| The high-intensity metabolism of lactating cows and the enhanced fat mobilization caused by the negative energy balance in prelactation can lead to excessive accumulation of free radicals in the body and oxidative stress,which in turn cause a decrease in milk production and milk quality.Chitosan(COS),as a natural antioxidant,has a catalytic role in antioxidant and anti-inflammatory effects in animals.Peripheral blood mononuclear cells(PBMC)are an important class of immune effector cells whose resistance to oxidative stress is closely related to the antioxidant and immune function of dairy cows.Therefore,an in-depth study on the mechanism of chitosan(COS)to alleviate oxidative stress in PBMC can provide a theoretical basis to alleviate oxidative stress through nutritional regulation and improve milk production performance of dairy cows.This thesis focuses on the mechanism of action of COS through NF-κB-IL-NO pathway to alleviate oxidative stress in PBMC.The thesis is divided into 3 parts.Trial 1 investigated the mitigating effect of IL-1Ra on LPS-injured PBMC using interleukin 1 receptor antagonist(IL-1Ra)to inhibit interleukin 1β(IL-1β)activity,using cell viability,antioxidant indexes and inflammatory factors as judgment indicators,using LPS as the stimulation source.A one-way completely randomized experimental design was used to randomize PBMC into seven treatment groups with six replicates each,the first group was the control group,incubated for 30 h without the addition of LPS and IL-1Ra;the second group was the LPS-damaged group,which was incubated for 6 h using medium without LPS and IL-1Ra working solution and continued for 24 h with the addition of 10 ug/m L LPS working solution;the third-seventh group was the IL-1Ra protection group with different doses(0.25,0.5,1,5,and 10 ng/m L)treated for 6h and continued incubation for 24 h with the addition of LPS working solution,in the order of LRa0.25,LRa0.5,LRa1,LRa5,and LRa10 groups.The results showed that cell viability,glutathione peroxidase(GPx),thioredoxin reductase(Trx R),total superoxide dismutase(T-SOD)and catalase(CAT)activities and total antioxidant capacity(T-AOC)were significantly lower in the LPS group compared to the CON group,malondialdehyde(MDA),IL-1β,interleukin 6(IL-6)content,nitric oxide(NO)concentration and inducible nitric oxide synthase(i NOS)activity were significantly increased.Compared with the LPS group,the LRa1 group significantly reversed the above-mentioned decrease in antioxidant activity and increase in inflammatory factor concentration caused by LPS,and the other LRa groups were less effective than the LRa1 group in reversing the above indices to varying degrees,it indicates that LPS induces oxidative damage in PBMC by producing large amounts of IL-1β,and the mitigating effect of IL-1Ra on LPS-induced oxidative damage is dose-dependent,with the best mitigation effect at an additive amount of 1 ng/m L.Trial 2 explored the mechanism of action of COS in protecting PBMC from oxidative stress damage from the IL-NO pathway using LPS as the stimulation source and IL-1Ra to inhibit the activity of IL-1β.A one-way completely randomized trial design was used to randomize PBMC into 8 treatments: control group,LPS injury group,COS group,IL-1ra group,LPS+IL-1ra group,COS+IL-1ra group,LPS+COS group and LPS+COS+IL-1ra group,with 6 replicates in each treatment group.The results showed that LPS-induced oxidative damage significantly decreased the activities of GPx,T-SOD,CAT and Trx R and T-AOC,and down-regulated the gene and protein expression of GPx and Trx R;increased the levels of MDA,NO and reactive oxygen species(ROS)and the gene and protein expression of NF-κBp65 compared with the control group,and enhanced the expression of their downstream inflammatory factors IL-1β,IL-6 and tumor necrosis factor(TNF-α)concentrations and their gene expression,and upregulated the protein expression of i NOS and IL-1β;these results suggest that LPS induced oxidative damage in PBMC and promoted the activation of NF-κB pathway with the release of inflammatory factor IL-1β,increased i NOS activity and NO content.Compared with the LPS group,both the LPS+IL-1ra group and the LPS+COS group reversed the changes in the above indices,indicating that LPS induced oxidative stress in cells by stimulating the excessive release of IL-1β,and COS slowed down the LPS-induced oxidative stress damage in cells by inhibiting the activity of IL-1β.Trial 3 explored the mechanism of COS to mitigate oxidative stress injury in PBMC from the NF-κB-IL-NO pathway using LPS as the stimulation source and pyrrolidine dithiocarbamate(PDTC)to inhibit NF-κB activity using a one-way completely randomized experimental design.The trial was randomly divided into 8 treatment groups,namely control,COS,LPS,PDTC,COS+PDTC,COS+LPS,LPS+PDTC,and COS+LPS+PDTC groups,with 6 replicates per treatment group.The results showed that the LPS group significantly decreased the activities of antioxidant enzymes GPx,T-SOD,CAT and Trx R and T-AOC and down-regulated the gene and protein expression of GPx and Trx R compared to the control group;increased MDA,ROS and NO content,significantly higher concentrations of i NOS activity,inflammatory factors IL-1β,IL-6and TNF-α and their gene expression,and upregulated gene and protein expression of NF-κBp65;these results suggest that LPS-induced oxidative damage in PBMC causes activation of NF-κB pathway and release of inflammatory factor IL-1β,i NOS activity and increase in NO content.The activity of the above antioxidant enzymes and their gene expression were significantly higher in the cells of the LPS+ PDTC and LPS+ COS groups compared to the LPS group,while the activity of the above inflammatory factors and their gene and protein expression were significantly lower in the cells of the LPS+PDTC and LPS+ COS groups along with the gene and protein expression of NF-κBp65,it is suggested that LPS promotes the release of IL-1β through activation of NF-κB signaling pathway and causes oxidative stress in cells,while COS reduces the release of IL-1β and thus NO concentration by inhibiting the activity of NF-κB signaling pathway,thus alleviating the oxidative damage in cells.In summary,this thesis interprets the mechanism by which COS alleviates cellular oxidative stress from the NF-κB-IL-NO pathway,i.e.,COS reduces IL-1β activity by inhibiting NF-κB signaling pathway activity and downregulates gene and protein expression of i NOS,thereby reducing NO overproduction and alleviating oxidative stress induced by LPS. |
