF-y and the anti-inflammatory cytokine at the same time would have an immunomodulatory bidirectional regulation[18].Xu,et al.observed that PPS could promote the proliferation of mouse spleen cells and the production of NO,IL-1β,TNF-αand TLR4 in peritoneal macrophages[19].It was speculated that PPS may activate mouse peritoneal macrophages through TLR4 and play a role in immune regulation.Sun,et al.found that PPS can increase the number of NK and LAK in the spleen of mice and promote the proliferation and differentiation of lymphocytes B and lymphocytes Tin themouse[20].Dai,et al.found that PPS can effectively activate B cells,macrophages,and tree-like cells[21].The polysaccharide epitopes are related to environmental antigens that can trigger adaptive humoral immune responses.PPS is the main component in the immune regulation of Zhuling.Pan WL,et al.found that the PPS had obvious amplification effect on umbilical cord blood hematopoietic stem cells [22].It could promote transplantation mice's immune hematopoietic reconstruction of cord blood hematopoietic stem cell.In the survival rate,blood recovery and the terms of time and effect,the PPStransplantation group was superior to the transplanted mouse group,and there were significant differences in the levels of CD3+,CD4+,CD8+,and CD19+.Zhang,et al.used injection of type IIcollagen to establish a rat model of arthritis(CIA)and isolated rat PP-lymphocyte(PPL),intestinal epithelial lymphocytes and lamina propria lymphocytes,co-cultured with different concentrations of PPS[23].The results showed that PPShad different immunomodulatory effects on rat's PPL,which could reduce the secretion of TNF-αby PPL and increased the secretion of IFN-γ.The levels of TNF-α and IFN-γsecreted by Intestinal Epithelial Lymphocytes and Lamina Propria Lymphocytes are reduced to different degrees.Li X,et al.found that PPS can strongly upregulate the function of macrophages,such as the production of NO and the expression of cytokines.PPS significantly stimulated the proliferation of splenocytes and the production of TNF-α,IL-1β and NO of peritoneal macrophages from C3H/HeN mice.The function blocking antibodies to TLR4,but not TLR2 and CR3,markedly suppressed PPS-mediated TNF-α and IL-1β production.Nuclear translocation and DNA binding activity of Nuclear factor kappa beta(NF-κB)were significantly induced by PPS.Their data suggest that PPS may exert its immunostimulatory effects through TLR-4 activation of thesignaling pathway[24].

Liver protection

PPS could reduce the damage of carbon tetrachloride to mice liver,cut down liver pathological damage,decreased serum alanine aminotransferase activity,prevented the liver 6-phosphoglucomutase and bound acid phosphatase activity decreasing[25].Huang DN,et al.demonstrated that the protective function of PPS used Carbon Tetrachloride(CCl4)-induced hepatic injury in mouse model.The effects of PPSwere evaluated by biochemical values and histopathological examinations;mRNA expression was measured by real-time Polymerase Chain Reaction(PCR).They found PPS dose-dependently alleviated hepatic injury manifested by the recovery of Lactate Dehydrogenase(LDH),Aspartate Transaminase(AST),Alanine Transaminase(ALT),Malondialdehyde(MDA),reduced GSH,Glutathione Peroxidase(GPx),Catalase(CAT)and Superoxide Dismutase(SOD)levels.Histopathological examination also confirmed the alleviation of hepatic injury.Meanwhile,the suppressed mRNA expression of GPx,CAT and SOD by CCl4 were restored by PPStreatment.These data indicated that PPS are protective against CCl4-induced hepatic injury,and the mechanism involves the upregulation of GPx,CAT and SOD expression[26].Liu XL,et al.established a model of alcoholic fatty liver in rats and studied that PPS can reduce liver cell inflammation in alcoholic fatty liver rats,confirming that PPScan repair liver tissue damage to some extent[27].Tao J,et al.found that the positive rate of serum HBsAb in patients with PPS combined with hepatitis B vaccine was higher than the rate of serum HBsAb in patients with hepatitis B vaccination alone,and the infection rate of hepatitis B was significantly lower,which shows that PPSis the main active ingredient in the lowering of blood fat and liver protection of piglet[28].Du JL,et al.found that PPS could inhibit hepatocyte injury caused by CCl4,decrease the activity of Alanine Transaminase, Aspartate Transaminase, and Malondialdehyde in hepatocytes and increase the survival rate of hepatocytes.At the same time,CYP3A mRNA expression was significantly induced by PPS,which has the effect of protecting hepatocytes[29].Liu XL et al.constructed a rat alcoholic fatty liver model and set up a small dose(0.3 g/kg),medium dose(1g/kg),high dose(3 g/kg)PPS group[30].The results showed that the levels of Cholesterol(TC)and Triglyceride(TG)in the serum of fatty liver rats in each dose group decreased,and the degree of hepatic cell steatosis was reduced.PPS has the effect of treating alcoholic fatty liver in rats.The curative effect of PPS on chronic hepatitis B is relatively certain.Whether it is used alone or combined with other traditional Chinese Medicines,Hepatitis B vaccine,interferon,lamivudine,et al.,it could effectively inhibit the replication of Hepatitis B virus and increase HBeAg and HBV-DNA's negative conversion rate and anti-HBe positive rate to improve liver function.Especially in combination with other drugs,it could play a therapeutic role in different aspects,such as improving efficacy,and some extent reduce the adverse reactions of other drugs,which opened up a new way for the combination of Chinese Medicine and Western Medicine treatment of chronic Hepatitis B[31].

Anti-tumor effect

Bladder cancer is one of the most malignant tumors closely associated with macrophages.PPS has shown excellent efficacy in treating bladder cancer with minimal side effects.PPS has been addressed to be highly effective in inhibiting bladder carcinogenesis in rats[32].In addition,Zhuling demonstrated clinical efficacy against bladder cancer [33]. Peroxisome Proliferator-activated Receptor Gamma(PPAR-γ)is a member of the nuclear receptor superfamily of ligand-activated transcription factors that has been associated with theinhibition of tumor cell growth.Li CX,et al.assessed the expression of PPAR-γin human T24 bladder cancer cells by immunocytochemistry,Real-time PCR,and Western blot.They concluded that PPS inhibited the growth of bladder cancer T24 cell.The PPAR-γantagonist biphenol A diglycidyl ether(BADGE)reversed PPS meditated cell growth inhibition[34].Studies have confirmed that PPS inhibits human bladder cancer cell line T24 cells,inhibits the proliferation of cells and prevents S-phase cells from entering G2/M phase.PPS inhibits cell proliferation by interfering and affecting T24 cell cycle.At the same time PPS can also make T24 cell morphology change,slow down cell proliferation,leading to the decrease of adherent cells and the increase of dead cells[35].Shen G,et al.treated the control group T24 cells normally,and the experimental group cells were incubated with different doses of PPS.Compared with the control group,they found that the cell apoptosis was obviously increased,the bcl-2 mRNA stability and bcl-2 mRNA and protein expression were decreased in the medium-and high-dose PPS groups,the total protein of Hu R was slightly changed,but the cytoplasm Hu R protein was down-regulated and the nucleus Hu R protein was up-regulated.They concluded that PPScould induce the apoptosis of T24 cells,which is possibly related with the down-regulation of the cytoplasm Hu R protein and with the decrease of bcl-2 m RNA stability and bcl-2 protein expression[36].Liu CP,et al.used macrophages cultured alone or with T24 human bladder cancer cell culture supernatant as study models.They found that PPS enhanced the activities of IFN stimulated RAW 264.7 macrophages,as shown by the release of INOS,secretion of TNF and Interleukin-6(IL-6),phagocytosis activity,as well as expression of M1 phenotype indicators,such as CD40,CD284 and CD86.PPS acted upstream in activation cascade of nuclear factor NF-κB signaling pathways by interfering with NF-κB phosphorylation.In addition,PPS regulated NF-κB P65 signaling by interfering with Toll-like receptor TLR-4,INOSand cyclooxygenase COX-2.Their results indicate that PPS activates macrophages through TLR4/NF-κB signaling pathways[37].

The anti-tumor effect of PPSwas found to be related to its promotion of NO release from macrophages,and the simultaneous stimulation of macrophages with rBCG-IL-2 and PPS was stronger than that stimulated with rBCG-IL-2 alone[38].Huang,D.N.et al.observed the production of NO,the activity and mRNA expression of INOSin peritoneal macrophages of mice administered with different dose of PPSby Griess reaction,fluorimetry assay and RT-PCR,respectively.They found that PPS could elevate the iNOS activity with dose-dependence and stimulate the iNOS mRNA expression of peritoneal macrophages in mice.As a result,they think that the regulation of PPS on the production of NO in peritoneal macrophages of mice may occur at transcriptional level of iNOS.This indicates that the mechanism of PPS's antitumor and immunoregulation functions may be related to increasing NO output of macrophages through stimulating iNOS's denovo synthesis[39].

It was found that PPS could promote the expression of CD14,TLR2,and TLR4 molecules and the expression of adhesion molecule CD11b on the surface of macrophages in the early stage of BCG synergy.Therefore,TLR molecules and signaling pathways may be used as one of the main pathways to mediate PPSand BCG-induced organism immunity and induce anti-tumor effects[40].It has also been found that rBCG can directly enter the cytoplasm of T24 cells,while PPShas the effect of promoting the transfering of rBCG from the cytoplasm to the nucleus of tumor cells[41].After T739 cells were treated with PPS,BCG and their combination,the changes in mRNA and protein expression of inhibitor of kappa B kinase beta(IKKβ),NF-κB subunit p65(NF-κB p65),ICAM1 and CCL2 in bladder cancer cell line T739 were determined by relative quantitative real-time PCR,Western blot and flow cytometry. NF-κB p65 DNA-binding activity in T739 cell was detected by biotinylated probe-ELISA,and NF-κB p65 nuclear expression in T739 cell was observed by immunohistochemistry.Wei JA et al.concluded that PPS could inhibit the over-activation of the NF-κB signaling pathway induced by BCG in bladder cancer cells and accordingly attenuate the adverse reactions to BCG therapy[42].In a study,Zhang G W et al.found that bladder cancer development in model rats exhibited significantly reduced cancer invasiveness with Zhu Ling PPS combined with BCG.Flow cytometric analysis showed expression of costimulatory molecules CD86,CD40 and TLR4/CD14 significantly increased with Zhu Ling PPS in combination with BCG.Their findings showed that Zhu Ling PPS strongly reduced side effects and displayed synergistic effects during BCG instillation in rat bladder cancer models[16].It was found that PPS combined with BCG can enhance the expression of CD11b,CD18 and costimulatory molecules[43].Zhang et al.found that PPS in combination with BCG could inhibit bladder cancer in rats and reduce the side effects of BCG in the body[16].At the same time,the expression of CD86,CD40,and TLR4/CD14 was increased in rat peritoneal macrophages and bladder epithelial cells.CD86 of cancer tissue epithelial cells was not expressed by cancer cells.Yang Dean,et al.used a carcinogen OH-BBN to make a bladder cancer model in female rats and fed 90 g/kg dry powder of swine fever.After 30 weeks,the rats were sacrificed.They observed the effects of Zhuling on bladder cancer rats.The results showed that the total tumor rate of the bladder reduced by 38.9%compared with the pathological control group.The tumor diameter per rat was significantly lower than the pathological control group.The cancer rate decreased by 66.7%compared with the pathological control group.It is shown that Zhuling has a significant inhibitory effect on the occurrence of OH-BBN bladder cancer,and there are no obvious side effects[44].Cui C,et al.studied in vitro the polysaccharide secreted by PPS to suppress the secretion of immunosuppressed molecules in tumor cells and found that PPS had an immunosuppression effect on the tumor in colorectal cancer Colon26 cells[45].Li CX,et al.used BBN and saccharin water to induce the establishment of Fisher344 rat bladder tumor model[46].Studies have shown that Zhuling and PPS can affect the occurrence and development of tumors by affecting the thymus,spleen index,and lymphocyte infiltration and CD86 expression in bladder tissue and paracancerous tissues of bladder cancer model rats.Qin GF,et al.showed that Zhuling had a significant inhibitory effect on the bladder cancer model induced by N-butyl-N-4-hydroxybutylnitrosamine(BBN)in a rat model of bladder cancer.[47]The expression of Aquaporin 1(AQP1)and Aquaporin 3(AQP3)protein indicates that the hydrophobilization efficacy of swine fever can inhibit the development of bladder cancer by inhibiting the expression of AQP1 and AQP3.Jiang ZB,et al.used macrophage RAW264.7 as a vector to induce the Inflammation of M1 subtype macrophages by IFN-γ[48].They studied the effect of PPS on M1 membrane surface protein expression and its related cytokines,and explored theinhibitory mechanism of PPSon tumors.The results showed that PPS has a bidirectional regulation effect and can polarize macrophages to M1 type and increase the expression of M1 inflammatory factor.Zeng,et al.have found that PPSmainly inhibit TLR4 signaling pathway by inhibiting the expression of related genes,NF-κB p65 DNA's binding activity,and nuclear expression to inhibit tumor growth[49].Studies have confirmed that the component ergone in Zhuling suppressesthe growth of human tumor cells HT-29(colon cancer),HeLa 229(cervix cancer),Hep3B(livercancer),and AGS(stomach cancer),and exhibitscytotoxicity[50].

Conclusion

As a commonly used medicinal fungus,PPS is extracted from Zhuling.In recent years,through modern pharmacological research,it hasbeen confirmed to have a variety of biological activities and pharmacological effects.It makes a major contribution to the clinical application of hepatitis B and plays a major role in anti-tumor bladder cancer. Through various pharmacological researches and studies,it provides a theoretical basis for its clinical application and has important guiding significance.

Acknowledgements

The authors wish to thank Guowei Zhang(M.D.and Ph.D.,China)for hisinvaluableassistance.

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