| Objective:1.Optimization of exopolysaccharide from Rhizopus nigricans?EPS?shake flask fermentation conditions.2.Study on the antioxidant activities in vitro of EPS.3.To investigate the inhibitory effect of EPS combined with oxaliplatin on dimethylhydrazine?DMH?-induced colon cancer in rats and its mechanism.Methods:1.On the basis of single factor experiment,orthogonal experiment was carried out to investigate the effects of fermentation time,the volume of liquid and shake flask speed on the production of EPS.The conditions of shaking fermentation of EPS were optimized,so as to improve the production efficiency of EPS shaking flask fermentation.2.The antioxidant activity of EPS in vitro was evaluated by measuring the scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl?DPPH?free radicals and hydroxyl free radicals?·OH?.3.Forty Wistar rats?80100 g?were randomly divided into 5 groups:control group,model group,EPS treatment group(150 mg·kg-1),oxaliplatin treatment group(10mg·kg-1)and EPS+oxaliplatin treatment group.In addition to the control group,rats were subcutaneousinjected with DMH(30 mg·kg-1)from the neck and back,once a week,continuously for 15 weeks to establish rat colon cancer model.At the same time,the rats were given medication according to the dosage and their weight was recorded weekly.At the same time,the rats were given medication according to the dosage and their weight was recorded weekly.4.In the 21st week,anesthetize and dissect the rats and the weight of the spleen and thymus were recorded.5.The histopathological changes of colon tissue in rats were observed by HE staining.6.Detection of Interleukin-1beta?IL-1??,Interleukin-2?IL-2?,Interleukin-6?IL-6?,tumor necrosis factor alpha?TNF-??and Interferon-gamma?IFN-??cytokines concentration in serum by enzyme-linked immunosorbent assay?ELISA?.7.The expression levels of cyclooxygenase2?Cox2?,peroxisome proliferator activated receptor gamma?PPAR??,nuclear factor-?B?NF-?B?,Caspase-3,Caspase-7 and Survivin proteins of colon tissue in rats were determined by Western blot and Immunohistochemistry.Results:1.The results of single factor test and orthogonal test show that the optimum conditions for EPS shake flask fermentation were 6 days of fermentation time,150 mL of liquid the volume,160 r/min of shake flask speed.2.The antioxidant activity of EPS in vitro showed that when the concentration of EPS was 5 mg/mL,the scavenging rates of DPPH free radicals and·OH free radicals could reach to 71.65%and 76.60%,respectively.3.Compared with the model group,the body weight,spleen and thymus index of rats in the treatment groups?EPS treatment group,oxaliplatin treatment group and EPS+oxaliplatin treatment group?elevated significantly?P<0.05?.4.H&E staining results showed that the damage degree of colon tissue could be significantly improved in the treatment groups.5.The ELISA results showed that compared with the model group,the cytokines concentration of IL-1?,IL-6 and TNF-?markedly decreased in the treatment groups but IL-2 and IFN-?cytokines concentration were significantly increased?P<0.05?.6.Western blot and Immunohistochemistry results showed,compared with the control group,the expression of Cox2,NF-?B and Survivin proteins were significantly upregulated and the expression levels of PPAR?,Caspase-3 and Caspase-7 were significantly downregulated in the model group?P<0.05?.Compared with the control group,the expression of Cox2,NF-?B and Survivin proteins were significantly downregulated and the expression of PPAR?,Caspase-3 and Caspase-7 were significantly upregulated in the treatment groups?P<0.05?.In particular,the EPS+oxaliplatin treatment group had the most significant improvement effect on the above indicators?P<0.05?.Conclusion:1.The production and efficiency of EPS fermentation were improved by optimizing shake flask fermentation conditions.2.EPS has great antioxidant capacity.3.Both EPS and oxaliplatin inhibited colon cancer in rats and show good synergistic inhibitory effect. |
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