The Effect And Mechanism Of Tiliroside On NLRP3 Inflammasome Activation And Endotoxin-Induced Acute Lung Injury In Mice

Objective: To ascertain Tiliroside’s effects on activating the NLRP3 inflammasome and endotoxin-induced acute lung injury(ALI)in mice,and to investigate any potential molecular pathways involved.Methods:1.Animal experiments: The experimental mice were separated into 4 groups: the vehicle group,the LPS group,the LPS+tiliroside low-dose(50 mg/kg)group,and the LPS+tiliroside high-dose(100 mg/kg)group.Mice in the administering group received the corresponding amount of tiliroside once daily through gavage,whereas mice in the other groups received an equivalent volume of 0.5% CMC-Na solution,nine days before modeling.To create an endotoxin-induced model of ALI in mice,intraperitoneal injection with 3 mg/kg LPS was administered two hours after gavage on day 9 of administration,and sample collection was completed 24 hours later.The number and percentage of peripheral blood neutrophils and monocytes were determined using the blood cell analyzer;Mice’s liver,kidneys,spleen,lungs,and spleen were gathered,and the organ/body weight ratio was computed;Calculate the lung tissue’s wet/dry weight(W/D)ratio after removing the top left lung;Hematoxylin-eosin(HE)staining was used to quantify the mean alveolar area,conduct lung histopathology scoring,and examine changes in lung tissue structure.BCA was used to measure the protein content in the bronchoalveolar lavage fluid(BALF)after it had been collected;m RNA expression levels of the inflammatory mediators Il-1β,Inos,Il-6,and Cxcl-2 in lung tissue may be measured using real-time quantitative PCR(q RT-PCR).NLRP3 inflammasome-related protein and AMPKphosphorylated protein expression levels in lung tissue were both discovered using Western blotting.IL-1β expression and CD68-labeled inflammatory macrophage infiltration in lung tissue are both observed using immunofluorescence.2.Cell experiment: The NLRP3 inflammasome model of THP-1 macrophages triggered by LPS and ATP was created,and the studies were separated into five groups: control,LPS+ATP,and LPS+ATP+tiliroside(3,10,30 μM)treatment.The protein levels of NLRP3,pro-caspase-1,pro-IL-1β,AMPKα,p-AMPKα(Thr172),and caspase-1 p20 and IL-1β were detected by Western blot in THP-1 cells stimulated with LPS+ATP or LPS.Inflammatory cytokines IL1 B,IL6,IL8,and TNFA m RNA expression levels are detected by q RT-PCR in THP-1 cells,and NLRP3 and ASC colocalize there,as seen by immunofluorescence.JC-1 labeling technique identifies changes in mitochondrial membrane potential;mito SOX labeling measures the generation of mitochondrial reactive oxygen species(mt ROS)in THP-1 cells.Results:1.Animal experiments: The organ index of lung,spleen,and liver was dramatically improved following LPS treatment compared to the vehicle group,but the index of lung and spleen declined significantly after tiliroside(100 mg/kg)intervention,and the index of liver and kidney organ also exhibited a lowering tendency.The categorization and counting of peripheral blood leukocytes revealed that,following LPS stimulation in mice,the proportion of neutrophils,neutrophils,and monocytes increased dramatically.The number of monocytes also exhibited an increasing trend,whereas the aforementioned indices revealed a descending trend following tiliroside pretreatment.The lung tissue of ALI model mice had a considerable thickening of the alveolar wall and septum,infiltration of many inflammatory cells in the lung,elevated score of pathological damage of lung histology,and loss of average alveolar area.Nevertheless,the application of tiliroside intervention could greatly lessen the pathological injury of the lung tissue.Tiliroside considerably decreased pulmonary tissue edema brought on by LPS,according to the findings of the W/D test on lung tissue.The measurement of protein content in BALF revealed that the protein concentration in BALF was highly regulated in the LPS group,but the protein content was dramatically decreased following tiliroside pretreatment.Tiliroside significantly decreased the expression of the inflammatory mediators Il-1β,Inos,Il-6,and Cxcl-2 in the lung tissues of ALI mice,according to the results of q RT-PCR.The Western blot results demonstrated that tiliroside dramatically increased the expression of p-AMPK proteins while downregulating the expression of pro-caspase-1,pro-IL-1β,and caspase-1 p20 in the lung tissues of ALI mice.Tiliroside significantly reduced LPS-induced CD68-tagged inflammatory macrophage infiltration and IL-1β expression in lung tissue immunofluorescence.Based on the reported key roles of the NLRP3 inflammasome and macrophages in acute lung injury,the above experimental results suggest that tiliroside may resist endotoxin-induced acute lung injury by inhibiting macrophage activation NLRP3 inflammasomes and thus improving the inflammatory response of lung tissue.2.Cell experiments: According to the CCK8 cytotoxicity findings,THP-1macrophage activity during 30 hours was unaffected by the dosage of tiliroside(0–30μM).The findings of Western blot indicated that tiliroside dose-dependently decreased the production of LPS+ATP-induced caspase-1 p20 and mature IL-1β in THP-1 macrophages,and the immunofluorescence results showed that tiliroside considerably prevented the colocalization of LPS+ATP-induced NLRP3 and ASCs in the perinuclear region,indicating that tiliroside effectively inhibited the activation of NLRP3 inflammasome in macrophages.The inflammatory factors IL1 B,IL6,IL8,and TNFA produced by LPS+ATP in THP-1 macrophages were similarly dramatically lowered by tiliroside,according to q RT-PCR data.When the levels of mt ROS and mitochondrial membrane potential were tested using the kit,it was shown that tiliroside could dramatically increase the phosphorylation level of AMPKα in THP-1macrophages while also greatly reducing the formation of mt ROS and improving the decrease of mitochondrial membrane potential caused by LPS+ATP.Based on the aforementioned experimental findings and the well-known critical function of the AMPK-mt ROS axis in the activation of NLRP3 inflammasomes,we hypothesize that tiliroside will likely prevent the activation of macrophage NLRP3 inflammasomes by increasing AMPK phosphorylation,decreasing mt ROS generation,and reducing mitochondrial damage.Conclusions:Tiliroside may suppress mt ROS generation by targeting the macrophage AMPK signaling pathway,lowering the activation of NLRP3 inflammasomes,regulating macrophage-mediated excessive inflammatory responses,and alleviating endotoxininduced acute lung damage in mice.