Experimental Study On Toxity Reducing And Efficacy Enhancing Effect Of Angelican Radix Hedysari Ultrafiltration Content Assisting ¹²C⁶⁺ HIB Treatment Of Liver Cancer

Objective: The study aims to certify the toxity reducing and efficacy enhancing effect of Anglica Radix Hedysari untrafiltration content in assisting ¹²C⁶⁺ HIB treatment on liver cancer,and probe into the mechanism for its effect in order to provide evidence for its future clinic application.Methods: Ultra performance liquid chromatography-mass spectrometry?UPLC-MS?is used to assay the difference on the untrafiltration and aqueou extract of Anglica Radix Hedysari untrafiltration.Annexin V-FITC/PI double stain,FCM,real-time PCR-based array analysis,LSCM and Western Blot are used respectively to analyze the function and mechanism of ¹²C⁶⁺ HIB radiation at various doses on human Hep G2.Mouse H22 liver cancer cells are primary cultured in vitro,and H22 ascitic tumor and solid tumor animal model is constructed.The content of blood WBC,RBC,PLT and HGB is checked respectively;Spleen index and thymus index are measured respectively;T-lymphocyte subgroup is assayed by FCM;serum complement C3,IFN-?,IL-4 and IFN-?/ IL-4 are tested by ELISA;Myelogram is conducted by bone marrow smear;HE staining of lung,cardiac,liver,spleen and kidney is pathomorphologically observed to find out the toxity reducing function of ARH ultrafiltration content after ¹²C⁶⁺ HIB treatment on H22 tumor-bearing mouse.Diameter of tumor is measured by vernier caliper;area of tumor is detected by small animal imaging technology;H22 tumor-bearing body of the mouse and tumor temperature are tested by infrared imaging system;tumor index and anti-tumor rate are measured respectively,HE stained tumor tissue is observed pathomorphologically;tumor tissue is observed micro-morphologically under electron microscope;The expression of TP53,Bax,Bcl-2,Survivin and VEGF is assayed respectively by immunohistochemistry method to verify the efficacy enhancing of ARH component aqueous extract in treating H22 tumor-bearing mouse by ¹²C⁶⁺ HIB radiation.Cell apoptosis detection,cell cycle analysis and Mouse Genome 430 2.0 plate ascitic cell genetic expression profile detecting technique are used to discuss the molecular mechanism of ARH ultrafiltration content in assisting toxcity reducing and efficacy enhancing after ¹²C⁶⁺ HIB radiation treatment of H22 tumor-bearing mouse.Results:?1?The determination of UPLC-MS is that the component of Anglica Radix Hedysari untrafiltration less than Anglica Radix Hedysari extract powder component,and the content of same component in Anglica Radix Hedysari untrafiltration is a certain increase?2?24 hours after ¹²C⁶⁺ HIB radiation at 6 Gy on human Hep G2 once,compared with the radiation dose at 0 Gy group,cellular late apoptosis rate and G2/M cell rate increase significantly?P < 0.01?.Cellular apoptosis and mitochondrion swelling are observed under electron microscope.31 genetic expressions take on significant change on p53 signaling pathway relating to DNA damage repair,apoptosis,cycle control,transcription regulation and angiogenesis.Compared with the control group?0 Gy?,the sample in the radiation group at 6 Gy after ¹²C⁶⁺ HIB radiation take on mitochondrial membrane potential disintegration expression,and high protein expression of FAS,TP53BP2,TP53AIP1 and CASP9?P < 0.01?.?3?Mouse H22 cells undergo adherent culture in vitro for multiple passages,and suspension cultured for about one week.Then the cultured H22 cells are made into cell suspension of 4×106 /m L,and injected into each mouse intraperitoneally so as to construct H22 tumor-bearing mouse ascetic tumor model.The ascitic cells from H22 tumor-bearing mouse are taken and made into cell suspension of 4×107 /m L by PBS,then 0.1 ml cell suspension is hypodermic injected under the right oxter of each mouse so as to construct H22 tumor-bearing mouse solid tumor model.The success rate of the above two models reachs 95% respectively.?4?Compared with the tumor model group,the number of WBC and PLT drop significantly in the pure radiation mouse group?P < 0.01?,spleen index and thymus index drop obviously?P< 0.05?,numbers of blood CD3+,CD4+,CD8+Tlymphocyte decrease greatly?P < 0.01?,CD4+/CD8+ value appears abnormal apparently?P < 0.01?,serum IFN-? and IL-4 content decline clearly?P<0.05?,IFN-?/IL-4 value shows abnormality clearly?P<0.05?,neutral band nucleus in myelogram drop evidently?P<0.01?,neutral Lobulated nuclear/neutral band nucleus rate is abnormal apparently?P<0.05?.HE staining pathomorphological observation finds out that ¹²C⁶⁺ HIB radiation can caused different degrees of damage on H22 tumor-bearing mouse liver,cardiac,liver,spleen and kidney.Compared with the pure radiation mouse group,the number of WBC and PLT,spleen index and thymus index,numbers of blood CD3+,CD4+,CD8+Tlymphocyte,CD4+/CD8+ value,serum IFN-? and IL-4 content,IFN-?/IL-4 value,neutral band nucleus in myelogram,and neutral Lobulated nuclear/neutral band nucleus rate in the medicine+ radiation group all have a certain degree of improvement.?5?Compared with tumor model group,the tumor diameter,area,temperature and index drop significantly in the pure radiation group?P< 0.05?;pathomorphology finds apoptosis and heterogeneity tendency;apoptosis and death can be seen frequently under electron microscope,immunohistochemical result shows that at the early stage after radiation treatment,ARH ultrafiltration content can down-regulate the expression of tumor tissue Bcl-2 and Survivin,and up-regulate Bax expression.When the combination of medicine and ¹²C⁶⁺ HIB radiation treatment is applied in H22 tumor-bearing mouse for 21 days,the tumor-inhibition rate is 59.51%,Q21value= 0.91;after 26 days of treatment,the tumor-inhibition rate is 71.72%,Q26value=1.01.?6?The pathway analysis result of differential genetic expression of the pure medicine group/tumor model group shows that: compared with the latter,the pure medicine group has 230 genetic expression up-regulations and 219 genetic expression down-regulations;differential expressions gather at antigen processing and presentation,p53 signaling pathway and PI3K-Akt signaling pathways;genetic expressions of ATR,Cyclin E,Sestrins,Hsp70,RTK,G?y and AMPK up-regulate,the expressions of MHCI and ECM down-regulate.The pathway analysis result of differential genetic expression of the pure radiation group/tumor model group shows that: compared with the latter,the pure radiation group has 874 genetic expression up-regulations and 445 expression down-regulations;the differential genetic expressions gather at Colorectal cancer signaling pathway,m TOR and AMPK signaling pathways,and chemotactic factor?CF?signaling transduction pathway;the expressions up-regulate of APC,?-catenin,TGF-? R?,SMAD1,DCC,KRAS,Raf,Rac/Rho,c-Jun,c-Fos,p53,BAX,AMPK,BRAF,CREB,FOS,Rab,Rho A,IKK,h MLH1,h MSH1,h MSH3 and h MSH6,the expressions down-regulate of PKB,Cyclin D1,HIF-1?,ATG1,MO25,TSC1,WASP,S6,CXCR5 and Gnai1.The KEGG pathway analysis result of differential genetic expression of the medicine+ radiation group/tumor model group shows that: compared with the latter,the medicine+ radiation group has 571 expression up-regulations and 410 expression down-regulations;the differential genetic expressions gather at P53 signaling pathway,glioma,cell cycle,m TOR signaling pathway,AMPK signal,renal cell carcinoma and PI3K-Akt signaling pathway;the expressions up-regulate of MDM-X,ATR,CHK1,2,Cyclin E,Siah,Sestrins,Bid,ATM,Hdac,Cyc E,CAM,p53,ORC,MCM,Bub3,PI3 K,AMPK,Hu R,CPT1,PDGF?,EGFR,FH,G?y,ECM,BHD,AMPK,RTK,Cyclin,IKK,MET and VHL;the expressions down-regulate of PKB,HNF-4?,HIF-1?,CUL2,IGFR,PAK,TSC1,S6K1/2,Rictor,Stag1/2,JAK,PTEN and TSP1.The results of gene ontology gathering analysis of differential genetic expression in medicine+ radiation group/ tumor model group are mainly displayed in terms relating to immunoregulation in the gene ontology database function clauses.Conclusion:?1?Ultrafiltration removes with the component of Anglica Radix Hedysari extract powder which molecular weight is above 10 KDa,and the component of Radix Astragali ultrafiltration less than those of Anglica Radix Hedysari extract powder.?2?¹²C⁶⁺ HIB radiation at 6 Gy on human Hep G2 can induce apoptosis and G2/M arrest significantly;during this process,series of genetic expressions take on characteristic change on the intracellular p53 signaling pathway relating to DNA damage repair,apoptosis,cycle control,transference,deterioration and radio-resistance;its apoptosis-inducing mechanism is caused by combined effort of extrinsic and intrinsic pathways;wild-type p53 takes part in the regulation of this process;its cancer-inhibition function repair relates to ASPP2 and TP53AIP1 high expressions.?3?Adherent culture in vitro is suitable for mouse H22 cancer cells.The H22 tumor-bearing mouse ascitic tumor animal model constructed by this study is suitable for series of experimental observation within 8 days;the H22 tumor-bearing mouse solid tumor animal model constructed by this study can be observed ideally 30 days after model-making.?4?¹²C⁶⁺ HIB radiation can cause immunologic function decline,myelosuppression and damage on lungs,cardiac,liver,spleen and kiney of H22 tumor-bearing mouse.ARH ultrafiltration content intervene can inhibit the above damages caused by ¹²C⁶⁺ HIB radiation to some extent.?5?Pure use of ARH ultrafiltration content can resist the tumor growth of H22 tumor-bearing mouse.¹²C⁶⁺ HIB radiation treatment can resist the tumor growth of H22 significantly.The combination treatment of ARH ultrafiltration content and ¹²C⁶⁺ HIB radiation can resist the tumor growth of H22 but the two treatments function additively rather than synergistically;their function mechnism both relate to p53 signaling pathway,but vary on activating specific target and time.?6?When ARH ultrafiltration content is used singly,it can regulate immune function,resist tumor and adjust energy metabolism.Its molecular mechanism may involves antigen processing and presentation,p53 and PI3K-Aktsignaling pathway;¹²C⁶⁺ HIB radiation treatment can resist tumor significantly,its molecular mechanism may involves colorectal cancer signaling pathway,m TOR and AMPK signaling pathways and chemokine signaling pathway;PKB?AKT?may be a key target spot for radiotherapy;ARH ultrafiltration content can assist ¹²C⁶⁺ HIB radiation treatment in toxicity reducing and efficacy enhancing with AMPK expression up-regulating;DNA damage repair genes relating to tumor radiation resistance such as h MLH1,h MSH1,h MSH3 and h MSH6 have their expressions down-regulate,which can be a target spot of molecular mechanism;the immune function improvement of tumor-bearing mouse can be one of these mechanisms.