| Background Sepsis-induced acute respiratory distress syndrome(ARDS)is a clinical pulmonary disease with high mortality,characterized by an excessive,uncontrolled pulmonary inflammatory response and cytokine storm mediated by immune cells.To date,clinical management of ARDS are mainly limited to supportive care,there is no available drug therapy to be proved.Itaconate is an immunomodulatory derivative accumulated during inflammatory macrophage activation and has attracted widespread attention for its potent anti-inflammatory and antioxidant properties.4-Octyl itaconate(4-OI)is a derivative of endogenous itaconate,and existing studies have pointed out the protective effect of 4-OI in acute lung injury,but its specific mechanism remains unclear.As the key of regulating the inflammatory response and local inflammatory microenvironment in lung tissues,alveolar macrophages are crucial to the development of ARDS.The purpose of this study was to explore the potential regulatory mechanisms of itaconate and investigate whether it could attenuate sepsis induced-ARDS via inhibiting alveolar macrophages pyroptosis.Methods(1)Animal model construction: C57BL/6 male mice(6-8 weeks old)were divided into three groups at random,A: CON group,B: LPS group,and C: treatment group(LPS+4-OI group).The model and treatment groups were established by intraperitoneal injection of 5 mg/kg lipopolysaccharide(LPS)for sepsis ARDS model,and the treatment group was injected with 4-OI 50 mg/kg intraperitoneally 24 h and 2 h before LPS injection.After 12 h of LPS stimulation,the degree of lung injury was scored according to HE staining results;lung tissues were immunohistochemically stained with F4/80 to observe the number of macrophages;immunofluorescence double staining with F4/80 and NLRP3/STING;immunofluorescence staining with TUNEL;alveolar lavage fluid(BALF)was taken to detect the protein concentration;the wet/dry weight ratio of lung tissues was calculated.The mRNA expression of inflammatory factors in lung tissues was measured by RT-qPCR;Western blot was used to detect the expression of pyroptosis and cGAS-STING pathway-related proteins in lung tissues.(2)Cell models: Mouse alveolar macrophage cell lines(MH-S cells)were cultured and modeled by 1 μg/m L LPS stimulation for 6 h.The effects of 4-OI pretreatment at different doses(10 μM,62.5 μM,125 μM,250 μM,500 μM,1000 μM,and 2000 μM)on cell viability were detected by CCK8 assay;mRNA expressions of inflammatory factors in MH-S cells were detected by RT-qPCR;the typical expression of scorched cells were observed by transmission electron microscopy(MMP);the expressions of pyroptosis and cGAS-STING pathway-related proteins were detected by western blot;the levels of inflammatory factors in cell culture medium were detected by ELISA assay;Calcein-AM/PI double staining to observe the percentage of dead cells and Mito SOX staining for mitochondrial ROS;JC-1 kit was used to detect mitochondrial membrane potential;the opening of m PTP was detected by mitochondrial permeability transition pore(m PTP)kit;RT-qPCR for level of mitochondrial DNA(mtDNA)in cytoplasm.Results(1)Compared with the control group,the model group mice had severe lung injury,HE staining showed obvious pathological injury changes,increased lung wet/dry specific gravity,obvious infiltration of macrophages,and significantly increased expression of inflammatory factor RNA in lung tissue.In the treated group,after 4-OI pretreatment,lung injury,macrophage infiltration and inflammatory response were significantly reduced compared with the model group.(2)LPS induction caused a significant increase in the expression of scorch death-related proteins(NLRP3,ASC,GSDMD-N,CASP-1 p20,Pro-IL-1β,mature IL-1β),and ELISA detection revealed an increased level of secretion of inflammatory factors IL-1β and IL-18 in BALF,serum and cell culture media,and a rise in the proportion of PI-stained cells.And 4-OI and NLRP3-specific inhibitor MCC950 could significantly reverse the above changes.(3)4-OI pretreatment significantly reduced LPS-stimulated mitochondrial ROS production,mitochondrial membrane potential deficit,m PTP opening and mtDNA cytoplasmic release.(4)LPS stimulation significantly increased the levels of cGAS-STING pathway-related proteins(cGAS,STING,p-TBK1,p-IRF3).And 4-OI was able to significantly inhibit the expression of the above proteins.To further investigate whether the inhibitory effect of 4-OI on cGAS-STING pathway is associated with AMs pyroptosis,we used C-176(STING inhibitor)and KIN1148(IRF3 agonist)to detect the expression of pyroptosis-related proteins,Calcein-AM/PI double staining,and the secretion levels of IL-1β and IL-18,which verified that 4-OI exerted anti-pyroptosis effects by inhibiting the activation of cGAS-STING pathway.Conclusion 4-OI inhibit alveolar macrophage pyroptosis by regulating mitochondrial function and downregulating the activation of cGAS-STING pathway,thus alleviating the lung injury in mice with sepsis-induced ARDS. |
