P38Regulate MRNA Stability Of Cytokines Induced By Zymosan A

Objectives：To study the expression of cytokine（TNF-α,CXCL1,IL-10and IL-23a）induced by Zymosan A and the mechanism of the ARE-mRNA stability regulated by p38.Providing the experimental basis for revealing the fungal pathogenesis,and looking outthe new target for treatment of fungal infections.Methods：Primary peritoneal macrophage were isolated and purificated from C57mice.Total protein was extracted from each sample using cell lysis buffer. The phosphorylationof p38,ERK and JNK were checked by western blot. Bioinformatic methods was used toanalyze the sequences of cytokine induced by Zymosan A and found that AREs-mRNAof these cytokines could be regulated in the post-transcription level. Total RNA wasextracted from each sample using TRIzol reagent and total RNA isolation systemaccording to the manufacturer. Total RNA was reverse transcribed using one-Step RT-PCR Kit according to the manufacturer’s instruction, then mRNA levels of TNF-α,CXCL1, IL-10and IL-23a induced by zymsonA were detected by real-time PCR. ThemRNA dagradation degree of TNF-α, CXCL1,IL-10and IL-23a was measured byreal-time PCR, and their mRNA stability was analyzed. Blocking p38,ERK and JNKkinases respectively with SB202190、PD98059and SP600125，then mRNA decay ofIL-10, IL-23a,CXCL1and TNF-α was measured through real-time PCR and thephosphorylation of MK2was detected by western blot. The phosphorylation of TTP wasdetected by enzyme experiment in invitro, the action of p38was determined throughZymosan A induced signaling pathway, the ARE-mRNA stability of cytokine wasregulated by p38through phosphorylating MK2and TTP.Results：When the concentration of Zymosan A was100μg/ml, MAPK signaling pathway was activited well, the p38,ERK and JNK could be phosphorylated effectually.The mRAN levels of TNF-α,CXCL1,IL-10,IL-23a were high at this concentration.Bioinformatic analyzing showed that the mRNA3’UTR of cytokine above had AREs.The ARE-mRNA of cytokines induced by Zymosan A decayed to50%-60%at4hoursafter transcription, but untreated samples decayed to10%, the relults expressed that theARE-mRNA of cytokines induced by Zymosan A was more stable than the untreatedgroup. TTP phosphorylation disappeared after adding λPP. The western blot showedthat phosphorylation of MK2and TTP was inhibited by SB202190which could block theactivity of p38, and real-time PCR revealed the mRNA levels of TNF-α, CXCL1,IL-10and IL-23a decreased significantly, the results indicated that the ARE-mRNA wasunstable after the treatment of p38inhibitor. The phosphorylation of ERK was inhibitedwith PD98059which blocked the activity of ERK, but the mRNA contents and stabilityof cytokines(TNF-α, CXCL1,IL-10and IL-23a) did not change.The phosphorylation ofc-Jun was inhibited with SP600125which blocked the activity of JNK, but also the ARE-mRNA contents and stability of TNF-α,CXCL1,IL-10and IL-23a didn’t change.Conclutions：1. MAPK signaling pathway was actived well by Zymosan A. Zymosan A couldeffectully induce phosphorylation of p38、ERK and JNK, and high expression ofTNF-α,CXCL1,IL-10and IL-23a.2. p38could enhance mRNA stability of cytokine induced by Zymosan A. Themechanism may be p38phosphorylated MK2and TTP, and the phosphorylated TTPcan’t degrade ARE-mRNA of TNF-α, CXCL1,IL-10and IL-23a, therefore theARE-mRNA became stable.