The Combination Effects Of Paracetamol And N-acetylcysteine On Inflammation Induced By Immunological Stress In Piglets

Immunological stress in piglets induced by pathogen infection or vaccination is very common and would bring great harm to the pig industry.There have not any effective drugs to control the stress. In this work, LPS-induced immunological stress models were established to evaluate the combination effects of paracetamol and N-acetylcysteine on inflammation of immunological Stress in piglets.Immunological stress model of porcine PBMC was established in vitro to investigate the combination effects of paracetamol and N-acetylcysteine. Method:Piglets PBMCs were separated and divided into five groups respectively:Control group, LPS-induced group, Single-treatment group (two drug concentrations both are0.0625-1mM), Combinated-treated group (AAP0.0625-0.5mM and NAC0.0625-0.5mM in combination). Incubation with drugs for1h, then LPS (the final concentration was1μg/mL) were added and continued to incubate. Cytokines (TNF-α, IL-1β, IL-6, IL-10), inflammatory mediators PGE2and COX-2activity and NF-κB activity were measured by ELISA assay. Results:Paracetamol and N-acetylcysteine could down-regulate the amount of Cytokines, inflammatory mediators and the activity of COX-2, inhibite NF-κB p65protein transfer from the cytoplasm to the nucleus. The IC50values of TNF-α, IL-1p, IL-6, PGE2and COX-2were0.359mM0.373mM,0.505mM,0.415mM and0.419mM respectively for paracetamol treatment,and these values were0.251mM,0.230mM,0.251mM,0.191mM and0.606mM respectively for N-acetylcysteine treatment. Combination use of NAC and AAP could enhance the inhibition actions of Cytokines and inflammatory mediators.There had no effect on up-regulate inhibition effect of NF-κB P65protein transfer from the cytoplasm to the nucleus.Piglets immunological stress model was also established in vivo to evaluate the combination effects of paracetamol and N-acetylcysteine. Methods:30healthy piglets (7.98±1.00kg) were randomly divided into①control group,②LPS group,③LPS+AAP group (mixed feed concentration of600mg/kg),④LPS+NAC group (mixed feeding concentration of1200mg/kg) and⑤LPS+AAP+NAC group (mixed feeding concentration of600mg/kg AAP+1200mg/kg of NAC). After pre-fed for10d, piglets were administrated with the drugs for5d, and then were stimulated by intraperitoneal injection at dose of100μg/kg.bw LPS. Blood samples were collected from piglets by anterior vena cava at3h and24h after LPS injection. The levels of cytokines, inflammatory mediators and the activity of COX-2in plasma and NF-κB activity in PBMC cytoplasmic and nuclear were measured by ELISA assay. Results:①Compared with the control group, the release of cytokines (TNF-a, IL-1β, IL-2, IL-6, IL-10) and inflammatory mediators PGE2in plasma of LPS group was significantly increased (P<0.01) at3h after LPS injection.NF-κB p65concentration ratio (The nucleus NF-κB p65divided by the cytoplasm NF-κB p65) was significantly increased (P<0.05), but COX-2activity in plasma showed no significant difference (P>0.05) at 3h after LPS injection. At24h after LPS injection, except the production of IL-10continued to rise, the levels of the other inflammatory cytokines and inflammatory mediator returned to normal levels.②Compared with LPS group, the production of IL-6in plasma of AAP group was significantly reduced (P<0.05) at3h after LPS injection, the production of IL-10significantly reduced (P<0.05) at24h after LPS injection.The release of inflammatory cytokine (TNF-α, IL-1β, IL-2, IL-6, IL-10) in plasma of NAC group was significantly lower at3h after LPS injection (P <0.05) and the release of PGE2was reduced,the production of IL-10was significantly decreased at24h after LPS injection (P<0.05); Paracetamol and acetylcysteine alone-treated could reduce NF-κB p65concentration ratio; The levels of cytokines (TNF-α, IL-1β, IL-2, IL-6, IL-10) and the levels of PGE2in plasma of AAP+NAC group were significantly lower (P <0.05) at3h after LPS injection. The level of IL-10were significantly lower (P<0.05) at24h after LPS injection, while the rest of the inflammatory cytokines were returned to normal levels; NF-κB p65concentration ratio had significantly reduced (P<0.05) when two drugs combinated-used, there is no significant differences in COX-2activity between control group and others groups in piglets plasma.In summary, acetylcysteine could enhance the anti-inflammatory effects of paracetamol.