The Combination Effects Of Acetaminophen And N-acetylcysteine On Inflammasome Signaling Pathway In Piglets Subjected To Immunological Stress

Immunological stress in piglets induced by pathogen infection or vaccination is verycommon and has brought great harm to the pig industry. In order to study and solve thisproblem,a series of experiments in vitro and in vivo had been completed in our projectteam. Lipopolysaccharide (LPS) is one of representative inducer of immune stress.In theearly work of our project team, LPS-induced immunological sress models were establishedsuccessfully to evaluate the combination effects of acetaminophen(AAP) andN-acetylcysteine(NAC) on inflammation from immunological stress in piglets,Whichshowed that acetaminophen and N-acetylcysteine could play a positive role in themitigation of inflammation from immunological stress in piglets by inhibiting theTLR4-NF-κB signaling pathway. Based on the work, a further research on the molecularmechanisms of the mitigation of inflammation from immunological stress in piglets byacetaminophen and N-acetylcysteine was completed to improve the molecular mechanismstheory. The inflammasomes-mediated caspase enzymes-1(caspase-1) signaling pathwayalso plays a very important role in inflammation from immunological stress by promotingthe maturation and secretion of inflammatory cytokines IL-1β and IL-18. Then, models invitro and in vivo of piglets were established in this study to evaluate the combinationeffects of acetaminophen and N-acetylcysteine on inflammasome signaling pathway inpiglets subjected to immunological stress.In vitro tests: LPS-induced immunological stress models of peripheral bloodmononuclear cells in piglets were established first to evaluate the combination effects ofacetaminophen and N-acetylcysteine on inflammasome signaling pathway in pigletsPBMCs by detecting the content of IL-1β, IL-18and TNF-α in the cell culture supernatant,the content of caspase-1and the mRNA of IL-1β, IL-18in the cell lysate. Method: pigletsPBMCs were separated and divided into five groups respectively: control group,LPS-induced group, AAP group (0.25,0.5,1mM), NAC group (0.25,0.5,1mM) andcombinated-treated group (AAP0.25-1mM and NAC0.25-1mM in combination).Incubation with drugs for1h, then LPS (the final concentration was1μg/mL) were addedand continued to incubate for3,10,16,20h. The content of IL-1β, IL-18and TNF-α in thecell culture supernatant after LPS-stimulation10,16,20h was determined by ELISA; Thecontent of cleaved casapse-1in the10h LPS-stimulated monocytes was determined bywestern blot; The mRNA expression of IL-1β and IL-18in the3h LPS-stimulatedmonocytes was determined by RT-PCR. Results:①10h after LPS stimulation, thecontent of IL-1β, IL-18and TNF-α in the cell culture medium was significantly increased(P<0.01). NAC with the concentration of0.25,0.5,1mM could significantly (P<0.01)enhance the inhibitory effect on the secretion of IL-1β, IL-18and TNF-α of AAP(0.25-1mM);16h after LPS stimulation, the content of IL-1β, IL-18and TNF-α in the cell culture medium was significantly increased (P<0.01). NAC with the concentration of0.25,0.5,1mM could significantly (P<0.01) enhance the inhibitory effect on the secretion ofIL-1β, IL-18and TNF-α of AAP (0.25-1mM);20h after LPS stimulation, the content ofIL-1β, IL-18and TNF-α in the cell culture medium was significantly increased (P<0.01).Ifonly the concentration was above0.5mM, NAC could significantly (P<0.01) enhance theinhibitory effect on the secretion of IL-1β of AAP (0.5-1mM), but NAC with theconcentration of0.25,0.5,1mM could significantly (P<0.01) enhance the inhibitory effecton the secretion of IL-18and TNF-α of AAP (0.25-1mM).②10h after LPS stimulation,the content of cleaved casapse-1was significantly increased (P<0.01) in the monocytes.NAC with the concentration of0.25,1mM could significantly (P<0.05) enhance theinhibitory effect on the biological activity of caspase-1of AAP (0.25,1mM) and NACwith the concentration of0.5mM could significantly (P<0.01) enhance the inhibitory effecton the biological activity of caspase-1of AAP (0.5mM).③3h after LPS stimulation, themRNA expression of IL-1β and IL-18in monocytes was significantly increased (P<0.01).NAC with the concentration of0.5,1mM could significantly (P<0.05) enhance theinhibitory effect on the mRNA expression of IL-1β of AAP (0.5-1mM), but could notenhance the inhibitory effect on the mRNA expression of IL-18of AAP (0.5-1mM).In vivo tests: immunological stress models in piglets was established first byintraperitoneal injection of lipopolysaccharide to evaluate the combination effects ofacetaminophen and N-acetylcysteine on inflammasome signaling pathway in piglets bydetecting the mRNA of IL-1β and IL-18in the cell lysate.Method:30healthy piglets (7.98±1.00kg) were randomly divided into five groups: control group, LPS group, LPS+AAPgroup (mixed concentration of600mg/kg), LPS+NAC group (mixed concentration of1200mg/kg) and LPS+AAP+NAC group (mixed concentration of AAP600mg/kg and ofNAC1200mg/kg). After pre-fed for10days, the piglets in treated groups were stimulatedby intraperitoneal injection of LPS at dose of100μg/kg.bw and the piglets in the controlgroup were stimulated by intraperitoneal injection of saline at the same dose.Bloodsamples were collected from the piglets by anterior vena cava at3h after LPS injection.The mRNA expression of IL-1β and IL-18in the3h LPS-stimulated peripheral bloodmonocytes was determined by RT-PCR. Results: the mRNA expression of IL-1β andIL-18in the monocytes of LPS group was significantly increased (P<0.01). NAC couldsignificantly (P<0.05) enhance the inhibitory effect on the expression of IL-1β mRNA ofAAP and significantly (P<0.01) enhance the inhibitory effect on the expression of IL-18mRNA of AAP.The results showed that acetaminophen combined with N-acetylcysteine could reduceLPS-induced immunological stress by inhibiting inflammasome signaling pathway inpiglets.