| Objective: To study the synergistic chemopreventive effects and underlying mechanism of combined use of ginsenoside Rd,folic Acid,metformin and vitamin D3 on mouse colitis-associated colon neoplasia.To evaluate the combined effect and explore possible mechanism of combined effect,and provide the experimental and theoretical evidence for the process of clinical therapy.Method: Colitis-associated colon neoplasia was established using ICR male mice via intraperitoneal administration(20 mg/kg body weight)of 1,2-dimethylhydrazine(DMH)and 1-week oral exposure(2% in drinking water)of a carcinogen,dextran sodium sulfate(DSS).A total of 195 ICR male mice,5-week old,were divided into 13 groups: 1)normal controls;2)DMH controls;3)DSS controls;4)DMH plus DSS controls;5)ginsenoside Rd group;6)metformin group;7)vitamin D3 group;8)vitamin D3 plus metformin group;9)ginsenoside Rd plus vitamin D3 plus metformin group;10)folic Acid group;11)ginsenoside Rd plus folic Acid group;12)folic Acid plus vitamin D3 plus metformin group;13)ginsenoside Rd plus folic Acid plus vitamin D3 plus metformin group.Different treatments were carried out from the 2th day after intraperitoneal administration of DMH,and lasted for 139 days.All mice were sacrificed at 20 th week,with large intestine and serum samples collected.Tumors were then counted and measured.Aberrant crypt foci(ACF)were observed under low power microscope after methylene blue staining.Expression levels of Phospho-AMPK?(Thr172),Phospho-Akt(Ser473),Phospho-m TOR(Ser2448),Phospho-p70 S6 Kinase(Thr389),Phospho-S6 Ribosomal Protein(Ser235/236),I?B?(44D4),?-Catenin(D10A8),Bcl-2,c-Myc and Cyclin D1 were determined using Western Blotting.Results: Compared with DMH+DSS group,ginsenoside Rd,folic Acid,vitamin D3,metformin,combined use of vitamin D3 and metformin,combined use of ginsenoside Rd and vitamin D3 and metformin,combined use of ginsenoside Rd and folic Acid,combined use of folic Acid and vitamin D3 and metformin,combined use of ginsenoside Rd and folic Acid and vitamin D3 and metformin significantly reduced the number of colon tumors,and also the numberCompared with metformin group,combined use of ginsenoside Rd and vitamin D3 and metformin significantly reduced the number of ACF.Compared with metformin group,combined use of ginsenoside Rd and folic Acid and vitamin D3 and metformin significantly reduced the number of ACF.Compared with vitamin D3 group,ginsenoside Rd,combined use of vitamin D3 and metformin significantly reduced the number of ACF.Compared with vitamin D3 group,combined use of ginsenoside Rd and vitamin D3 and metformin,folic Acid,combined use of ginsenoside Rd and folic Acid,combined use of ginsenoside Rd and folic Acid and vitamin D3 and metformin significantly reduced the number of ACF.Compared with vitamin D3 group,metformin,combined use of folic Acid and vitamin D3 and metformin significantly reduced the number of ACF.Compared with normal control group,expression levels of p-m TOR,p-P70S6 K,p-S6 P,?-Catenin,Bcl-2/Bax,c-Myc and Cyclin D1 in DMH+DSS group significantly increased,whereas the expression level of p-AMPK and I?B? significantly decreased.Compared with DMH+DSS group,ginsenoside Rd,combined use of VD3 and metformin,combined use of ginsenoside Rd and VD3(200 IU/kg/day)and metformin increased the expression level of p-AMPK and I?B?,decreased that of p-AKT,p-m TOR,p-P70S6 K,p-S6 P,?-Catenin,Bcl-2/Bax,c-Myc and Cyclin D1 significantly.Compared with ginsenoside Rd group,combined use of ginsenoside Rd and vitamin D3 and metformin increased the expression level of I?B?,decreased that of p-P70S6 K and p-S6 P significantly.Compared with combination group(vitamin D3 plus metformin),combined use of ginsenoside Rd and VD3 and metformin decreased that of p-AKT and c-Myc significantly.Conclusion:Inhibitory effects of ginsenoside Rd,folic Acid,vitamin D3,metformin,combined use of vitamin D3 and metformin,combined use of ginsenoside Rd and vitamin D3 and metformin,combined use of ginsenoside Rd and folic Acid,combined use of folic Acid and vitamin D3 and metformin,combined use of ginsenoside Rd and folic Acid and vitamin D3 and metformin on mouse colitis-associated colon neoplasia induced by 1,2-dimethylhydrazine(DMH)and dextran sodium sulfate(DSS)were obvious and stable.Inhibitory effects of folic Acid on mouse colitis-associated colon neoplasia were better than combined use of ginsenoside Rd and folic Acid.Inhibitory effects of folic Acid on mouse colitis-associated colon neoplasia were better than combined use of ginsenoside Rd and folic Acid and vitamin D3 and metformin.Inhibitory effects of combined use of ginsenoside Rd and vitamin D3 and metformin on mouse colitis-associated colon neoplasia were better than vitamin D3 alone or metformin alone.Inhibitory effects of ginsenoside Rd,folic Acid alone on mouse colitis-associated colon neoplasia were better than vitamin D3 alone.The underlying anti-tumor mechanism of ginsenoside Rd may be related to activate expression of p-AMPK and I?B?,as well as inhibited expressions of p-m TOR,p-P70S6 K,p-S6 P,?-Catenin,Bcl-2,c-Myc and Cyclin D1.The anti-tumor effect of ginsenoside Rd was related to regulate of cell cycle,promote apoptosis of tumor cells and inhibit the Wnt/beta-catenin,NF-kappa B and m TOR signaling pathway.The underlying anti-tumor mechanism of combined use of vitamin D3 and metformin may be related to activate expression of p-AMPK and I?B?,as well as inhibited expressions of p-m TOR,p-P70S6 K,p-S6 P,?-Catenin,Bcl-2,c-Myc and Cyclin D1.The anti-tumor effect of combined use of vitamin D3 and metformin was related to regulate of cell cycle,promote apoptosis of tumor cells and inhibit the Wnt/beta-catenin,NF-kappa B and m TOR signaling pathway.Compared with ginsenoside Rd group or combination group(vitamin D3 plus metformin),combined use of ginsenoside Rd and vitamin D3 and metformin significantly inhibited aberrant crypt foci(ACF)formation and colitis-associated colon neoplasia.The underlying anti-tumor mechanism may be related to activate expression of p-AMPK and I?B?,as well as inhibited expressions of p-m TOR,p-P70S6 K,p-S6 P,?-Catenin,Bcl-2,c-Myc and Cyclin D1.Its anti-tumor mechanism may be related to the following aspects:(1)Inhibitied Wnt/beta-catenin,NF-kappa B and m TOR signaling pathway.(2)Regulated of cell cycle and promoted apoptosis of tumor cells. |
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