Mechanism Of Chitosan Regulating Antioxidant Function And Anti-inflammatory Response Of Dairy Cows By NF-?B Signaling Pathway

During the lactation stage of dairy cows,due to high nutritional requirements and metabolic levels,it is usually accompanied by an increase in nitric oxide(NO)and other free radical metabolites,which exacerbates the inflammatory reaction of the dairy cows and causes a reduction in milk production and milk quality.Chitosan has biological activities such as anti-oxidation,anti-tumor and anti-inflammatory,which is widely used in food,pharmaceutical,pig and poultry farming and other fields.Limited data reported that chitosan can improve the antioxidant function and anti-inflammatory of dairy cows,but its mechanism is not yet clear.This paper investigated the regulation of dietary supplementation of chitosan on milk performance,antioxidant function and anti-inflammatory response of dairy cows.In vitro tests,NO and lipopolysaccharide(LPS)were used to induce oxidative damage to peripheral blood mononuclear cells(PBMC)of cows,and gene silencing technology was used to study the regulatory mechanism of chitosan on the antioxidant function and anti-inflammatory response of dairy cows.Study provided theoretical basis for improving milk production and milk quality.The study is divided into 7 trials:Trial 1 adopted a completely randomized trial design,and 40 high-yielding dairy cows(200±15 days in milk,34.80±4.95kg/d of milk yield,2 or 3 parity)were randomly assigned to five treaments with 8 replicates each:CON:control treatment without CHI addition;or C500,C1000,C1500 and C2000 group,with CHI addition of 500,1000,1500,2000 mg/kg DMI to the diet.Daily record of dairy cows' feed intake and milk production.Milk samples were collected at weeks 0,2,4,6,and 8 of the test period for milk quality analysis.Tail vein blood was collected on day 60 of the test period,and serum and PBMC were separated for analysis of blood antioxidant and immune functions.The results showed that the addition of chitosan to the diet of high-yielding cows increased the dry matter intake,milk production,energy correction milk,milk protein and lactose content of the cows,and reduced the somatic cell score;Dietary supplementation with chitosan had a dose-dependent effect on the antioxidant capacity and immune function,which significantly increased the activity of superoxide dismutase(SOD)and catalase(CAT)in serum and reduce the content of malondialdehyde;It significantly increased the proportion of CD4+ and CD8+ in lymphocytes,and the ratio of CD3+ and CD4+/CD8+both showed an upward trend.Dietary supplementation of chitosan reduced NO content in the serum by a dose-dependent manner,significantly reduced iNOS activity,and the content of interleukin-1?(IL-1?)showed a downward trend;It reduced the genes and protein expression of IL-1?,iNOS and P-NF-?Bp65 in PBMC.Based on the above indicators,the addition of chitosan to the diet promoted the milk performance,antioxidant capacity and immune function of high-yielding dairy cows.The addition of 1500mg/kg chitosan had a better improvement effect.The mechanism by which chitosan regulated the antioxidant capacity and inflammatory response of dairy cows may be related to its inhibition of NF-?B signaling pathway and reduced iNOS activity,thereby reducing excessive NO production.Tria12 used a single-factor randomized test design,which explored the effect of COS on PBMC's antioxidant activity and inflammatory factors content in vitro.The isolated PBMC of cows were randomly divided into 5 groups as follows:control group(CON)without COS in the cell culture medium;COS 40,COS 80,COS 160 and COS320 groups,with 40,80,160 or 320 ?g/mL of COS in the cell culture medium,respectively.The results showed that the addition of COS promote the antioxidant function of PBMC in dose depend.It could enhance the activity of CAT and SOD,and inhibit the content of IL-1?,IL-6,TNF-?,and NO.The addition of 160 ug/mLCOS showed a better effect.The addition of COS inhibited the gene expression of NF-?Bp50 and NF-?Bp65.These results implied that COS may reduce the gene expression of iNOS and inflammatory factors by inhibiting the activity of the NF-?B signaling pathway,and reduce the production of NO,thereby promoting the antioxidant capacity.Tria13 used diethylenetriamine/nitric oxide polymer(DETA/NO)to establish the PBMC in vitro oxidative damage model.The addition of DETA/NO to cell culture solution(0,50,100,200,300,and 500 ?mol/L)for 2,4,6,8,and 12 h respectively to screen the appropriate dosage and time testing cell viability and enzyme activity.The results showed that 200 ?mol/L of DETA/NO acting on PBMC for 4 h can be used as a suitable condition for establishing a model of PBMC oxidative damage.Excessive NO caused PBMC oxidative damage,and decreased the antioxidant capacity,and increased inflammatory factors IL-1? and NO content.Tria14 used a single factor completely random design,and the isolated PBMC were allocated to receive one of treatments:CTR:control treatment without COS addition,COS40,COS80,COS160 and COS320,which received 40,80,160,and 320?g/mL of COS,and then treated with 200 ?mol/L DETA/NO for 4 h.The results showed that COS had a dose-dependent effect on antioxidant function,and inhibited the release of inflammatory factors IL-1?,IL-6 and TNF-?,and the activity of the NF-?B pathway.Tria15 consists of two parts.The first trial was conducted to screen the damage time and dose of LPS.A single-factor completely randomized trial design was used.The LPS concentration included 5 treatments:0,0.1,1,10,and 20 ?g/mL,and the 0 ?g/mL group was used as the control group.The time of LPS acting on the cells including 6,12 and 24h.The results showed that 10?g/mL of LPS for 24 h could be used as a suitable condition for LPS-induced oxidative damage to PBMC.The second part of the trial assigned the PBMC into 6 treatment:control group,no COS and LPS treatment;LPS group,LPS treatment;CL40,CL80,CL160 and CL320 groups,which recived 40,80,160 and 320 ?g/mL COS treatment,then treated with LPS.The results showed that LPS could induce PBMC oxidative stress and increase the phosphorylation level of NF-kBp65,resulting in a decrease in antioxidant function and enhanced expression of proinflammatory factors.COS had a dose-dependent protective effect on the oxidative damage induced by LPS,which increased the activity of antioxidant enzymes GPx and SOD,reduced the proinflammatory factors such as IL-1?,IL-6,TNF-? and NO,and 160?g/mL COS had better effect.Tria16 was based on the trial 1.At the end of the feeding experiment(Experiment 1),PBMC was separated from the cows fed with different doses of chitosan(0,500,1000,1500,and 2000 mg/kg),and the test was divided into 6 treatment groups.The PBMC sample from cows without feeding chitosan is divided into 2 parts,one part is the control group without LPS treatment,and another part is the LPS injury group with the LPS treatment.At the same time,the PBMC isolated from the other chitosan feeding groups in experiment 1 were all suffered LPS injury and were set as CL500,CL1000,CL1500,and CL2000 groups respectively.The results showed that PBMC in the control group without feeding chitosan induced oxidative damage under the induction of LPS;PBMC of cows fed with chitosan had a mitigative effect on the oxidative damage induced by LPS in vitro,and showed a dose effect.It caused a decrease in the phosphorylation level of NF-?Bp65,iNOS enzyme activity and the expression of its genes and proteins,and the production of NO.The mechanism by which chitosan regulates the antioxidant capacity and anti-inflammatory response of dairy cows may be related to pathway of NF-?B-NO.Tria17 was divided into two parts.In the first part of test used siRNA to silence the NF-?Bp65 gene of PBMC,and selected the interference sequence with the best silencing effect.The results showed that siRNA successfully silenced the gene expression of NF-?Bp65 in cow PBMC with a silencing efficiency of 61%and the phosphorylation expression of NF-?Bp65 protein reduced by 38.5%.In the second part of test we used siRNA to silence the NF-?Bp65 gene to reveal the role of NF-?Bp65 gene in COS relieving oxidative stress of PBMC induced by LPS.The experiment was divided into 8 treatment groups:control group,LPS treatment group,siRNA group,COS group,siRNA+COS group,siRNA+LPS group,COS+LPS group,and siRNA+COS+LPS group.The results showed that siRNA silencing NF-?Bp65 reduced the oxidative damage caused by LPS,down-regulated the expression of iNOS genes and proteins,and promoted the antioxidant capacity of cells.Results indicated that NF-?B is a key pathway that causes oxidative damage to cells,and NF-?Bp6 5 is a key gene for COS to regulate the antioxidant capacity of dairy cows.COS reduced the gene and protein expression of iNOS by inhibiting the NF-?B pathway,and relieved the oxidative stress and inflammatory response induced by LPS.In summary,the addition of chitosan to the diet could promote the milk production performance,antioxidant capacity and anti-inflammatory response of dairy cows,of which 1500mg/kg chitosan had a better promotion effect This article illustrated the mechanism of the influence from the NF-?Bp65-NO pathway.That is,chitosan reduces the expression of iNOS genes and proteins by inhibiting the NF-?B pathway,reduces the excessive production of NO,inhibits the inflammatory response and promotes the antioxidant function of dairy cows.