β防御素124沉默通过p38MAPK/AP1通路调控山羊附睾头细胞趋化因子和细胞因子的表达

doi:10.11843/j.issn.0366-6964.2020.009

Abstract

Abstract: The study was conducted to investigate the effects of silencing goat beta-defensin 124 (gBD124) on the expression of key genes in p38MAPK/AP1 pathway and their downstream target genes of the cytokines and chemokines. Three designed gBD124-shRNAs were loaded into LV10-U6/RFP&Puro shuttle plasmid, then cotransfected into 293T cell with packaging plasmid for lentivirus packaging. Virus titer was detected using the method of suspension gradient dilution. Three recombinant LV10-gBD124 vectors and LV10-gBD124-NC were transfected into epididymal caput cells of Taihang goat, respectively for screening the effective silent vectors. The effective recombinant LV10-gBD124 vectors were co-transfected into epididymal caput cells, and the blank cell group, LV10-NC group and effective silent vector group were setup, respectively. The epididymal caput cells and culture mediums were collected separately after screening by 2 μg·mL^-1 puromycin. The mRNA and protein expression levels of gBD124, some key genes in MAPK signaling pathway, cytokines and chemokines were detected by qRT-PCR, Western blot and the high-specific ELISA kits, respectively. The results showed that LV10-gBD124 recombinant vectors were constructed and two valid recombinant vectors of LV10-gBD124-51 and LV10-gBD124-161 were screened for silencing gBD124. The epididymal caput cells with gBD124 silenced were successfully constructed. The results of qRT-PCR indicated that silencing of gBD124 in epididymal caput cells significantly decreased the expressions of MAPK1, c-JUN and c-FOS(P<0.05),and significantly increased the expression of RASA1 in the MAPK signaling pathway(P<0.05). The result of Western blot showed that protein levels of total p38MAPK, total c-JUN, total c-FOS, phosphor-p38MAPK and phosphor-c-JUN were significantly reduced in epididymal caput cells with gBD124 silenced (P<0.05). The expressions of IL-1β, IL-1R2, IL-8, CCL6, CCL21 were markedly promoted (P<0.05), the expressions of CCL5 and IL-1α were significantly reduced in epididymal caput cells with gBD124 silenced (P<0.05). The concentration of CCL5 in the culture medium was significantly reduced in the group of silencing gBD124 (P<0.05). Compared with the blank control group, the concentrations of IL-1β and IL-8 in the culture medium were enhanced, and the concentration of IL-1α was significantly decreased in the group of silencing gBD124(P<0.05). The silencing of gBD124 in epididymal caput cells could regulate the expression of cytokines and chemokines through inhibiting the p38MAPK/AP1 signaling pathway.

Key words: epididymal caput cell, beta-defensin 124, shRNA-lentivirus vector, p38MAPK/AP1 pathway, goat

CLC Number:

S826.2

MENG Fanrong, TAI Miaomiao, DONG Fucheng, REN Youshe, LIU Wenzhong, QIAO Liying, ZHANG Chunxiang. Silencing of Goat Beta-defensin 124 Regulate the Expression of Cytokines and Chemokines in Epididymal Caput Cell by the p38MAPK/AP1 Signaling Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(6): 1248-1259.

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Silencing of Goat Beta-defensin 124 Regulate the Expression of Cytokines and Chemokines in Epididymal Caput Cell by the p38MAPK/AP1 Signaling Pathway

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