| Background There are many biological activities of Grifola frondosa polysaccharide(GFP),among which immunomodulatory activity is one of its important biological activities.At present,there were a large number of animal experimental studies about the effect of GFP on the immune regulatory function in animal disease models at home and abroad,but most of them had some limitations in terms of methodology.For example,some of the studies did not conducted random grouping of animals,and a few had conducted random grouping studies,however,they had not paid attention to the hiding of random sequence.In addition,there were more or less deficiencies in the important measures and steps that would affect the quality of an experimental method,such as randomized placement of experimental animals,blind method for breeders or researchers or outcome evaluators,and random selection of animals for outcome evaluation,which might affect the authenticity of the experimental results.Objective 1.Through the comprehensive retrieval,quality evaluation and data synthesis of the existing research on the regulation of immune function in animal disease model by GFP,we will understand the current research status and existing problems in this field,and provide reliable evidence-based medicine for the follow-up experimental research.2.Through the rigorous design and scientific implementation of animal experimental research methods,the successful mouse liver cancer disease model will be constructed by the intervention of GFP,and the related immunological indexes in vivo will detect to verify the effect of GFP on the immune regulation in vivo of the liver cancer model of mice.Methods 1.The effect of GFP on immune regulation in animal disease model is studied by the method of Meta-analysis.2.By the method of animal experiment in vivo,according to the principle of random distribution,BALB/c mice are divided into three groups randomly,12 mice in each group,GFP(GFP intragastric administration),negative control group(saline intragastric administration)and positive control group(5-FU intraperitoneal injection),subcutaneously inoculate with H22 cells of mouse hepatoma for 24 hours,after that,each group are given corresponding drug treatment.After 14 days of administration,the mice are killed,the proliferation of immune cells(spleen T and B cells)are detected by flow cytometry,and the secretion of cytokines(INF-γ,IL-12 and TNF-α)in the peripheral blood of tumor bearing mice are detected by enzyme-linked immunosorbent assay(ELISA),so as to verify the effect of GFP on the immune regulation in the liver cancer model of mice.Results The results of Meta-analysis show that:totally 21 animal experimental studies are included.(1)The percentage of CD4~+T cells in spleen:the percentage of CD4~+T cells is higher in GFP group[MD=1.89,95%CI(0.94,2.83),P<0.0001]than that of the control group,the difference is statistically significant.(2)The percentage of CD8~+T cells in spleen:the percentage of CD8~+T cells of GFP group[MD=8.46,95%CI(5.93,11.00),P<0.0001]is higher than that of the control group,with a statistically significant.(3)The percentage of CD19~+B cells in spleen:the percentage of CD19~+B cells[MD=-1.73,95%CI(-6.10,2.64),P=0.44]has no significant difference between the two groups.(4)The percentage of NK cells:the percentage of NK cells in GFP group[MD=2.67,95%CI(0.23,5.11),P=0.03]is higher than that of the control group,the difference is statistically significant.(5)The percentage of macrophages:the percentage of macrophages in GFP group[MD=14.09,95%CI(0.84,27.34),P=0.04]is higher than that of the control group,with a statistically significant.(6)The secretion capacity of INF-γ(pg/ml):the secretion of INF-γ(pg/ml)in cells are significantly higher in GFP group[SMD=5.34,95%CI(3.42,7.26),P<0.0001]than that of the control group,with a statistically significant difference between the two groups.(7)The secretion of TNF-α(pg/ml):the secretion of TNF-α(pg/ml)both in cells[SMD=15.92,95%CI(9.07,22.76),P<0.05]and serum[SMD=2.82,95%CI(1.19,4.45),P<0.05]of the GFP groups are higher than those of the control groups,both with statistically significant.(8)The secretion of IL-12(pg/ml):the secretion of IL-12(pg/ml)in cells[SMD=3.63,95%CI(2.34,4.92),P<0.05]and in serum[MD=7.29,95%CI(4.77,9.82),P<0.05]of the GFP groups are both higher than those of the control groups,all with statistically significant.Among the 21 animal experiments,only 5 studies were randomly divided,but it is not clear whether they use the correct random method and whether they hide the random sequence.None of the included studies reported randomized placement of animals,blinding of breeders or researchers or outcome evaluators,or random selection of animals for outcome evaluation.2.The in vivo experimental results show that:(1)Tumor weight:the average tumor weight of the control group,GFP group and 5-FU group are 3.86±0.85g,2.20±0.54g and 2.49±1.02g,respectively,GFP group and the control group have statistical difference(P<0.001),and 5-FU group and the control group have statistical difference(P<0.01).(2)Proliferation activity of spleen T cells:the percentage of CD4~+T cells are 7.42±1.26 and 12.91±2.90 in the control group and GFP group,respectively,with a statistically significant difference between the two groups(t=4.25,P=0.002);the percentage of CD8~+T cells are 1.54±0.32 and 2.37±0.49,respectively,with statistical significance(t=3.47,P=0.006);the CD4/CD8 are 4.96±1.11 and 5.71±2.00,respectively,with no significant difference between the two groups(t=0.80,P=0.443).(3)Proliferation activity of spleen B cells:the percentage of CD19~+CD49~+B cells are 2.06±0.74 and 3.37±0.59 in the control group and GFP group,respectively,with a statistically significant difference between the two groups(t=3.39,P=0.007).(4)The secretion IFN-γ(pg/ml):the IFN-γ(pg/ml)secretion of tumor bearing mice serum in the control group and GFP group are 3.61±1.28(pg/ml)and 6.38±2.11(pg/ml),respectively,with a statistically significant difference between the two groups(t=3.90,P=0.001).(5)The secretion of IL-12 p70(pg/ml):the IL-12 p70(pg/ml)secretion in serum of tumor bearing mice in the control group and GFP group are 15.64±6.96(pg/ml)and 21.46±6.94(pg/ml),respectively,with no significant difference between the two groups(t=2.05,P=0.052).(6)The secretion of TNF-α(pg/ml):the TNF-α(pg/ml)secretion in serum of tumor bearing mice in the control group and GFP group are 12.07±3.91(pg/ml)and 16.00±3.04(pg/ml),respectively,with statistical significance(t=2.75,P=0.012).(7)The results of hematoxylin eosin staining of liver and kidney show that GFP does not cause further damage to the liver and kidney of the tumor bearing mice.Conclusions Both meta-analysis and animal experimental studies reveal that GFP can regulate the immune function of animal disease model by influencing the proliferation of immune cells and the secretion of immune factors.For the tumor bearing mice model,it can enhance its immune activity by enhancing the proliferation of CD4~+T cells,CD8~+T cells,CD19~+CD49~+B cells and NK cells of the spleen,as well as increasing the secretion of immune factors INF-γ,TNF-αand IL-12,which has a certain inhibitory effect on tumor growth.However,for the arthritis mouse model,there is only one research focus at present,and further research is needed to verify in the future.The risk of bias is higher of the 21 included animal studies in meta-analysis,only 24%studies are randomly divided,but it is not clear whether they use the correct random method and whether they hide the random sequence.None of the included studies reported randomized placement of animals,blinding of breeders or researchers or outcome evaluators,or random selection of animals for outcome evaluation.Therefore,in the future animal experiments,we should pay attention to the random grouping,the concealment of random sequence and the implementation of blind method,so as to improve the quality of the method. |
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