Preliminary Study On The Expression And Function Of DUSP1 In Hypoxic Macrophages

Background:High altitude disease?HAD?is a kind of high altitude idiopathic diseases caused by hypobaric hypoxia,which can be classified into acute altitude diseases and chronic altitude diseases based on the clinical onset.Recent studies have shown that hypoxia-induced inflammation is involved in the occurrence and development of HAD.Macrophage is an important innate immune cell,which exists widely in different organs and tissues of the body and performs various functions,including fighting against invading pathogens and tumor cells,coordinating wound healing,and participating in antigen presentation in acquired immune response,etc.Many of the pathological processes which macrophage is involved in?such as inflammation,wound healing,atherosclerosis,and tumors?are also characterized by hypoxia.Macrophages are very sensitive to hypoxia,and activated macrophages can activate and recruit other inflammatory cells by expressing and secreting certain inflammatory cytokines and chemokines,so as to participate in hypoxia induced tissue and body injury or remodeling.Hypoxia induced macrophage inflammatory immune response has been shown closely related to the onset and development of HAD.Therefore,it is of great significance to deeply understand the mechanism of the hypoxia induced macrophage inflammatory immune response for further elucidating the pathogenesis of HAD and developing prevention and treatment drugs.So far,the specific mechanism of the hypoxia-induced macrophage inflammatory immune response is still not fully understood.Dual specificity phosphatase-1?DUSP1?,also known as Mitogen-activated protein kinase phosphatase-1?MKP-1?,is the earliest identified DUSP member and also the most important one.DUSP1 catalyzes the hydrolysis of specific motif TXY phosphate groups in activated MAPK family members?p38,JNK and ERK?,thereby inhibiting their activity and blocking their downstream biological reactions.The Dusp1 gene was initially identified as one of a group of genes expressed during G0/G1 transformation in cultured murine cells.Various stress factors such as ultraviolet radiation,pathogen protein and hypoxia can induce the up-regulation of DUSP1 expression.Our results about whole blood transcriptome study of people showed that the transcription level of DUSP1 increased significantly after entering5300m plateau.The biological significance of increased DUSP1 expression in the occurrence and development of HAD is not clear.In view of DUSP1's important role in negatively regulating Toll-like receptors?TLR?ligand-induced macrophage inflammation and the important role of hypoxia-induced macrophage inflammation in the occurrence and development of HAD,DUSP1 may be involved in the occurrence and development of HAD by regulating hypoxia-induced macrophage inflammation.Currently,the mechanism of hypoxia induced DUSP1 expression and whether DUSP1 is involved in the regulation of hypoxia-induced macrophage inflammation remain unclear.Our results of bioinformatics analysis show that Activating transcription factor 3?ATF3?may participate in the regulation of Dusp1 expression.Therefore,we speculated that hypoxic macrophages may up-regulate DUSP1 expression through transcription factor ATF3,and then regulate the expression of some inflammation-related cytokines,so as to prevent their own excessive inflammatory immune responses.In this study,mouse RAW264.7 macrophages,which was widely used in the study of inflammatory response were selected as a cell model to preliminarily explore the expression mechanism of DUSP1 in hypoxic macrophage and the role of DUSP1 in the regulation of hypoxia-driven macrophage inflammatory immune response,Aimed at to provide a new idea and some theoretical and experimental basis for the pathogenesis and prevention of hypoxia induced injury in high altitude.Methods:Mouse RAW264.7 macrophage was used as the research model.According to different experimental purposes,they were divided into normoxia?21%O₂?group,hypoxia?1%O₂?group,hypoxia+small interfering RNA?siRNA?control transfection group?si-C?and hypoxia+siRNA transfection group.Hypoxic cells were cultured in a hypoxic incubator?1%O₂?,while the normoxic cells were cultured in an ordinary incubator.The mRNA and protein expression of DUSP1 and ATF3 in hypoxic RAW264.7 cells was detected by Quantitative Real-time Polymerase Chain Reaction?qRT-PCR?and Western Blot?WB?.The mRNA expression and secretion level of inflammation-related cytokines IL-1?,IL-10 and TNF-?were detected by qRT-PCR and Enzyme linked immunosorbent assay?ELISA?.The Dusp1 expression was interfered by siRNA,and the interference efficiency was detected by qRT-PCR and WB.The mRNA expression and secretion level of IL-1?,IL-10 and TNF-?in hypoxic RAW264.7 cells transfected with si-Dusp1 was detected by qRT-PCR and ELISA.The expression of Atf3 was interfered by siRNA,and the interference efficiency was detected by qRT-PCR and WB.DUSP1 expression in hypoxic RAW264.7 cells transfected with si-Atf3 was detected by qRT-PCR and WB.The mRNA expression and secretion level of IL-1?,IL-10 and TNF-?in hypoxic RAW264.7 cells transfected with si-Atf3 was detected by qRT-PCR and ELISA.The p38 phosphorylation level was detected by WB.Results:1.Effects of hypoxia on DUSP1 expression in RAW264.7 macrophages:The results of qRT-PCR and WB showed that,compared with the normoxia group,the mRNA and protein expression levels of DUSP1 in 12h,24h and 36h hypoxic RAW264.7 cells all increased significantly?P<0.05?.2.The regulatory role of DUSP1 in the expression of some inflammatory-related cytokines in hypoxic RAW264.7 macrophages:Compared with the normoxia group,there were no significant changes in Il1b and Il10 mRNA expression in RAW264.7 macrophages after 12h of hypoxia exposure?P>0.05?,while the mRNA expression of Il1b and Il10 after24h and 36h of hypoxia exposure increased significantly?P<0.05?.Compared with the normoxia group,the mRNA expression of Tnfa after 12h of hypoxia exposure reduced significantly?P<0.05?,while the mRNA expression of Tnfa after 24h and 36h of hypoxia exposure did not changed significantly?P>0.05?.Compared with the normoxia group,the secretion level of IL-1?in three hypoxia group all increased significantly?P<0.05?.Compared with the normoxia group,the p38 phosphorylation level in hypoxic RAW264.7cells increased,and the p38 phosphorylation level after 24h of exposure increased significantly?P<0.05?.Compared with the si-C group,the mRNA expression level of Il1b increased significantly?P<0.05?and the Tnfa mRNA expression level decreased significantly?P<0.05?in hypoxic RAW264.7 cells transfected with si-Dusp1,while the Il10 mRNA expression level did not changed significantly?P>0.05?.Compared with the si-C group,the secretion level of IL-1?and IL-10 in hypoxic RAW264.7 cell transfected with si-Dusp1increased significantly?P<0.05?and the TNF-?secretion level decreased significantly?P<0.05?.Compared with the si-C group,the p38 phosphorylation level in hypoxic RAW264.7 cell transfected with si-Dusp1 increased significantly?P<0.05?.3.The regulatory role of ATF3 in the expression of DUSP1 and aforesaid inflammation-related cytokines in hypoxic RAW264.7 macrophages:The results of qRT-PCR and WB showed that,compared with the normoxia group,the mRNA and protein expression levels of ATF3 in RAW264.7 cells after 12h,24h and 36h hypoxia exposure all increased significantly?P<0.05?.qRT-PCR and WB results showed that compared with the si-C group,DUSP1 expression did not changed significantly in hypoxic RAW264.7 cells transfected with si-Atf3?P>0.05?.Compared with the si-C group,The mRNA expression and secretion level of IL-1?,IL-10 and TNF-?increased significantly in hypoxic RAW264.7 cells transfected with si-Atf3?P<0.05?.Compared with the si-C group,the p38 phosphorylation level in hypoxic RAW264.7 cell transfected with si-Atf3 increased significantly?P<0.05?.Conclusions:1.Hypoxia can induce the up-regulation of DUSP1 expression in RAW264.7macrophages.2.Hypoxia can induce the up-regulation of inflammation-related cytokines Il1b and Il10mRNA expression in RAW264.7 macrophages,and can also cause an up-regulation of IL-1?secretion level.Hypoxia can induce the up-regulation of p38 phosphorylation level in RAW264.7 cell.DUSP1 participates in the hypoxia-induced RAW264.7 macrophages inflammatory immune response by regulating the mRNA expression and/or secretion of IL-1?,IL-10 and TNF-?,and the mechanism may be relevant with the regulation of p38phosphorylation3.Hypoxia can induce the up-regulation of transcription factor ATF3 expression in RAW264.7 macrophages.ATF3 may not be involved in the up-regulation of DUSP1expression in hypoxic RAW264.7 cells.ATF3 participates in the hypoxia-induced RAW264.7macrophages inflammatory immune response by regulating the mRNA expression and secretion of IL-1?,IL-10 and TNF-?,and the mechanism may be relevant with the regulation of p38 phosphorylation.