The Protective Effect Of Paeatoluenesulfonyl Vildagliptin On Acute Lung Injury And Its Anti-inflammatory Mechanism

OBJECTIVE: To investigate the protective effect of Paratoluenesulfonyl Vildagliptin in LPS-induced acute lung injury(ALI),and LPS were used to construct an inflammatory proliferation model in mouse RAW264.7 macrophages to explore its possible anti-inflammatory mechanism.METHODS: In this experiment,Balb/c male mice(18-22g)were selected in random,and LPS(20mg/kg)was used to replicate the acute lung injury model by intraperitoneal injection.Mice in each group were intragastric administrated with solvent(0.5% sodium carboxymethyl cellulose solution containing 4%,1,2-propanediol)or corresponding drugs respectively(dexamethasone: 1.5mg/kg;PV: 458μg/kg and229μg/kg).After 12 hours of modeling,the blood of the mice were collected and serum(1006xg,4 ℃,centrifugation for 10min)were separated to measure the release of TNF-α,IL-6 and NO or other cytokines.The lung and heart,liver,spleen,kidney tissues of the mice were collected for subsequent experiments.Computer fitting and proteomics were used to find the possible anti-inflammatory targets of PV,molecular signal pathways were determined based on differential protein expression in proteomics and the molecular docking constructed basing on PV pharmacophore.The levels of JAK phosphorylation and phosphorylated NF-κ B,caspase-3 and caspase-1 protein levels in lung tissues of mice were detected by Western blot.The expression of caspase-1 in lung tissues of mice was detected by immunohistochemistry.Mouse RAW264.7 macrophages were cultured,and the inflammatory proliferative model was stimulated by LPS.Different concentration of PV were added to intervene.The cell viability of RAW264.7 was detected by MTT assay.Cell apoptosis of RAW264.7was detected by flow cytometry.The concentrations of TNF-α,IL-1β and IL-6 were detected by ELISA.JAK phosphorylation and phosphorylated NF-κB,caspase-3 and caspase-1 protein levels were detected by Western blot.The m RNA expressions of NLRP3,caspase-1,IL-1β,IL-18,caspase-3 and Bax were detected by RT-q PCR.JAK2 and JAK3 were silenced or overexpressed in RAW264.7 cells using small interfering RNA plasmids,and the protein levels of p JAK2/JAK2,p JAK3/JAK3 and phosphorylated NF-κB,caspase-3 and caspase-1were detected by Western blot.RESULTS:1.The protective effect of Paratoluenesulfonyl Vildagliptin on acute lung injury1)PV significantly improved the survival rate of mice with acute lung injury induced by LPS(P <0.05);At the same time,compared with the normal group,there was no significant difference in the index of each organ in PV(458μg/kg)group(P > 0.05),suggesting that PV had no obvious toxicity to mice.2)PV significantly improved the pathological damage of lung tissue in mice with LPS-induced acute lung injury,decreased the infiltration of inflammatory cells in the alveoli and reduced pulmonary edema(P <0.01);Significantly reduced the activity of MPO and the production of MDA in lung tissues of mice(P <0.01);Significantly decreased the production of inflammatory factors(TNF-α,IL-1β,and IL-6)and NO in the serum of mice(P <0.05),suggesting that PV could protect mice in LPS-induced acute lung injury.2.The anti-inflammatory mechanism of Paratoluenesulfonyl Vildagliptin and the changes of related pathway protein levels1)Proteomics studies showed that the highly differential protein expression in JAK and NF-κB signaling pathways,suggesting that both JAK and NF-κB may be the targets of PV.Computed molecular fitting experiments also suggested that 195 potential targets were calculated in the PV pharmacophore and ALI disease model,including JAK kinase,caspase-3 and NF-κB signaling pathways.2)PV significantly inhibited the phosphorylation of JAK2 and JAK3 in lung tissues of mice(P <0.05),significantly reduced the production of phosphorylated NF-κB protein(P <0.01),and at the same time significantly decreased the expression of pyroptosis marker caspase-1 and apoptosis marker caspase-3(P <0.05),improved the pathological apoptosis and inflammatory pyroptosis of lung tissue caused by LPS,and reduced the occurrence of inflammation.3)PV significantly inhibited the inflammatory proliferation of RAW264.7 macrophages induced by LPS in a concentration-dependent manner(P <0.01),significantly promoted the apoptosis of RAW264.7macrophages induced with LPS(P <0.01),and reduced the levels of inflammatory factors(TNF-α,IL-1β,IL-6)in the culture medium of RAW264.7 macrophages after LPS induction(P <0.05).4)PV significantly inhibited the phosphorylation of JAK2 and JAK3 kinases in RAW264.7 macrophages induced by LPS(P <0.01),and the phosphorylation of NF-κB protein(P <0.01).PV also significantly inhibited the level of pyroptosis protein(caspase-1)and the expression of pyroptosis protein m RNA(caspase-1,NLRP3,IL-1β,IL-18);Significantly promoted the expression of apoptosis protein(caspase-3)and apoptosis protein m RNA(caspase-3 and bax)(P <0.05).3.Changes in protein levels of related signaling pathways after transfection of Si RNA plasmids1)PV inhibited the phosphorylation of JAK2 and JAK3 in macrophages that were silenced or overexpressed(P <0.05).2)PV inhibited the phosphorylation of JAK2 and JAK3,at the same time,inhibited the activation of NF-κB protein(P <0.05)and the expression of caspase-1 protein(P <0.05),and promoted the expression of caspase-3 protein(P <0.05).CONCLUSIONS:1)Paratoluenesulfonyl Vildagliptin can alleviate LPS-induced acute lung injury(ALI),and inhibit the occurrence of inflammatory cell pyroptosis and pathological apoptosis in lung tissue.2)Paratoluenesulfonyl Vildagliptin can promote the apoptosis of macrophages induced by LPS and inhibit the occurrence of pyroptosis,which may be related to the selective inhibition of phosphorylation modification of JAK2 and JAK3,thereby affecting NF-κB-NLRP3-caspase-1 signaling pathway.