Study On The Material Basis Of Anti-tumor Effect And Mechanism Of The Dioscorea Septemloba Thunb.

The Dioscorea septemloba Thunb.（DS）recorded in"China Pharmacopoeia",with the function of removing dampness,clearing away turbidness and dispelling wind to remove pain,and has been widely used in stranguria marked by chyluria,joint stiffness and rheumatic arthralgia.It has obvious curative effect and broad application prospect.DS mainly containing steroids,diarylhepanes,lignans,organic acids,esters,polysaccharides,mucus and tannin and other chemical components,with anti-tumor,anti-osteoporosis,uric acid,hypolipidemic,anti-fungal,anti-myocardial ischemia and prevention of atherosclerosis and other pharmacological effects.The fraction of ethyl acetate of Dioscorea septemloba Thunb.（DSE）showed strong antitumor activity in vitro.In this paper,the modern separation technology was used to isolate,purifie for the chemical composition of DS,and to study its anti-tumor activity.The modern extraction techniques and spectral analysis were used to study the chemical constituents of DSE.Human hepatocellular carcinoma cells HepG2,Lung cancer cells A549,Breast cancer cells MCF-7 and Macrophages cells RAW264.7 for activity screening.To study on the effects of extracts,components and monomer components of DS on the proliferation,apoptosis and cycle of tumor cells.The inhibitory effect of DSE on tumor cells was examined by MTT.Mitochondrial membrane potential was detected by flow cytometry.Detection of reactive oxygen species by flow cytometry.Hoechst 33258 staining and the morphological changes of tumor cells were observed by fluorescence microscopy.Flow cytometry analysis of tumor cell apoptosis and cycle.Colony formation assay was used to examine the growth of tumor clonal proto-cells by DSE.Protein expression of Bcl-2,Bax,Caspase3,STAT3,p-STAT3,Bcl-xl,Mcl-1,NF-κB,p-NF-κB was detected by Western Blot.To clarify DS anti-tumor effect and mechanism in vitro.Mouse hepatoma tumor cells h₂₂2 was used to establish solid tumor and ascites tumor model.The pathological changes of tumor tissue were observed by HE staining.The expression of apoptosis-related proteins in tumor tissue was detected by WB.Immunohistochemistry was used to detect the changes in the tissues of related proteins in tumor tissues.To study anti-tumor effect and mechanism of DSE in vivo.16 monomer components were isolated from DSE.DSE was qualitatively and quantitatively analyzed by HPLC,The results showed that DSE antitumor activity of the material may be diarylheptanone,dioscin,prosapogenin B of dioscin.The results showed that DSE has strong anti-tumor activity in vivo and in vitro.The proliferation of HepG2,A549,MCF-7,RAW264.7 cells significantly inhibited by DSE.The inhibitory effect on HepG-2 cell proliferation had more selective,DSE（50μg/mL）administered 4 h,DSE could reduce the mitochondrial membrane potential.DSE（50μg/mL）could promote the production of reactive oxygen species（ROS）.DSE（50μg/mL）for 12 h,cell cycle arrested,DSE（50μg/mL）for 24 h,the cells were significantly apoptotic and had apoptotic bodies.DSE（50μg/mL）administered 48 h,the apoptosis rate was75%.DSE could significantly inhibit the growth of tumor cloned progenitor cells.DSE could promote the expression of Cleaved-Caspase 3 protein in tumor cells and reduced Bcl-2/Bax ratio,reduced protein expression of p-STAT3,Bcl-xl,Mcl-1,p-NF-κB.In animal experiments,DSE had a significant inhibitory effect on the growth of solid tumor in H₂₂2 hepatoma mice.And the mouse liver,kidney,immune system,blood parameters did not show significant affect,DSE could prolong the lifetime of H₂₂2 hepatocellular carcinoma ascites tumor mice.The apoptotic bodies appeared in the tumor tissue of DSE-treated mice.DSE could promote the expression of Cleaved-Caspase3protein in tumor cells and reduced Bcl-2/Bax ratio,reduced protein expression of p-STAT3,Bcl-xl,Mcl-1,p-NF-κB.The results of immunohistochemistry for protein expression were consistent with the results of Western Blot.DSE may exert anti-tumor effect by activating mitochondria pathway to promote tumor cell apoptosis and inhibiting the phosphorylation of STAT3 and NF-κB.Modern research shows that inflammation and tumor occurrence and development are closely related.Such as the development of hepatitis to liver cancer,enteritis developed liver cancer,the literature shows that saponins have significant anti-inflammatory activity.In this paper,The modern extraction techniques and spectral analysis were used to study the chemical constituents of Total saponins of Dioscorea septemloba Thunb.（TSDS）,two kinds of pain models were established,to detect the analgesic activity of total saponins from Dioscorea septemloba Thunb.Xylene-induced ear swelling in mice and carrageenan-induced inflammatory model of rat foot swelling inflammatory model were established to detect the anti-inflammatory activity of total saponins from Dioscorea septemloba Thunb.At the same time detection of liver and kidney function index and organ index.6 compounds were obtained and identified.The TSDS had a certain effect on the pain caused by hot plate,could effectively reduce the number of writhing reactions caused by acetic acid,P-xylene-induced auricle swelling,the carrageenan-induced rat foot swelling had a certain inhibitory effect.Results showed that the TSDS have a certain anti-inflammatory and analgesic effects,and no obvious side effects.Based on TSDS had obvious anti-inflammatory activity,The protective effect of TSDS on acetaminophen-induced liver injury was further investigated.A model of liver injury induced by AP was established to observe the protective effect of TSDS on liver injury.Kunming mice were randomly divided into control group,model group（AP,400 mg/kg）TSDS（100,200,400 mg/kg）,By calculating organ index,Serum Liver function indicators（ALT,AST）,oxidative stress indicators（GSH,MDA,SOD）in liver tissues and Inflammatory factors（TNF-α,IL-6,IL-1β）were detected by kits,Liver tissue sections to observe the pathological changes,Western blot detection of HO-1,Bcl-xl,Mcl-1,STAT3,p-STAT3,NF-κB,p-NF-κB protein expression.Results showed that compared with the model group,low,medium and high doses of TSDS significantly inhibited serum ALT,AST,GSH content in tissues and SOD activity increased,MDA content decreased.Inflammatory cytokines TNF-α,IL-6,IL-1βlevels were gradually decreased.Histopathology HE staining showed that TSDS can significantly improve liver necrosis and apoptosis,and reduce the necrotic area,reduced the inflammatory infiltration.TSDS could inhibit oxidative stress and inflammatory response.TSDS could protect AP-induced acute liver injury.The mechanism may be related to inhibition of oxidative stress,reduction of inflammatory reaction and inhibition of cell signal transduction and transcriptional activators.