Anti-hepatoma Active Components Screening Of Extracts From Astragalus-Curcumae And Its Mechanism

Objective:Liver cancer,which has a high incidence rate in China,is one of the most popular and deadliest cancer in the world.Liver cancer is mainly treated in early surgical resection and chemotherapy,but the early surgical resection rate is very low,and the negative side effects of chemotherapy drugs is serious.So the anti-cancer research of traditional Chinese medicine has become a hot spot.Astragalus and Curcuma as the traditional Chinese medicine,both have obvious anti-tumor effect,but there are few studies on the anti-tumor effect and its mechanism for the compatible Astragalus-Curcuma.This study is divided into two parts.In the first part,anti-tumor compoents of Astragalus-Curcuma were screened,and the immunoregulation,apoptosis and VEGF expression on H22 transplanted hepatocarcinoma mice was detected to explore its possible anti-tumor mechanism.In the second part,the inhibitory effects of components on the expression of Caspase-3,Caspase-8,Caspase-9,Bax and Bcl-2 in human HepG2 hepatocarcinoma cells in vitro was studied and the prossible mechanism of incducing apoptosis of human HepG2 hepatocarcinoma cells was also discuss.This work provides a theoretical basis for the clinical anti-tumor study of Astragalus-Curcuma,which can make the two drugs better used compatibly in clinical practice.Methods:1.?1?The volatile oil and thelow polar lipid soluble components in Astragalus-Curcuma were extracted with 95% alcohol as component of EH1.The dregs was extracted with water extraction and alcohol precipitation as component of EH5,and the supernatant of alcohol precipitation was concentrated and dried as component of EH6.?2?The components of triterpenoids,saponins and flavonoids in Astragalus-Curcuma were extracted with 50% alcohol as component of EH2.The dregs was ectracted with water extraction and alcohol precipitation as component of EH7,and the supernatant of alcohol precipitation was concentrated and dried as component of EH8.?3?The crude polysaccharides in Astragalus-Curcuma were extracted with water extraction and alcohol precipitation as component of EH3,and the supernatant was concentrated and dried as component of EH9.?4?The Astragalus-Curcuma was extracted with traditional water decoction as component of EH4.2.The effective anti-tumor components of EH1-EH9 were screened in vitro and in vivo.?1?Screened experiment in vitro: nine different components were applied to H22 cells,cultured 24 h,and the proliferation inhibition rate detected with CCK-8 method was used to screen the effective anti-tumor components.?2?Screened experiment in vivo: the H22 tumor bearing mice model were established,and the experimental groups were administrated with nine different extraction components for 11 d continuously.Then the tumor tissues were weighed and tumor inhibition rates were calculated to screen the effective anti-tumor component.3.The effective anti-tumor components were selected and then applied to H22 cells in vitro.After cultured 24 h,the apoptosis of H22 cells was detected by flow cytometry.4.The changes of CD₄⁺T / CD₈⁺T cells in the peripheral blood and the levels of serum IL-2?IL-4?IL-18?TNF-? and IFN-? were detected by flow cytometry and ELISA assay,respectively,to preliminary study the effects of anti-tumor components of Astragalus-Curcuma on immune function in H22 tumor-bearing mice.5.To further explain the intervention effects of effective components of Astragalus-Cu rcumae on H22 tumor bearing mice and its possible mechanisms,HE staining was used to detect the changes of tumor tissues in active groups,TUNEL method was used to detect the effect of effective extraction components on tumor cell apoptosis in vivo and the expression of VEGF and NF-?B-p65 were detected by immunohistochemistry.6.The Human HepG2 Hepatocellular Carcinoma cells were treated with the above effective anti-tumor components and cultured 24 h,then the growth inhibition ratios and apoptosis ratios in vitro of HepG2 cells were detected by CCK-8 and flow cytometry.7.The expression of Caspase-3,Caspase-8,Caspase-9,Bax and Bcl-2 in HepG2 cells were detected by Western blotting.Results:1.In vitro and vivo results showed that only the components of EH1?EH2?EH3 and EH4 of Astragalus-Curcuma have obvious anti-tumor effects.The vitro and vivo tumor inhibition ratios were 66.97%?54.86%?50.60%?45.75% and 47.55%?36.12%?29.96%?34.68%,respectively.2.The flow cytometry results showed that components of EH1-EH4 had the effects of inducing H22 cell apoptosis with the apoptosis ratios were 63.4%?53.1%?53.1%?60.1%,respectively.3.The results of blood routine showed that compared with control group,the number of white blood cells,lymphocytes and eosinophils in EH1 group were significantly increased?P <0.05?;the number of lymphocytes in EH2 group was significantly increased?P <0.01?;the number of leukocytes,lymphocytes and mononuclear cells in EH4 group were significantly increased?P <0.05?.4.The results of levels of serum CD₄⁺T,CD₈⁺T,IL-2,IL-4,IL-18,TNF-? and IFN-? showed that compared with control group,the number of CD₄⁺T cells in each group did not increase significantly?P >0.05?;the number of CD₈⁺T cells in EH3 and EH4 groups were significantly decreased?P <0.05?;the level of IL-2 in EH3 and EH4 groups were significantly increased?P <0.05?;the levels of IL-4?IL-18 and TNF-? in each groups were no significantly increased?P >0.05?;the level of IFN-? in EH3 group was significantly increased?P <0.05?.5.HE staining results showed that compared with the control group,the H22 tumor bearing cells arranged in treatment groups were relatively neat,uniform size and had a large number of bad areas.6.The results of apoptosis ratio and expression of VEGF and NF-?B in H22 tumor tissue were showed by the average integral optical density,the higher the value,the higher the expression levels.Compared with the control group,the TUNEL apoptosis and VEGF test results showed that:?1?EH1 and EH2 groups were significantly apoptotic;?2?the expression of VEGF in EH1?EH2 and EH3 groups were significantly decreased?P <0.05?;?3?the expression of NF-?B p65 in EH1 and EH2 groups were significantly decreased?P <0.05?.7.The results of CCK-8 showed that the highest inhibitory ratios and apoptosis ratios of HepG2 hepatoma cells in the EH1,EH2,EH3 and EH4 groups were 89.13%?78.36%?20.38%?18.78% and 67.8%?65.5%?23.4%?24.9%,respectively.The results suggested that only EH1 and EH2 groups had obvious anti-tumor activity and could induce apoptosis in human HepG2 hepatoma cells.8.Western blot results showed that compared with control group,the expression of Caspase-3,Caspase-8,Caspase-9 and Bax protein in EH1 group were significantly increased?P <0.05?,and the expression of Bcl-2 protein was significantly decreased?P <0.05?.The expression of Bax in EH2 group was significantly increased?P <0.05?,and the expression of Bcl-2 protein was significantly decreased?P <0.05?.Conclusion:1.The four components of Astragalus-Curcumae of EH1,EH2,EH3 and EH4 have anti-H22 tumor effect.2.?1?The anti-tumor effects of EH1 component may be related to significantly increase the number of white blood cells,lymphocytes and eosinophils,induce H22 tumor-bearing cells apoptosis and inhibit the expression of VEGF and NF-?B p65.?2?The anti-tumor effects of EH2 component may be related to significantly increase the number of lymphocytes,induce H22 tumor-bearing cells apoptosis and inhibit the expression of VEGF and NF-?B p65.?3?The anti-tumor effects of EH3 component may be related to significantly increase the number of lymphocytes,monocytes,IL-2,TNF-?,decrease the level of CD₈⁺T and inhibit expression of VEGF.?4?The anti-tumor effects of EH4 component may be related to significantly increase levels of white blood cells,lymphocytes,mononuclear granulocytes,IL-2,CD₄⁺T /CD₈⁺T.3.?1?EH1 and EH2 components could significantly inhibite HepG2 cell growth and induce apoptosis while EH3 and EH4 components not significantly.?2?EH1 component could induce HepG2 cell apoptosis that may be related to activate Caspase-3,Caspase-8,Caspase-9 and elevate Bax/Bcl ratio,and EH2 component may be related to elevate Bax/Bcl ratio.