Lycium Barbarum Polysaccharide Enhances The Antitumor Effect Of Doxorubicine In Hepatoma Mice

ObjectiveThe incidence and motality rates for hepatocellular carcinoma(HCC)rank high on the top three of all the cancer cases in China,and the cases account for about half of those in the world.Surgery and local ablation are the best treatment options for early-stage HCC.However,most HCC are at the late stage when diagnosed and chemotherapy is one of the best treatment options.Hepatocarcinogenesis is a multistage process under a multifactorial etiology,the cancer cells get the persistent ability of proliferation and metastasis by a variety of signaling pathways and the tumor microenvironment is complex.So,single drug treatment strategy proved to be poor efficacy in HCC treatment and combination therapy strategies may be the effective treatment options for HCC.Doxorubicin is a drug commonly used for chemotherapy of HCC.However,doxorubicin as well as most chemotherapy drugs are accompanied by severe side effects,such as bone marrow suppression and immune suppression.To reduce the toxicity of doxorubicin can help to enhance its anti-tumor effects for HCC.Lycium barbarum polysaccharide extracted from traditional Chinese medicine Lycium barbarum has multiple biological effects,such as enhancing immune function,anti-tumor,anti-oxidation.It also can reduce the side effect of bone marrow suppression induced by chemotherapy and radiotherapy.It suggests that LBP acts as anti-tumor and chemoprotection reagents.LBP may reduce the immunosuppression toxicity and enhance the anti-tumor effects in doxorubicin-treated HCC mice.Polysaccharide is consisting of a series of different molecular weight fragments that each fragment may have different biological activities.However,the active fraction and mechanism of LBP in anti-tumor and immunomodulatory is unclear.The purposes of this study is as shown below.(1)To find out the main active fragment of LBP that has the function of antitumor and immunomodulatory effects(2)To further explore the immunomechanism of anti-tumor effect of LBP.(3)To investigate whether LBP has the ability to reduce the immunosuppression toxicity and enhance the anti-tumor effects in doxorubicin-treated HCC mic.Methods and results1.The analysis of ultraviolet spectrum,infrared spectrum and ELISA kit for LBP fragementThe qualitative analysis of nucleic acids and proteins in the five LBP fragments with different molecular weight were assayed by ultraviolet spectrum.The glycosidic bond configuration and other functional groups were analyzed by infrared spectrum in LBP.LPS content was assayed by ELISA kit.The results showed that all the LBP fragments contain protein but without nucleic acid.Special infrared absorption peaks of functional groups in polysaccharide fragments were found.The glycosidic bond of LBP-2/3/5 were alpha configuration,but alpha and beta configuration were both found in LBP-4.2.To find out the main active fragment of LBP that has the function of antitumor and immunomodulatory effects2.1 Effects of different molecular weights LBPs on H22 cells proliferationCells activity was detected by MTT assay,the proliferation of H22 was analyzed with flow cytometry,cells apoptosis was detected with Annexin V/PI apoptosis detection kit,membrane potential was detected by rhodamine 123(Rho123),PI staining analysis of the cell cycle was also detected with flow cytometry.The results showed that different molecular LBPs can reduce the activity of H22 cells,inhibit their proliferation,in which LBP-3 shows the most significant role.Further studies showed that LBP-3 inhibit the H22 cell cycle arrest in S phase,decline the mitochondrial membrane potential resulting in apoptosis in dose dependent manner.2.2 Effects of LBP fractions on the function of lymphocytesThe spleen cells were stimulated by LBPS,and the cytoactive was detected by MTT assay.LBPS-treated mouse spleen cells were harvested and treated with Rhol23 to detect mitochondrial membrane potential.CD69 expressions and the proliferation treated with CFSE were quantified by flow cytometry.Cytokine secretion was measured by Cytometric Bead Assay(CBA).The results showed that four fractions of LBPS won't restrain the cytoactive of spleen cells while LPB-3,LBP-4 and LBP-5 increase that.LBP-2 and LBP-4 have no obvious effect on lymphocyte mitochondrial membrane but the mitochondrial membrane increases by treated LBP-3 and LBP-5.LBP-2 does not increases expression of CD69 while LBP-3,LBP-4,LBP-5 do and LBP-3 has stronger activity.Although LBP-3 stimulates the activation both of CD4-CD3 +T cells and CD4+CD3+ T cells,especially for the CD4-CD3+T cells.LBP-3 has stronger effect than LBP-5 on the proliferation with a dose-dependent manner.LBP-3 and LBP-5 stimulate proliferation of CD 19+B cell except for the CD3+T cells.LBP-3 increases the secretion of IL-6?IL-10?TNF and IFN-y except for IL-2 and IL-4 with a dose-dependent manner.2.3 Effects of LBP fractions on the function of macrophageMacrophages(RAW264.7)and LBP fraction(LBP-2,LBP-3,LBP-4 and LBP-5)were incubated for 24h.The molecule expression levels of CD 14,CD86 and MHC-II on cell surface were measured by flow cytometry.The level of released NO was detected by Griess reagent and the mRNA expression of iNOS was assayed by RT-PCR.Reactive oxygen species(ROS)was detected by DCFH-DA in the cells.Cytokine level of IL-6;TNF,IL-10 were measured by Cytometric Bead Assay(CBA).Fluorescent microspheres phagocytosed by macrophage were measured by using fluorescence and flow cytometry.The results shown that LBP-2,LBP-3,LBP-4 and LBP-5 improved the expression level of CD86 and MHC-? on cell surface,and LBP-3 improved the expression of CD 14,CD86 and MHC-? with a dose-dependent manner.LBP-3,LBP-4 and LBP-5 stimulated the release of NO in Raw264.7 and primary peritoneal macrophages.LBP-3 promted the mRNA expression of iNOS,and it promoted the production of NO in a time-dose dependent manner in RAW264.7.LBP3,LBP4 and LBP5 also significantly improved the production of ROS in RAW264.7.LBP-3 improved the release of IL-6,IL-10,TNF and enhanced the phagocytosis of RAW264.7 in a dose-dependent manner.2.4 Effects of LBP fractions on the tumor growth of H22 miceBALB/c mice inoculated armpit hepatoma H22 cells to build a hepatic carcinoma graft model,oral administration of LBP-1,LBP-2,LBP-3,LBP-4,LBP-5 respectively in a dose 250mg/kg treating H22 tumor-bearing mice,or with different doses of LBP-3 for treating H22 tumor-bearing mice.Mice fed once a day for ten days.Mice were observed daily living conditions,after the end of the experiment,tumors were isolated and weighed to calculate the inhibition rate;isolated thymus and spleen to calculate the organ index;detected the number of leukocytes in peripheral blood;ELISA kit used to test the level of serum IL-2,IL-10,IFN-y.The results showed that LBP-3 effectively improve the living conditions of liver cancer in mice.In five polysaccharide fragments,LBP-3 can inhibit tumor growth in mice,showing a significant reduction in tumor mass,compared with the model group The difference was statistically significant(P<0.05),tumor inhibition rate is 37.97%,much higher than other polysaccharide fragments(table 2-8,Figure 2-22).There were no significant differences of spleen and thymus index in tumor-bearing mice with different molecular weights LBPs compaired with the model group,on the contrary,has a trend with varying degrees of increase thymus index(Table 2-8).LBP-2,LBP-5 groups mice serum IL-2 content is higher compared with the model group,and LBP-1,LBP-3 groups is lower than the model group,the difference was statistically significant(P<0.001,Fig.2-23A).IL-10 levels of LBP-3 group was significantly lower than the model group(P<0.01),while LBP-5 group was significantly higher than the model group(P<0.001,Fig.2-23B).Serum IFN-y levels of LBP-3,LBP-5 groups were significantly lower than the model group(P<0.01,Figure 2-13C).Further research found that different doses of LBP-3 have different degrees of inhibition xenografts in mice in a dose-dependent manner(Table 2-9),LBP-3 without effects of inhibiting thymus and bone marrow of tumor-bearing mice,in contrast,in certain doses can improve the tumor-bearing mouse thymus and bone marrow suppression status,increase the number of leukocytes in peripheral blood(Figure 2-24,Figure 2-25).3.The immunological mechanism of LBP inhibit the development of liver cancer in miceBALB/C mice were divided into control group and model group which inoculated with H22 cells in armpit to build the hepatic carcinoma metastatic tumors model,intervened with 250mg/kg LBP-3.Flow cytometry was used to detect expression level of CD3/CD 19/CD8/CD4/CD69/PD-1/CD25 molecules on cell surface;Elisa kit was used to test content of TGF-?in serum;Incubated CFSE-labeled H22 with spleen cells?TDLN lymphocytes and peritoneal macrophages,detected the PI positive proportion of H22 by flow cytometry.The results showed that LBP-3 significantly increased CD3+T cells proportion in peripheral blood?TDLN and tumor of liver cancer mice.Further analysis showed that the proportion of CD8+T cells and CD4+T cells in T cells didn't change in the peripheral blood and TDLN of tumor-bearing mice,LBP-3 did not change the proportion of T cell subsets of these parts also(Fig 2-25B/C?Fig 2-26B/C).However,LBP-3 could increase CD8+T cells and significantly improved CD4+T cells in tumor of tumor-bearing mice(P<0.001,Fig 2-27B/C).LBP-3 could not only greatly improve T cells activation level in tumor tissue,but also TDLN CD8+T cells and CD69 expression level of tumor CD8'CD3+T cells in tumor tissue as well(Fig 2-28B?Fig 2-29C).LBP-3 significantly decreased PD-1 expressing proportion of CD3+T cells in TDLN and tumor tissue of hepatic carcinoma mice(Fig 2-26?Fig 2-27B).LBP-3 could reduce PD-1 +CD25-CD4+Tregs proportion(P<0.001)and PD-1-CD25+CD4+Tregs proportion(P<0.01)in TDLN and tumor tissue.LBP-3 could lower serum TGF-?1 level of hepatic carcinoma mice with statistical significance difference compared to the model group(P<0.01).Further analysis revealed LBP-3 significantly enhanced function of spleen cells(mainly NK cells)?TDLN cells(mainly CTL)and macrophages in hepatic carcinoma mice which made the mortality of H22 obviously higher than the model group(P<0.05 or 0.001).4.Enhancing efficacy of LBP-3 on anti-tumor effect of doxorubicin in HCC mice4.1 Effect of lycium barbarum polysaccharides(LBP)on immunity of doxorubicin treated miceThe doxorubicin chemotherapy model of mice was induced by injection of doxorubicin(5 mg/kg)into the intraperitonium,then the mice were administrated with lycium barbarum polysaccharides by the concentration of 250 mg/kg and 50 mg/kg for 10 days to intervene and treat.The body weight and the condition of whether the mice is alive or dead were recorded,and the mice's activity state,the colour of the mice and characteristics of feces and other conditions were observed everyday.The relative number of peripheral white blood cells(WBC)was counted by flow cytometry,the thymic index was calculated by the separation of thymic and PI positive proportion of H22 cells was detected to investigate NK cells' function by flow cytometry after incubating the spleen cells with H22 cells labeled by CFSE for 24 and 48 h.The cycle of the bone marrow cells,which was obtained from the normal control group,the Dox group and LBP-3,250 mg/kg group mice,was detected.The results show that LBP-3 can significantly improve the living status of doxorubicin chemotherapy mice,promote the body weight to go back to normal.LBP-3 can improve the situation of thymus and bone marrow inhibition induced by Dox,raise the thymus index,promote the number of peripheral blood lymphocytes in mice to recover.The chemotherapy effect of Dox can inhibit the killing activity of NK cells in mice.The ability of killing activity of NK cells of the two dose of LBP-3 groups in mice returns to normal roughly,which shows significantly improvement compared with Dox group,but there is no statistical significance when compared with normal group,the experimental results prompt that LBP-3 can improve the inhibitory states of NK cells in Dox chemotherapy mice.4.2 Effect of LBP on the tumor growth of doxorubicin treated H22 miceBALB/C mice inoculated armpit hepatoma H22 cells to build a xenograft model,divided into model group,doxorubicin treatment group(Dox group),LBP-3 at 250mg/kg treatment group(LBP-3 group)and the group combined treatment with doxorubicin and LBP-3(combined treatment group),intraperitoneal injection doxorubicin for 2 times at 5mg/kg/times,orally administered LBP-3 for 10 consecutive days.Weigh and record the weight of mice to observe the general condition of the mice active,coat,stool,and other daily survival.Stripped end of the experiment were weighed and tumor inhibitory rate was calculated;isolated thymus and calculated thymus index;peripheral blood lymphocyte subsets and their relative number of cells were detected by the flow cytometry;splenocytes were stimulated by ConA and LPS respectively,and their relative number of cells were detected by the flow cytometry,also their cell proliferation index was calculated simultaneously;mouse spleen cells,TDLN cell and the H22 cells labeled with CFSE were incubated 36h,the percentage of PI-positive cells H22 were examined by flow cytometry to investigate the function of NK cells and CTL killing.The results showed that LBP-3 effectively improve the living conditions of doxorubicin chemotherapy in mice,showed the hair shiny,active,fecal nature compared with doxorubicin alone-treated group.Compared with the model group,the tumor mass of Dox group,LBP-3 group,combined treatment group were significantly decreased in mice,and the difference was statistically significant.In the three drug-treated group,the tumor mass in mice of the combined treatment group was less weight than the Dox group or LBP-3 group,the difference was statistically significant,the inhibition rate was 79.06%,significantly higher than the other two groups.Under the experimental conditions LBP-3 was no significant improvement in Dox-induced inhibition of thymus in liver cancer mouse(Figure 3-7),but it can improve the status of mouse bone marrow suppression,manifested as the relative number of peripheral blood lymphocyte and their subsets in the combined treatment group were both more increased than Dox group which the difference compared with Dox group in the relative number of total lymphocytes,CD3 + T cells,CD 19 + B cells,CD8 + CD3+ T cells and CD8-CD3 + T cells were statistically significant.LBP-3 improved T cell proliferation in liver cancer mouse with Dox chemotherapy,but had no effect on B cell proliferation.The cytotoxic function of spleen cell was decreased obviously in Dox group,and the difference compared with the model group was statistical significance.The cytotoxic function of mice spleen cell in LBP-3 group and combined treatment group was significantly higher than Dox group,and the difference was statistically significant.The results suggest that LBP-3 enhances the ability of doxorubicin killing H22 hepatoma cells in mouse.Furthermore,the cytotoxic function of CTL in Dox group and model groupthe have no significant difference,but LBP-3 group and the combined treatment group were higher than Dox group,and the differences were statistically significance.In addition,results suggest that doxorubicin have no significant inhibition of the killing activity of CTL tumor draining lymph nodes in liver cancer mice,but LBP-3 significantly enhance CTL killing activity in liver cancer mice.ConclusionLBP-3 is the active fragment of LBP in anti-tumor and immunomodulatory.In vitro,experiments indicated that LBP-3 could inhibit H22 cells proliferation and induce cells apoptosis,promote the activation and secretion of a variety of cytokines of lymphocytes and macrophages;In vivo,the results showed that LBP-3 have shown higher activity in inhibiting the growth of hepatic carcinoma metastatic tumors compared to other molecular fragment of LBP.Further studies showed that LBP-3 could reduce the proportion of PD-1+CD25-CD4+Tregs and PD-1-CD25+CD4+Tregs in TDLN and tumor tissue of hepatic carcinoma mice,enhance anti-tumor activity of spleen cells(mainly NK cells)?TDLN cells(mainly CTL)and macrophages.The results indicate that LBP-3 inhibit hepatic carcinoma metastatic tumors by breaking the immunosuppressive microenvironment of tumor and enhancing the anti-tumor immune response.Studies also show that LBP-3 can effectively reduce immunosuppression of doxorubicin chemotherapeutic mice,enhance anti-tumor effect of doxorubicin in combination with doxorubicin and reduce immunosupression status of hepatic carcinoma mice.