Effect Of Bioactive Peptides On Immune Function Of Tumor-bearing Nude Mice And Its Mechanism

OBJECTIVE:Bioactive peptides,as an emerging anti-tumor preparation in the medical field,have a regulating effect on various physiological functions of human body,and have certain potential value in the research of regulating immune function of tumor-bearing organisms.This paper aims to investigate the effect of bioactive peptides?BAP?on the immune function of gastric cancer-bearing nude mice,and to conduct a preliminary study on its mechanism,providing theoretical and experimental basis for the development and application of bioactive peptides.METHODS:The effect of BAP on the immune function of nude mice:60 SPF-class nude mice weighing half?12±2?g were selected for adaptive feeding for 4 days,and then randomly divided into 5 groups:blank control group?CON?,model group?MG?,low dose active peptide group?LBAP:0.047 mg/10 g?,medium dose active peptide group?MBAP:0.094 mg/10 g?,high dose active peptide group?HBAP:0.188 mg/10 g?.In vitro cultured BGC-823 human gastric cancer cells?1x107cells/ml?were implanted in the back of nude mice in MG group,LBAP group,MBCP group and HBAP group.On the 4th day after implantation,the tumor-bearing nude mice could touch the size of the tumor.Pathological analysis confirmed the successful modeling.From day 5,all nude mice were administered intraperitoneally with equal volume of drug?1 time/day?,and the dose was gradually increased with the increase in body weight.After continuous administration for 2 weeks,24 hours after the last administration,the tumor-bearing nude mice were bled and removed by cervical dislocation.?1?Observing the general state of nude mice during the treatment:including feeding,drinking water,activity,body weight,length and short diameter of the tumor,and tumor rupture;?2?2 weeks after the neck was sacrificed,the spleen tissue and tumor were removed.The body tissue was weighed to determine the spleen coefficient and tumor inhibition rate;?3?the percentage of NK cells and the percentage of B lymphocytes in the venous blood of each group were determined by flow cytometry;?4?Hematoxylin-I The morphological changes of spleen tissue and tumor tissue of nude mice were observed by hematoxylin and eosin stain?H&E stain?.The effect of BAP on the immune function of nude mice:Real-time quantitative PCR?PCR?and immunohistochemistry were used to detect the target gene NF-?B and downstream gene TNF-?in the spleen of nude mice.And changes in the expression level of protein function.RESULTS:1.Quality of life of tumor-bearing nude mice:There was no significant change in the activity of the tumor-bearing nude mice in each group.Compared with the CON group,there was no significant difference in food intake between the MG group and the nude mice,which was not statistically significant?P?0.05?.Compared with the MG group,there was no significant difference in food intake between the LBAP group,the MBAP group and the HBAP group,and there was no statistical significance?P?0.05?.Compared with the CON group,there was no significant difference in drinking water between the MG group and the nude mice,which was not statistically significant?P?0.05?.Compared with the MG group,the water consumption of the tumor-bearing nude mice in the LBAP group,the MBAP group and the HBAP group increased significantly,but there was no statistical significance?P?0.05?.2.Tumor:Compared with the MG group,there was no significant difference in tumor volume and tumor inhibition rate between the LBAP group and the HBAP group?P?0.05?,and the tumor volume of the tumor-bearing nude mice in the MBAP group decreased.There was a significant difference in the rate?P<0.05?,suggesting that a certain dose of BAP has a good inhibitory effect on gastric cancer cells.3.Spleen coefficient and percentage of immune cells:Compared with CON group,the spleen coefficient of tumor-bearing nude mice in MG group increased,the difference was statistically significant?P<0.05?.Compared with MG group,there was no significant difference in spleen coefficient between LBAP group and HBAP group?P?0.05?.The spleen coefficient of MBAP group was significantly increased,the difference was statistically significant?P<0.05?.Compared with the CON group,the percentage of NK cells in the MG group was significantly increased?P<0.05?;compared with the MG group,the percentage of NK cells in the LBAP group,MBAP group and HBAP group was significantly increased?P<0.05?.Compared with the CON group,the percentage of B lymphocytes in the MG group was significantly increased?P<0.05?;compared with the MG group,the percentage of B lymphocytes in the MBAP group and the HBAP group was significantly increased?P<0.05?.It indicates that MBAP can improve the immune function of tumor-bearing nude mice to some extent.4.HE staining:There was no significant difference in the general structure of spleen tissue,which indicated that short-term continuous use of BAP did not cause morphological changes in the spleen of tumor-bearing nude mice.In the HE staining of tumor tissue,the area of inflammatory infiltration of tumor tissue in each administration group gradually increased with the dose of administration,and the area of?inflammatory infiltration in the MBAP group was significant.There is a complex correlation between inflammation and immunity during tumor growth.Studies have shown that in the early stages of tumors,tumor growth induces inflammation,and inflammation helps the immune response inhibit tumor growth^[1].In this experiment,the tumor-bearing nude mice were administered for 2 weeks,and no morphological changes were observed in the spleen tissue.It was concluded that the tumor was still in the early stage,indicating that a certain dose of BAP can enhance the inflammatory response of the gastric cancer-bearing nude mice.Improve the level of immune response.5.Real-time PCR analysis of gene expression:Compared with the CON group,the relative expression of NF-?B and TNF-?in the MG group was significantly increased?P<0.05?.Compared with MG group,the relative expression of NF-?B in LBAP group and MBAP group had no significant difference?P?0.05?,and the relative expression of NF-?B in HBAP group decreased significantly,the difference was statistically significant?P<0.05?.Compared with MG group,the relative expression of TNF-?in LBAP group,MBAP group and HBAP group decreased significantly,the difference was statistically significant?P<0.05?,suggesting that a certain dose of BAP can activate tumor-bearing nude mice.B cell receptor signaling pathway inhibits NF-?B and TNF-?,and enhances the immune function of tumor-bearing nude mice,thereby inhibiting tumor growth.Immunohistochemical analysis of protein expression:Compared with CON group,the expression of NF-?B and TNF-?protein in MG group were increased,the difference was statistically significant?P<0.05?.Compared with MG group,there was no significant difference in NF-?B protein expression between LBAP group and MBAP group?P?0.05?,and there was significant difference in NF-?B protein expression between HBAP group?P<0.05?.Compared with the MG group,there was no significant difference in TNF-?protein expression between the LBAP group?P?0.05?.There was a significant difference in the expression of TNF-?protein between the MBAP group and the HBAP group?P<0.05?.Studies have shown that NF-?B can mediate the physiological functions of immune responses and also promote tumor growth.Stimulation of tumor cells on the body can induce NF-?B activation to enhance the body's immunity,but when the stimulation persists,the NF-?B activity increases too fast,which will cause a large amount of synthesis and release of inflammatory factors such as TNF-?,and stimulate again by positive feedback.The rapid increase of NF-?B activity induces a larger number of proinflammatory factors to form a“waterfall effect”,leading to SIRS characterized by self-destruction,so that the function of phagocytic cells is weakened to inhibit the immune function of the body^[2].This indicates that a certain dose of BAP can activate the B cell receptor signaling pathway in tumor-bearing nude mice to reduce the expression of NF-?B and TNF-?in the spleen,and to control the activation of NF-?B within a certain range to enhance the immunity of tumor-bearing nude mice.Functional effects can also inhibit tumor growth.IN CONCLUSION:?1?Continuous use of bioactive peptides has the effect of enhancing the humoral immune function of tumor-bearing nude mice.?2?Continuous use of bioactive peptides has a certain inhibitory effect on tumor growth in tumor-bearing nude mice.?3?The effect of bioactive peptides on the immune function of tumor-bearing nude mice may enhance the immune function through NF?B-TNF-?signaling pathway.