Goat Beta-defensin 124 Modulate Expression Of Cytokines And Chemokins By The P38MAPK/AP1 Signaling Pathway

Beta-defensin is a kind of small molecule cationic polypeptide,which plays an important role in the immune regulation of organism.The study was conducted to investigate the silence and overexpression of goat beta-defensin 124(g BD124)on key genes in p38MAPK/AP1 pathway and their downstream target genes of the cytokines and chemokines.In this experiment,the effective recombinant LV10-gBD124-sh RNA vectors were transfected into epididymial caput cells as treatment group,negative control group transfected by LV10-NC lentivirus vector(LV10-NC),blank control of caput cells,respectively.The epididymial caput cells and culture mediums were collected after selecting by 2 μg·m L-1puromycin.The m RNA and protein expression levels of g BD124,some critical genes in MAPK signaling pathway,cytokines and chemokins were detected by q RT-PCR,western blot and the high-specific ELISA kits,respectively.The results showed as follows:1.The transfection efficiency of LV10-gBD124-51 and LV10-g BD124-161 combined transfection group and LV10-NC group exceeded 80%.The blank control group had no fluorescence.The results of RT-PCR indicated that silencing of g BD124 in caput cells decreased significantly the expression of MAPK1,c-JUN and c-FOS of AP-1 transcription factor subunit and increased the expression of RASA1 in the MAPK signaling pathway(P <0.05).The result of western blot suggested that total p38 MAPK,total c-JUN,total c-FOS,phosphor-p38 MAPK and phosphor-c-JUN were significantly reduced with silencing of g BD124 in caput cells(P < 0.05).The expression of IL-1β,IL-1R2,IL-8 of cytokines,CCL6,CCL21 of chemokines in the MAPK downstream genes were markedly promoted(P < 0.05),and CCL5 of chemokine and IL-1α of cytokine were reduced by the silencing of g BD124 in caput cells(P < 0.05).The concentration of CCL5 in the culture medium was lowered in the group of the silencing of g BD124.In comparison with the blank control group,the concentration of IL-1β and IL-8 in the culture medium was enhanced,and the concentration of IL-1α was decreased in the group of the silencing of g BD124(P < 0.05).2.The transfection efficiency of LV5-gBD124-overexpression group and LV5-NC group was more than 80%.There was no fluorescence in the blank control group.The expression levels of m RNA and protein of g BD124 in the overexpression group increased significantly(P < 0.05).Compared with LV5-NC group,the overexpression of g BD124 significantly reduced the expression of TAB2,MAPK1,MAPK2,MAPK8 IP,RASA1 and RAGRP1(P <0.05);The overexpression of g BD124 significantly increased the expression of the total protein of p38 MAPK and c-FOS,and the phosphorylation of p38MAPK(P < 0.05);The overexpression of g BD124 significantly increased the expression of CCL21 and IL-1R2(P <0.05),and decreased the expression of CCL5 and CCL6(P < 0.05);In the culture medium the overexpression of g BD124 significantly increased the concentration of IL-1α(P < 0.05).Conclusion: The silencing of gBD124 in caput cells could modulate the expression of cytokines and chemokins by inhibiting the p38MAPK/AP1 signaling pathway;The overexpression of g BD124 regulates the expression of chemokines and cytokines in epididymal caput cells by upregulating the expression of key genes in the p38 MAPK / AP1 signaling pathway.