Study On Antipyretic And Antitumor Activities Of Polysaccharide From Aerial Parts Of Tetrastigma Hemsleyanum

Objective The research object of this paper is the aerial parts of Tetrastigma hemsleyanum.Polysaccharide from aerial parts of Tetrastigma Hemsleyanum(PTH)was screened out as its main active ingredient.Through the research on separation and purification,structure analysis,antipyretic and anti-tumor effect and mechanism of PTH,it is aim to promote the development and utilization of the aerial parts of Tetrastigma hemsleyanum,and to provide theoretical basis for the development of the new type of Chinese medicine preparation.Methods1.Dry yeast induced fever model and H22 hepatocellular carcinoma model were established to compare the effects of different extracts from aerial and underground parts of Tetrastigma hemsleyanum.The main active components from the aerial parts of Tetrastigma hemsleyanum were screened out.2.PTH was extracted by water-extraction and alcohol-precipitation method.PTH was purified by DEAE Sepharse Fast Flow anion exchange resin and Superdex-200 gel column.HPGPC,GC-MS and IR were used to analyze the relative molecular weight and monosaccharide composition of RP1-2.3.By means of methylation and partial acid hydrolysis,the structure of RP1-2 was analyzed by IR,GC-MS and NMR.4.Through the establishment of dry yeast induced fever model,Various cytokines(including TNF-α,IL-1βand IL-6),temperature regulation mediators(including PGE2,cAMP,T3,TSH and TRH),and energy metabolism related indexes(including Glu,PA,LD,TG,NEFA,LDH and Na~+-K~+-ATPase)were measured.The Antipyretic effect and mechanism of PTH were studied.5.The effects of PTH on the proliferation of H22,B16 tumor cells and spleen lymphocytes were detected by MTS.Through the establishment of Lewis lung cancer,S180sarcoma,H22 liver cancer and 4T1 breast cancer tumor model,the tumor inhibition activity of PTH was observed.The synergistic and attenuated effect of PTH combined with chemotherapy drug CTX or ADM,was evaluated by the measurement of serum and myocardial tissue indicators(including AST,CK,LDH,ATP and PA).By establishing H22tumor model,the cytokines in serum and tissues(including TNF-α,IL-6 and IFN-γ)and energy metabolism related indexes(including Glu,LD,PA,TG,ATP,LDH,PK,HK,PFK and SDH)were measured.The anti-tumor mechanism of PTH was discussed.6.Using TAK-242 as the TLR4 inhibitor and LPS as the positive control,by determining the secretion of TNF-αand IP-10 in RAW264.7 and THP-1 macrophages,it was discussed whether PTH could play a role through TLR4 signaling pathway.Through the establishment of LPS infected animal model and determination of inflammatory cytokines in serum(including IL-6,IL-10,IFN-γ,TNF-αand IL-12)by flow cytometry,it was discussed whether PTH could play a dual function to relieve heat and inflammation and immune regulation through TLR4 signaling pathway.Results1.The aqueous extract from aerial and underground and preciptation part of ethanol subsiding method from aerial parts of Tetrastigma hemsleyanum could significantly reduce the temperature of the fever mouse model induced by dry yeast.The aqueous extract and the crude polysaccharide component from aerial parts of Tetrastigma hemsleyanum had obvious antitumor effect on H22 hepatocarcinoma mice,with an inhibition rate of 33.1%and 32.3%respectively.2.The rate of PTH was 11.9%.PTH was refined to obtain RPTH,and RPTH was removed impurities such as protein and sulfate,and increased the content of glucose and uronic acid.PTH was further purified to obtain RP1-2.The relative molecular weight of RP1-2 was 78473.6,which was mainly composed of rhamnose,arabinose,mannose,glucose and galactose.3.The main chain of RP1-2 primary structure was composed of rhamnose,mannose and galactose,and the branch chain was consisted of glucose and galactose.The specific structure of its main chain and branch chain was as follows:4.PTH could reduce the temperature of dry yeast induced fever model,and could significantly prolong the survival time of mice in the model group.PTH could reduce the content of the cytokines(including TNF-α,IL-1β,IL-6),temperature regulation mediators(including PGE2,cAMP,T3 and TSH)and energy metabolism related indexes(including Glu,PA,LD,TG,NEFA,LDH and Na~+-K~+-ATPase)in the serum and tissues of the model group.5.PTH does not inhibit the proliferation of tumor cells in vitro,but could promote the proliferation of splenic lymphocytes.PTH had the effect of inhibiting tumor growth in Lewis lung cancer,H22 liver cancer and 4T1 breast cancer tumor model,and could significantly prolong the survival time of H22 ascites tumor mice.PTH could significantly increase the levels of red blood cells and hemoglobin in peripheral blood of tumor mice,improve the myocardial damage caused by ADM,and reduce the content of AST,CK,LDH in serum and ATP,PA in myocardial tissue.PTH could improve the levels of TNF-α,IL-6,IFN-γin serum of H22 tumor mice,and reduce Glu,LD,PA,TG,ATP,LDH,PK,HK,PFK and other contents in serum and tumor tissues.PTH could increase the proportion of CD4~+T cells in the spleen and CD4~+,CD8~+T cells in the thymus tissues of 4T1 breast cancer model,and inhibit the percentage of CD4~+CD25~+Foxp3~+Treg cells in serum.6.PTH could induce the secretion of TNF-αand IP-10 in RAW264.7 and THP-1macrophages significantly.However,when the TLR4 receptors were blocked by TAK-242before the PTH,the secretion of TNF-αand IP-10 was significantly lower than those not treated with TAK-242 cells.PTH could significantly improve the survival rate of LPS infected mice in 24 and 48 h with preventive or therapeutic administration.PTH could promote the secretion of inflammatory cytokines such as IL-6,IL-10,IFN-γ,TNF-α,IL-12,through administration by gavage and intraperitoneal injection alone.PTH was given to mice before LPS infected,which could significantly reduce the content of IL-6 and IFN-γin the serum.PTH could also significantly reduce IFN-γcontent in serum of LPS infected mice after LPS exposure.Conclusion1.PTH was the main active component from the aerial parts of Tetrastigma hemsleyanum.2.PTH was refined and purified to obtain RP1-2.The relative molecular weight of RP1-2 was 78473.6,which was mainly composed of rhamnose,arabinose,mannose,glucose and galactose.3.The main chain of RP1-2 primary structure was composed of rhamnose,mannose and galactose,and the branch chain was consisted of glucose and galactose.The specific structure of its main chain and branch chain was as follows:4.PTH had antipyretic and anti-inflammatory effects by reducing the heating cytokines,temperature regulation mediators,and inhibiting the energy metabolism of the body.5.PTH had no anti-tumor activity in vitro,but it had anti-tumor activities in vivo by regulating the level of cytokines,inhibiting the level of glycolysis of tumor cells and regulating the proportion of T lymphocyte subsets in the body.And it had the effect of reducing toxicity to the chemotherapy drugs.6.PTH may play the role of antipyretic,anti-inflammatory and anti-tumor effects by TLR4 signaling pathway.