A Experimental Model Of Acid Gastric Contents And LPS Induced Aspiration Lung Injury In Rats And The Role Of Pyroptosis

Objective:By intratracheally instilled with CASP followed by another instillation of LPS,to investigate whether synergy between the aspiration of gastric contents and bacterial products(LPS)amplifies injury severity in rat,so as to provide an experimental model of aspiration pneumonia,and explore the effect of aspiration pneumonia on pyroptosis signalling pathway.Methods :74 healthy SD male rats,aged 6-8 weeks,weight 220 士 20 g,were selected,10 rats were used to extract the stomach contents.Combined acid and small particles was prepared from stomach contents by filtration,utoclave sterilization,centrifugation,and p H adjusting.The rest 64 rats were randomly divided into 4 groups:one instillation of saline followed by one instillation of saline 2 h later(NS/NS group),one instillation of CASP followed by one instillation of saline 2 h later(CASP/NS group),one instillation of saline followed by one instillation of LPS 2 h later(NS/LPS group),and one instillation of saline followed by one instillation of LPS 2 h later(CASP/LPS group).All rats in those 4 groups were euthanatized at a specific time point(6 or 24 hours after the first instillation),by exsanguination through the abdominal aorta.The activity of myeloperoxidase(MPO)content in the lung tissue were measured with biochemistry methods;The levels of IL-1β in the lung tissue were detected using enzyme linked immunosorbent assay(ELISA);The expression of protein GSDMD and NLRP3 were determined by Western blot and immunohistochemistry;The wet/dry weight ratio(W / D)of lung,the levels of total proteins,the total cell count,in bronchoalveolar lavage fluid(BALF)were examined.The pathological change of lung tissue was observed by light microscope.Results:1.HE stain: The lung tissue of NS/NS group was approximately normal.Lung pathological injury and inflammatory responses could be seen in CASP/NS、NS/LPS、CASP/LPS group,especially in CASP/LPS group at 24 h.2.The activity of myeloperoxidase(MPO)content in the lung tissue: After aspiration lung injury was formed 6,24 hours later,compared with NS/NS group CASP/NS group,and NS/LPS group,the MPO in CASP/LPS group were significantly higher;Compared with NS/NS group the count of total cell in CASP/NS group were significantly higher;3.The levels of IL-1β in the lung tissue: After aspiration lung injury was formed 6 hours later,compared with NS/NS group,the levels of IL-1β in CASP/NS group,CASP/LPS group were significantly higher,compared with NS/LPS group and CASP/NS group,the levels of IL-1β in CASP/LPS group were significantly higher;At 24 h,compared with NS/NS group,the levels of IL-1β in CASP/NS group,NS/LPS group and CASP/LPS group were significantly higher,compared with CASP/NS group,the levels of IL-1β in CASP/LPS group were significantly higher.4.The expression of protein GSDMD and NLRP3:Western blot: After aspiration lung injury was formed 6,24 hours later,compared with NS/NS group CASP/NS group,and NS/LPS group,the expression of GSDMD in CASP/LPS group were significantly higher;After aspiration lung injury was formed 6 hours later,compared with NS/NS group,the expression of NLRP3 in CASP/LPS group were significantly higher.At 24 h,compared with NS/NS group,CASP/NS group and NS/ LPS group,the expression of NLRP3 in CASP/LPS group were significantly higher.IHC:In NS/NS group,GSDMD was hardly any expression,compared with NS/NS group,the expression of GSDMD in CASP/NS group,and CASP/LPS group were significantly higher,especially in CASP/LPS group.5.The wet/dry weight ratio(W/D): After aspiration lung injury was formed 6 hours later,compared with NS/NS group,the W / D of lung inCASP/NS group,NS/LPS group,CASP/LPS group were significantly higher,compared with CASP/NS group and NS/LPS group,the W / D of lung in CASP/LPS group were significantly higher;At 24 h,compared with NS/NS group,CASP/NS group and NS/LPS group,the W/D of lung in CASP/LPS group were significantly higher.6.The levels of total proteins: After aspiration lung injury was formed 6hours later,compared with NS/NS group,the levels of total proteins in CASP/NS group,CASP/LPS group were significantly higher,Compared with NS/LPS group,the levels of total proteins in CASP/LPS group were significantly higher;At 24 h,compared with NS/NS group,the levels of total proteins in CASP/NS group,CASP/LPS group were significantly higher,compared with CASP/NS group and NS/LPS group,the levels of total proteins in CASP/LPS group were significantly higher.7.The total cell count in bronchoalveolar lavage fluid(BALF): After aspiration lung injury was formed 6,24 hours later,compared with NS/NS group,the count of total cell in CASP/NS group,NS/LPS group,CASP/LPS group were significantly higher,Compared with CASP/NS group and NS/LPS group,the count of total cell in CASP/LPS group were significantly higher.Conclusion:The animal model of aspiration pneumonia in rats induce by acid gastric contents with LPS instillation can induce aspiration lung injury in rats,which is more serious than acid gastric contents or LPS instillation alone.The expression of GSDMD,NLRP3 and IL-1 β were induced by acid gastric contents with LPS instillation were up-regulated.Acid gastric contents combined with LPS instillation may activate NLRP3 inflammasome signaling pathway,cause cell pyroptosis and cause more serious lung injury.