Regnase-1 Participates In The Immune Response To Hepatic Ischemia/reperfusion Injury(IRI)by Regulating Macrophage Polarization

Hepatic IRI is an acute and dynamic process.In the ischemic stage,liver metabolism imbalance,severe tissue hypoxia,ATP depletion,mitochondrial dysfunction,oxidative stress and endoplasmic reticulum stress occur,and a large number of reactive oxygen species(ROS)are produced,which lead to organelle damage,and then initiate cell necrosis or apoptosis.However,liver dysfunction and failure mainly take place in the reperfusion stage.Hepatic IRI has always been a major challenge to clinical practice.Liver IRI is an aseptic inflammatory response in nature.During this process,both the innate and adaptive immune responses are enhanced,with the preponderance of innate immunity,which involve a series of complex cellular and molecular signaling pathways.Although the knowledge of liver IRI is expanding,how injury signals are transformed from metabolic stress to inflammation still deserves attention.macrophages(m?)are one important member of the innate immune system.During hepatic IRI,m? has been considered as the major contributor to oxidative stress and inflammatory response after early ischemia and as the main source of pro-inflammatory cytokines.However,m? will show both pro-inflammatory and anti-inflammatory reactions during hepatic IRI,which is called polarization.M1/M2 polarization exerts a significant effect on excessive inflammatory response in the process of liver IRI.Regnase-1,also known as MCPIP1 and ZC3H12 A,has attracted increasing attention due to its immunoregulatory activity.Regnase-1 is mainly expressed in monocytes and m?.It can recognize and bind to the specific secondary structure of target genes,namely the "stem loop" structure,and degrade a group of m RNAs encoding inflammatory cytokines through its endoribonuclease(RNase)activity,thus acting as an inhibitor of cytokine signaling to reduce inflammation.It is a negative regulator of m? activation,and is essential for maintaining immune homeostasis and preventing immune system overactivation under inflammatory conditions.However,the role of Regnase-1 in the course of liver IRI remains unclear.Regarding its immunoregulatory properties,this research aimed to explore the inhibitory influence of Regnase-1 on the inflammatory responses and the regulation of m? polarization during hepatic IRI,and to further clarify its potential mechanism on this basis.1.In vivo study of Regnase-1 regulating macrophage polarization during liver IRIA mouse liver IRI model was established successfully.Compared with the Sham group,the liver function and Suzuki's score in the IRI group were exacerbated,which turned reversed in the Clodronate group.RT-PCR and Western blot analysis showed that the gene and protein expressions of Regnase-1 were significantly increased in IRI group,which diminished in Clodronate group.Subsequently,double immunofluorescence staining confirmed that Regnase-1 was mainly expressed in m?.Additionally,in the CLO+BMDM group,the affected lesions were once again deteriorated with infusion of BMDM,which became more worsened with transfusion of BMDM with MCPIP knockdown in the Clo+LV MCPIP BMDM group.The gene expressions of M1 and M2 markers were detected by RT-PCR,suggesting that MCPIP deficiency tended to favor the M1 polarization.2.Ex vivo study of the regulation of macrophage polarization by Regnase-1during liver IRIBy isolating murine primary hepatocytes,we tried to simulate hepatic hypoxia and reoxygenation(H/R)model ex vivo.We used LPS to stimulate BMDM to induce M1 transformation,and used IL-4 to induce M2.Flow cytometry analysis showed that BMDM exhibited M1 and M2 states to some extent when stimulated by H/R supernatant,mostly being M1.However,for LV-MCPIP BMDM,M1 increased with LPS stimulation while M2 decreased with IL-4 induction.Under H/R supernatant stimulation,the M1/M2 ratio was markedly higher than that of BMDM.Subsequently,we detected the gene expression of M1 and M2 markers in all groups of cells by RT-PCR,which indicated that LV-MCPIP BMDM was more inclined to promote M1 polarization.3.Mechanism research on the modulatory role of regnase-1 in M1/M2 transformation through the NF-?B and CCAAT/enhancer binding protein ?(C/EBP?)and peroxisome proliferators-activated receptor ?(PPAR?)signaling pathwayWestern blot analysis showed enhanced NF-?B p65 levels in both BMDM and LV-MCPIP BMDM after H/R supernatant stimulation in vitro,while it was lower in BMDM.However,when NF-?B specific inhibitor BAY-11-7082 was added to both groups,the differences in M1/M2 phenotypic markers and flow cytometrical parameters disappeared.In vivo,the level of NF-?B p65 in the IRI group was significantly higher than that in the Sham group,which was reversed in Clodronate group.Phosphorylation of NF-?B p65 was increased once again in the CLO+BMDM group,which was more obvious in CLO+LV-MCPIP BMDM group.In addition,CST-PPAR?(C26H12)levels in both BMDM and LV-MCPIP BMDM were also enhanced in vitro after H/R supernatant stimulation,but it was higher in BMDM.When PPAR? specific inhibitor GW9662 was added to both groups,the differences in M1/M2 markers and flow cytometrical parameters were absent,too.In vivo,CST-PPAR?(C26H12)expressions displayed a first upregulated,then abated,and then elevated,but finally reduced tendency among these five groups.Meanwhile,C/EBP?(LAP)protein shared the same manifestations with CST-PPAR?(C26H12)basically.Collectively,this study demonstrated that Regnase-1 has an important immunosuppressive impact on liver IRI.Regnase-1 participates in the immune response of hepatic IRI by manipulating m? polarization,while Regnase-1knockdown tends to favor M1.Regnase-1 is involved in M1/M2 polarization by inhibiting NF-?B and inducing C/EBP? and PPAR? signaling pathways.