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

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

旨在研究山羊β防御素124(goat beta-defensin 124,gBD124)沉默对p38MAPK/AP1通路及下游细胞趋化因子和细胞因子表达的影响。本研究将设计的3条gBD124-shRNAs载入LV10-U6/RFP&Puro质粒载体,与包装质粒共转染到293T细胞中,用梯度稀释法测定病毒原液的滴度;将构建的3个LV10-gBD124和LV10-gBD124-NC载体转染到山羊附睾头细胞中,筛选有效沉默载体;然后用有效沉默载体联合转染附睾头细胞,设置了空白细胞对照组、LV10-NC组、有效沉默载体组,用2μg·mL^-1嘌呤霉素筛选后分别收集细胞和培养液,采用qRT-PCR、Western blot和ELISA检测gBD124、MAPK信号通路关键蛋白、细胞因子及趋化因子基因及蛋白表达情况。本研究构建了gBD124沉默慢病毒载体,筛选出LV10-gBD124-51和LV10-gBD124-161两个有效载体,并成功构建了gBD124沉默稳定转染的附睾头细胞株。与NC对照组相比,gBD124沉默显著降低了山羊附睾头细胞MAPK通路中MAPK1以及AP1的两个亚型c-JUN和c-FOS基因表达(P<0.05),显著增加了RASA1基因表达(P<0.05);gBD124沉默显著降低了总p38MAPK、总c-JUN和总c-FOS蛋白表达,以及磷酸化p38MAPK和c-JUN蛋白表达(P<0.05);gBD124沉默显著上调了MAPK通路下游IL-1β及其受体IL-1R2、IL-8和趋化因子CCL6和CCL21基因表达(P<0.05),下调了CCL5和IL-1α基因表达(P<0.05);gBD124沉默显著降低了细胞培养液中CCL5浓度(P<0.05)。与空白对照组相比,gBD124沉默显著增加了培养液中IL-1β和IL-8浓度,降低了IL-1α浓度(P<0.05)。gBD124基因沉默通过抑制p38MAPK/AP1信号通路调控附睾头细胞趋化因子和细胞因子的表达。

The study was conducted to investigate the effects of silencing goat beta-defensin 124(gBD 124)on the expression of key genes in p38MAPK/AP1 pathway and their downstream target genes of the cytokines and chemokines.Three designed gBD 124-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-gBD 124 vectors and LV10-gBD 124-NC were transfected into epididymal caput cells of Taihang goat,respectively for screening the effective silent vectors.The effective recombinant LV10-gBD 124 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 2μg·mL-1 puromycin.The mRNA and protein expression levels of gBD 124,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-gBD 124 recombinant vectors were constructed and two valid recombinant vectors of LV10-gBD 124-51 and LV10-gBD 124-161 were screened for silencing gBD 124.The epididymal caput cells with gBD 124 silenced were successfully constructed.The results of qRT-PCR indicated that silencing of gBD 124 in epididymal caput cells significantly decreased the expressions of MAPK 1,c-JUN and c-FOS(P<0.05),and significantly increased the expression of RASA 1 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 gBD 124 silenced(P<0.05).The expressions of IL-1β,IL-1R2,IL-8,CCL 6,CCL 21 were markedly promoted(P<0.05),the expressions of CCL 5 and IL-1αwere significantly reduced in epididymal caput cells with gBD 124 silenced(P<0.05).The concentration of CCL5 in the culture medium was significantly reduced in the group of silencing gBD 124(P<0.05).Compared with the blank control group,the concentrations of IL-1βand IL-8 in the culture medium were enhanced,and the concentration of IL-1αwas significantly decreased in the group of silencing gBD 124(P<0.05).The silencing of gBD 124 in epididymal caput cells could regulate the expression of cytokines and chemokines through inhibiting the p38MAPK/AP1 signaling pathway.