Ghrelin Inhibits Alveolar Macrophages Pyroptosis And Subsequently Attenuates Sepsis-induced Acute Lung Injury Through TLR4/NLRP3 Inflammatory Signaling Pathway

Background: Sepsis is delimited as a life-threatening organ dysfunction led to a dysregulated host response to infection,ALI is one of the most common complications of sepsis.Sepsis leads to the rapid onset of ALI with a mortality of up to 40%.Ghrelin is secreted in organs such as lung,kidney and placenta,immune organs and immune cells such as lymphocytes,neutrophils and monocytes,and has anti-inflammatory effects.Studies have shown that ghrelin can reduce sepsis induced-ALI,but the exact mechanism is not clear.Aims: This study aimed to investigate whether exogenous ghrelin can protect sepsis-induced ALI by regulating pyroptosis activation of alveolar macrophages via TLR4/NLRP3 pathway.Methods: The lung injury model of sepsis in mice was established by cecal ligation and puncture(CLP)method.The rats were divided into sham operation group(sham group),CLP group,CLP+ghrelin group,CLP+ghrelin/[D-Lys-3]-GHRP-6 group,CLP+ghrelin/TAK-242 group).The effect of ghrelin on lung structural damage in sepsis mice was detected by H&E staining,total protein level and cell number in BALF,and the survival time of mice was recorded for 120 hours.Inflammatory factors(TNF-α,IL-6,IL-10,IL-1 β and IL-18)in bronchoalveolar lavage fluid(BALF)and plasma were detected by ELISA.The expression of TLR4 and GSDMD were detected by immunofluorescence staining.The m RNA of TLR4/NLRP3 pathway and NLRP3,ASC,Caspase-1,GSDMD pathway were test by q PCR,and the protein expression of TLR4/NLRP3 pathway and NLRP3,ASC,Caspase-1,GSDMD pathway were evaluated by using western blotting.Cell experiment: the model of RAW264.7 macrophage sepsis was induced by LPS.They were divided into Control group,Con + ghre lin group,LPS group,LPS + ghrelin group,LPS+ghrelin/[D-Lys-3]-G HRP6 group,LPS + ghrelin/TAK-242 group.The survival rate of RA W264.7 cells was detected by CCK8,and the apoptosis of RAW264.7 cells was detected by mitochondrial membrane potential and flow c ytometry.The inflammatory cytokines(TNF-α,IL-6,IL-10,IL-1β and IL-18)in the supernatant of cell culture were detected by ELISA.The expression of TLR4 and GSDMD were detected by immunofluor escence staining.The m RNA of TLR4/NLRP3 pathway and NLRP3,ASC,Caspase-1,GSDMD pathway were test by q PCR,and the prote in expression of TLR4/NLRP3 pathway and NLRP3,ASC,Caspase-1,GSDMD pathway were evaluated by using western blotting.Results: Animal experiment: Ghrelin can significantly reduce the mortality of sepsis lung injury mice induced by CLP.Ghrelin intervention decreased the total protein and cell number in bronchoalveolar lavage fluid of sepsis induced-ALI mice,and ghrelin improved the pathological changes of lung tissue in sepsis mice.Compared with sham group,IL-10,TNF-αand IL-6 were significantly increased in CLP group,the m RNA and protein expression of TLR4/NLRP3 pathway and pyroptosis pathway(NLRP3,ASC,Caspase-1,GSDMD)were significantly increased in CLP group,while ghrelin decreased the expression of pro-inflammatory factors TNF-αand IL-6 and promoted the expression of anti-inflammatory factor IL-10.Ghrelin decreased TLR4/NLRP3 pathway and the expression of m RNA and protein of NLRP3,ASC,Caspase-1 and GSDMD pathway in sepsis lung injury mice.Immunofluorescence results showed that the expression of TLR4 and GSDMD in ghrelin group was significantly lower than that in CLP group.In addition,Ghrelin decreased the release of scorch related factors IL-1β and IL-18.The expression of downstream pathway(NLRP3,ASC,Caspase-1,GSDMD)was significantly increased after the administration of TLR4 receptor antagonist TAK-242.Cell experiment: Ghrelin significantly increased the survival rate of cells in LPS group;ghrelin group decreased the loss of mitochondrial membrane potential and apoptosis rate of RAW264.7 alveolar macrophages induced by LPS;compared with LPS group,ghrelin decreased the expression of pro-inflammatory cytokines TNF-α and IL-6 in the supernatant and increased the expression of anti-inflammatory cytokines IL-10.Compared with Control group,the expression of TLR4/NLRP3 pathway and pyroptosis pathway(NLRP3,ASC,Caspase-1,GSDMD)in LPS group was increased,and ghrelin decreased the expression of TLR4/NLRP3 pathway and NLRP3,ASC,Caspase-1,GSDMD induced by LPS in Raw264.7 cells.The results of immunofluorescence showed that the expression of TLR4 and GSDMD in ghrelin group was significantly lower than that in CLP group.In addition,ghrelin decreased the release of pyroptosis related factors IL-1β and IL-18.The expression of downstream pathway(NLRP3,ASC,Caspase-1,GSDMD)was significantly increased after the administration of TLR4 receptor antagonist TAK-242.Conclusion: Ghrelin improved the survival rate of sepsis lung injury mice and alleviated lung histopathological changes,ghrelin increased the survival rate of RAW264.7 cells induced by LPS,significantly decreased the loss of mitochondrial membrane potential and apoptosis rate of RAW264.7 cells induced by LPS,and ghrelin decreased the expression of inflammatory factors such as TNF-α and IL-6 in sepsis lung injury.The results show that ghrelin has a protective effect on sepsis lung injury.Ghrelin may regulate the pyroptosis of alveolar macrophages through TLR4/NLRP3 inflammatory signal pathway,thus reducing ALI caused by sepsis.