The Combination Effects Of Acetaminophen And N-acetylcysteine On MAPK And NF-κB Signaling Pathways In Piglets Subjected To Immunological Stress

Oxidative stress and inflammation induced by immunological stress in pigletshave brought great harm to the pig industry. Studies have shown that the MAPKsignaling pathway plays a vital role in stress response and cell apoptosis, and theTLR4-NF-κB signaling pathway could induce cytokine expression and immunestress inflammation. Our project team has proved that acetaminophen (AAP)combined with acetylcysteine (NAC) has a positive effect on inflammation inpiglets subjected to immunological stress. This study will evaluate the combinationeffects of AAP and NAC on MAPK and NF-κB signaling pathway in pigletssubjected to immunological stress.1. The effect of acetaminophen alone and combined with N-acetylcysteine onMAPK signaling pathway in piglets subjected to immunological stress.In vitro test. Methods: piglets PBMCs were isolated and divided into fivegroups: control group, LPS-induced group, AAP group (0.5,1mM), NAC group(0.5,1mM) and combinated-treated group (AAP0.5-1mM and NAC0.5-1mM incombination).After incubation with drugs for1h, LPS (the final concentration was1μg/mL) were added and continued to incubate for0.5,3h.The concentration ofJNK, p-JNK, p-p38, and p-ERK in the0.5h LPS-stimulated monocytes wasdetermined by western blot. The mRNA expression of Bax、Fasl、Bcl-xl in the3hLPS-stimulated monocytes was determined by RT-PCR. The concentration ofcleaved casapse-3in the3h LPS-stimulated monocytes was determined by westernblot. Results：①0.5h after LPS stimulation, the concentration of JNK increasedinsignificant, while the concentration of p-JNK, p-p38, p-ERK increasedsignificantly (P<0.01). NAC (0.5and1mM) could not significantly (P>0.05)enhance the AAP (0.25-1mM) inhibitory effect on the secretion of JNK. NAC (0.5and1mM) could significantly (P<0.01) enhance the AAP (0.25-1mM) inhibitoryeffect on the secretion of p-p38and p-ERK. NAC (0.5mM) could significantly(P<0.01) enhance the inhibitory effect on the AAP (0.5mM) secretion of p-JNK.NAC (1mM) could not significantly (P>0.05) enhance the AAP (0.25-1mM)inhibitory effect on the secretion of p-JNK.②3h after the LPS stimulation, theexpression of Bax, Fasl, and Bcl-xl were significantly increased (P<0.01). NAC(0.5mM) could significantly (P<0.01) enhance the AAP (0.5mM) inhibitory effecton the secretion of Bax mRNA. NAC (1mM) could significantly (P<0.01) enhancethe AAP (1mM) inhibitory effect on the secretion of Bax mRNA. NAC (0.5and1mM) could significantly (P<0.01) enhance the AAP (0.5-1mM) inhibitory effect onthe secretion of Bcl-xL mRNA. NAC (0.5and1mM) could significantly (P<0.05)enhance the AAP (0.5-1mM) inhibitory effect on the secretion of Fasl mRNA.③3h after the LPS stimulation, cleaved casapse-3was significantly increased (P<0.01).NAC (0.5mM) could significantly (P<0.01) enhance the AAP (0.5mM) inhibitoryeffect on the secretion of cleaved casapse-3. NAC (1mM) could not significantly(P>0.05) enhance the AAP (1mM) inhibitory effect on the secretion of cleavedcasapse-3.In vivo test. Methods:30healthy piglets (7.981.00kg) were randomlydivided into five groups：control group, LPS group, LPS+AAP (600mg/kg) group,LPS+NAC (1200mg/kg) group, and LPS+AAP plus NAC group (AAP were600mg/kg and NAC were1200mg/kg in combination). After pre-fed for10days,the piglets in treated groups were stimulated by intraperitoneal injection of LPS at100μg/kg.bw and piglets in the control group received intraperitonealadministration of the same volume of sterile saline. Blood samples were collectedfrom the anterior vena cava3h after the LPS injection. The mRNA expression ofBax, Bcl-xL, and Fasl in peripheral blood monocytes3h after the LPS injection wasdetermined by RT-PCR. Results: the mRNA expression of Bax, Bcl-xL, and Fasl inthe monocytes of LPS group was significantly increased (P<0.01). NAC couldsignificantly (P<0.05) enhance the AAP inhibitory effects on the expression of Bax,Bcl-xL, and Fasl mRNA.2. The effect of AAP alone and combined with NAC on NF-κB signalingpathway in LPS-challenged piglets.In vitro test. Methods：piglets PBMCs were isolated and divided into fivegroups: control group, LPS-induced group, AAP group (0.5and1mM), NAC group(0.5and1mM) and combinated-treated group (AAP were0.5-1mM and NAC were0.5-1mM). After incubation with drugs for1h, LPS (the final concentration was1μg/mL) were added and incubated for3h and20h. The concentration of TNF-α inthe cell culture supernatant3h after the LPS-stimulation and the concentration ofIL-6and IL-10in the cell culture supernatant20h after the LPS-stimulation weredetermined by ELISA. The mRNA expression of IL-6, IL-10, and TNF-α in the3hLPS-stimulated monocytes was determined by RT-PCR. Results：3h after the LPSstimulation, the concentration of TNF-α in the cell culture medium wassignificantly increased (P<0.01). NAC (0.5mM) could significantly (P<0.01)enhance the AAP (0.5-1mM) inhibitory effect on TNF-α.20h after the LPSstimulation, the concentration of IL-6and IL-10in the cell culture medium wassignificantly increased (P<0.01). NAC (0.5mM) could significantly (P<0.01)enhance the AAP (0.5-1mM) inhibitory effect on IL-6and IL-10. The mRNAexpression of TNF-α, IL-6, and IL-10in the monocytes was significantly increased(P<0.01)3h after the LPS stimulation. NAC (0.5and1mM) could significantly(P<0.01) enhance the AAP (0.5-1mM) inhibitory effects on the mRNA expression of TNF-α, IL-6, and IL-10.In vivo test. Method: Blood samples were collected from the piglets byanterior vena cava3h after the LPS injection. The mRNA expressions of IL-6,IL-10, and TNF-α in the peripheral blood monocytes3h after the LPS-stimulationwere determined by RT-PCR. Results: the mRNA expression of IL-6, IL-10, andTNF-α in the monocytes of LPS group was significantly increased (P<0.01). NACcould significantly (P<0.01) enhance the AAP inhibitory effects on the mRNAexpression of TNF-α, IL-6, and IL-10.The results showed that the combination use of AAP and NAC could inhibit theMAPK signaling pathway and NF-κB signaling pathway in LPS-induced piglets.NAC could enhance the AAP inhibitory effects.