Effect And Mechanism Of Cecropin A-magainin Hybrid Peptide On Small Intestinal Mucosal Immune Function

Objective This experiment was to study the effect of cecropin A-magainin hybrid peptide on small intestinal mucosal immune function in mice and to investigate the acting mechanism.Methods To express cecropin A-magainin hybrid peptide in Pichia pastoris, determine the antibacterial activity and the protein concentration after mixing. The eighteen SPF BALB/c mice who were six weeks old with similar weight were randomly divided into three groups: control group(given 0.75 m L saline water with 0.9% by Gastric lavage), low dose of hybrid peptide group(given 0.75 m L cec A-mag peptide with half protein concentration of dope by Gastric lavage), high dose of hybrid peptide group(given 0.75 m L cec A-mag peptide with the same protein concentration as dope by Gastric lavage). The feeding experiment lasted for 6 weeks. We recorded the body weight on an empty stomach before all gastric lavage and the ending,calculated the average daily gain of body weight in mice of each group within the 6 weeks. To study the effect of cecropin A-magainin hybrid peptide on small intestinal mucosal structure. The number of intestinal microflora was measured through Plate clounting. To observe the effect of cecropin A-magainin hybrid peptide on the proliferation of T or B lymphocytes from payer’ patches with the method of Brdu incorporation. We detected the positive expression of Ig A by immunohistochemical staining. The content of IL-2、IFN-γ and IL-4 from intestinal tissue homogenates was measured through ELISA and IFN –γ/IL-4 was calculated. Using q Real-time PCR to detect the change of nuclear transcription factor NF-КB m RNA level in intestinal tissue and the expression of NF-КB protein in the small intestine tissue homogenates was measured by ELISA.Results The protein concentration of cecropin A-magainin hybrid peptide is 0.52 mg/ m L. After gavaged, the weight of weight in peptide groups was significantly higher than the control group(P<0.05). The villous length in duodenum of peptide groups was higher than in control group(P<0.05); the crypt depth in small intestinal of peptide groups was significantly lower than control group(P<0.05); V/C in small intestinal was significantly higher in peptide groups than in control group(P<0.05). The number of Escherichia coli in cecal contents of peptide groups was significantly lower than that in control group(P<0.05), the number of Bifidobacterium and Lactobacillus in peptide groups was significantly higher than in control group(P<0.05), and it showed a dose-dependent manner. The number of CD3+Brd U+ cells in CD3+T cells or B220+B cells in B220+Brd U+ cells from payer’ patches in peptide groups was significantly higher than that in control group(P<0.05). In contract with the control group, the positive expression of Ig A in duodenum, jejunum, ileum was significantly higher in peptide groups(P<0.05). IL-2, IFN-γ and IL-4 in peptide groups was significantly higher than in control group(P<0.05). IFN-γ/IL-4 was not significantly different among different groups(P>0.05). The level of nuclear transcription factor NF-КB m RNA in intestinal tissue and the expression of NF-КB protein was significantly higher than in control group(P<0.05).Conclusions Cecropin A-magainin hybrid peptide can significantly increase weight of mice and promote humoral immunity. The acting mechanism may be that cecropin A-magainin hybrid peptide improve the intestinal mucosa structure, enhance intestinal barrier function and improve the release of NF-КB, activate the transcription of NF-КB, induct the proliferation of T lymphocytes and B lymphocytes from Peyer’s patches and the secret of Ig A, improve the expression of IL-2, IFN-γ and IL-4 in intestinal mucosa to promote humoral immunity and cell-mediated immunity and the balance between Th1 and Th2 also can be kept.