Alleviating Of Vitamin A On The Decline Of Milk Protein Synthesisin By NO-Induced Damage In Bmecs Through Inhibiting NF-κB Pathway

Bovine mammary epithelial cells(BMECs)are the main sites of milk protein synthesis.Oxidative stress is one of the main reasons for reducing milk protein synthesis and affecting milk yield and milk quality of dairy cows.This thesis aims to explore the mitigating effect and mechanism of vitamin A(VA)throughc inhibiting the activity of NF-κB pathway of nitric oxide(NO)injuries caused by falling within BMECs Milk Protein Synthesisin relief research It provides theoretical basis for reducing oxidative stress of mammary gland,improving mammary gland health,increasing milk yield and improving milk quality of dairy cows.This thesis is divided into two experiments:The first experiment was designed with a single factor completely randomized trial.NO is used to establish the BMECs oxidative stress model,to research the alleviated effect of different concentrations VA on the reduction of milk protein synthesis in BMECs induced by oxidative stress.The experiment was divided into 9 groups,which were CON group,NO damage group and NO+VA pre-protection group.The VA concentrations were0.05(NA0.05),0.1(NA0.1),0.2(NA0.2),0.5(NA0.5),1 μg/m L(NA1),2 μg/m L(NA2)and 4μg/m L(NA4).The results showed that NO-induced oxidative damage significantly decreased the BMECs proliferation,the activity of glutathione peroxidase(GPx),catalase(CAT),thioredoxin reductase(Trx R),the total oxidation capacity(T-AOC),expression ofαs1-casein(CSN1S1),β-casein(CSN2),κ-casein(CSN3),GPx1,Trx R1,m TOR,S6K1,eukaryotic initiation factor 4E(e IF4E),eukaryotic initiation factor 4E-binding protein 1(4EBP1),and signal transduction and transcriptional activator 5(STAT5)(P<0.05),and significantly increased levels of reactive oxygen species(ROS)nitric oxide(NO)and the activity of inducible nitric oxide synthase(P<0.05).Compared with NO group,the activities of Trx R,T-AOC,CAT and GPX in NA0.2 to NA4 groups were significantly up-regulated(P<0.05);The expression of CSN1S1 gene was significantly up-regulated in NA0.2-NA1 group(P<0.05);NA1 group significantly increased S6K1 enzyme activity and GPx1,CSN2 and JAK2 gene expression(P<0.05),significantly decreased the contents of NO and ROS(P<0.05);NA4 significantly up-regulated the expression of m TOR,S6K1,4EBP1 and e IF4 E genes(P<0.05)and down-regulated the activity of i NOS(P<0.05);NA2 and NA4 groups significantly up-regulated m TOR content,Trx R and STAT5 gene expression(P<0.05).The results showed that the cell proliferation,antioxidant function,milk protein synthesis-related enzyme activities and gene expression of BMECs were decreased after cells injured by NO.especially 1.00μg/m LVA could alleviate the decrease of milk protein synthesis in BMECs induced by NO,and the effect of VA is in a quadratic dose-dependent response effect with VA supplemental dose,0.20-2.00 μg/m L group is better,the optimum concentration was 1.00μg/m L.The experiment 2 was divided into two parts.In the first part,it was investigated effect of different concentrations of NF-κB inhibitor(PDTC)on cells proliferation and NF-κB expression.In the first part,the experiment consisted of 6 treatment groups,which were respectively control group(CON,without PDTC)and five experiment groups with different PDTC concentrations: 200,400,600,800 and 1000μmol/L(P200,P400,P600,P800 and P1000),6 replicates per group,cell proliferation and NF-κBp65 gene expression were determined after 6h treatment.The results showed that compared with CON group,the cell proliferation and gene expression of NF-κBp65 in P400 and P600 groups were significantly decreased(P<0.001),the inhibition efficiency reached25%-30%.So 400μmol/L PDTC dose was selected for subsequent tests.The second part,NF-κB pathway was inhibited by PDTC to study whether VA alleviates the reduction of milk protein synthesis caused by oxidative damage through inhibiting NF-κB pathway.The experiment was divided into 6 treatment groups,which were CON group,NO damage group(NO group),PDTC group,VA pre-protection group(VA+NO group),NO+PDTC group and VA+NO+PDTC group with 4 replicates in each group.The dose of VA was 1μg/m L.The results showed that compared with CON group,the activities of antioxidant enzymes Trx R and GPx and gene expression were significantly decreased(P<0.05),significantly increased the contents of NO,ROS,enzyme activity of i NOS,gene expression of NF-κBp65,IL-1,i NOS and protein expression of NF-κBp65 in NO group(P<0.05).and significantly decreased casein content and gene expression of JAK2,STAT5 and PI3 K,m TOR,e IF4 E,4EBP1(P<0.05).Compared with CON group,PDTC group GPx activity and Trx R gene expression were significantly increased(P<0.05),the gene expressions of CSN1S1,CSN3,PI3 K and4EBP1 were significantly increased and the phosphorylation levels of m TOR and e IF4 E,and protein expression of 4EBP1 were significantly increased(P<0.05),while the gene expressions of NF-κBp65 and IL-1 were significantly decreased and the protein expression of NF-κBp65 were significantly decreased(P<0.05).Compared with NO group,the addition of VA preprotection significantly alleviated the increase of inflammatory factors,the decrease of antioxidant activity and gene expression of casein synthesis,and genes related to m TOR pathway and JAK2/STAT5 signaling pathway induced by NO injury.Compared with VA+NO group,the content of CSN1S1,gene expressions of NF-κBp65,GPx1,CSN3,S6K1,JAK2 and STAT5 in VA+NO+PDTC group were significantly decreased(P<0.05).The contents of NO,ROS,i NOS,Trx R enzyme activities,and gene expressions of Trx R,CSN2,m TOR and 4EBP1 were significantly increased(P<0.05).These results indicate that VA promotes the activation of m TOR pathway related factors and JAK-STAT pathway by inhibiting NF-κB,thus alleviating the reduction of milk protein synthesis caused by oxidative damage.In conclusion,the VA by inhibiting the expression of the NF-κB,promote GPx activity and reduce the content of NO and ROS,the enzyme activity of i NOS,relieve oxidative stress caused by the NO,Furthermore,up-regulate the expression of JAK2/STAT5 and m TOR pathway S6K1,e IF4 E and promote the synthesis of casein gene.This in turn mitigated the decline in casein synthesis in NO-induced damage BMECs.