Mechanism Of Vitamin A Regulating Milk Fat And Protein Synthesis In Bovine Mammary Epithelial Cells Induced By H₂O₂

Bovine mammary epithelial cells（BMECs）are the main sites for the synthesis and secretion of milk fat and milk protein.The occurrence of oxidative stress will reduce the synthesis of milk fat and milk protein,and affect the yield and quality of milk.This thesis aims to explore the mitigating effect and mechnism of vitamin A（VA）on the reduction of milk fat and milk protein synthesis in hydrogen peroxide（H₂O₂）-damaged BMECs;and these results are to slow down the oxidative stress of the mammary glands of dairy cows,improve mammary glands health and provide a theoretical basis for improving the quality of raw milk.The thesis is divided into 3 experiments.The first experiment was designed with a single factor completely randomized trial,with H₂O₂ as the oxidative stress source,to investigate the inhibitory effects of VA on the reduction of milk fat and milk protein synthesis in BMECs caused by oxidative stress.The experiment was divided into nine treatment groups,including CON group,H₂O₂damage group and seven pre-protection groups with different concentrations of VA（VA concentration is 0.05（0.05VAH group）,0.1（0.1VAH group）,0.2（0.2VAH group）,0.5（0.5VAH group）,1（1VAH group）,2（2VAH group）and 4（4VAH group）μg/m L）.The results showed that H₂O₂-induced oxidative damage significantly reduced BMECs viability,glutathione peroxidase（GPx）,superoxide dismutase（SOD）,catalase（CAT）,thioredoxin reductase（Trx R）and total antioxidant capacity（T-AOC）,significantly reduced gene expression of GPx and Trx R1（P<0.05）,and significantly increased levels of reactive oxygen species（ROS）（P<0.05）.The H₂O₂-injured group significantly reduced triglyceride（TG）content and the activities of acetyl coenzyme A carboxylase（ACACA）,stearoyl coenzyme A desaturase（SCD）,lipoprotein esterase（LPL）（P<0.05）,peroxisome proliferator-activated receptor gamma（PPARγ）,sterol regulatory element binding protein1（SREBP1）,ACACA and fatty acid synthetase（FASN）gene expression（P<0.05）.The H₂O₂-injured group significantly reduced the enzyme content ofαs1-casein（CSN1S1）,β-casein（CSN2）,target of rapamycin（m TOR）and decreased the gene expression of CSN1S1,m TOR,e IF4E,JAK2,STAT5（P<0.05）.VA pre-protection from 0.2 to 4μg/m L can reverse those changes of the above indicators caused by H₂O₂,up-regulate the gene expression of transcription factors PPARγand SREBP1,promote gene expression and enzyme activity related to FA de novo synthesis,uptake and transport,and up-regulate m TOR and expression of genes related to the JAK2/STAT5 pathway slows down the reduction of milk fat and milk protein synthesis caused by oxidative stress.And the cell viability,enzyme activity and gene expression of GPx and gene expression of FASN,CSN3 and STAT5 were significantly higher in the VA pre-protection group at 1μg/m L than in the other VA pre-protection and H₂O₂ damage group（P<0.05）,and the activities of T-AOC,SOD,CAT,FASN,S6K1 and the contents of TG,CSN2 and the gene expression of Trx R1,SCD,LPL,CSN1S1,CSN2 were numerically higher than those of the other VA pre-protection and H₂O₂ damage group.Based on the above indicators,it is concluded that 1μg/m L VA has the best alleviating effect.The experiment 2 was divided into two parts,both adopt a single factor completely randomized trial.In the first part,by adding different concentrations of PPARγinhibitor（GW9662）,using cell viability as an indicator to determine the appropriate concentration of GW9662 added to BMECs is 20μmol/L.The second part uses GW9662 to inhibit the activity of PPARγto study whether VA can improve the activity of PPARγto alleviate the reduction of milk fat and milk protein synthesis caused by oxidative damage.The experiment was divided into seven treatment groups,CON group,H₂O₂ damage group,VA group,VA+H₂O₂ group,GW group（PPARγinhibition group,concentration determined according to the first part）,VA+GW group,VA+H₂O₂+GW group.The dose of VA is 1μg/m L.The results showed that compared with the CON group,the antioxidant enzymes GPx,Trx R,SOD,T-AOC and CAT activity and the gene and protein expression of GPx and Trx R1 in the GW group were significantly decreased（P<0.05）,and TG and milk protein content,the expression of milk fat synthesis related genes FASN,ACACA,LPL,SCD,FABP3,SREBP1,the expression of milk protein synthesis related genes CSN1S1,CSN2,CSN3,m TOR,4EBP1,STAT5（P<0.05）,protein expression of PPARγ,SREBP1 and phosphorylation levels of m TOR signaling pathway factors were significantly reduced（P<0.05）.Compared with the VA+H₂O₂ group,the above indicators of the VA+H₂O₂+GW group were significantly reduced,and the content of ROS was significantly increased（P<0.05）;indicating that VA promotes the de novo synthesis,uptake and transport of fatty acids by activating PPARγ,and promotes m TOR and JAK-Activation of the STAT pathway,thereby alleviating the reduction of milk fat and milk protein synthesis caused by oxidative damage.Using the si RNA silencing Trx R1 technique,to study whether Trx R1 is the key gene for VA to alleviate the oxidative damage of BMECs and the reduction of milk fat and milk protein synthesis caused by oxidative damage.The trial 3 was divided into seven treatment groups:CON group,H₂O₂ damage group,VA group（concentration was determined according to experiment 1）,VA+H₂O₂ group,si Trx R group,si Trx R+VA group and si Trx R+VA+H₂O₂ group.To investigate whether Trx R1 is a key gene for VA to mitigate oxidative damage in BMEC and the reduction in milk fat and milk protein synthesis caused by oxidative damage.The results showed that compared with the CON groups,the antioxidant enzymes GPx,Trx R,SOD,T-AOC,CAT activity and the gene and protein expression of GPx and Trx R1 in the si Trx R group were significantly decreased（P<0.05）;TG and milk protein content,PPARγand SREBP1 gene and protein expression were significantly reduced（P<0.05）,and the expression of the milk fat synthesis-related gene FASN,ACACA,LPL,FABP3,SREBP1 and the expression of the milk protein synthesis-related gene CSN1S1,CSN2,CSN3,m TOR,S6K1,e IF4E,4EBP1,JAK2,STAT5 were significantly reduced（P<0.05）.The phosphorylation levels of the milk protein synthesis signaling pathway factors m TOR,e IF4E and 4EBP1 were also significantly reduced（P<0.05）.Compared with the VA+H₂O₂ group,si Trx R+VA+H₂O₂group with the Trx R1 gene silenced significantly reduced the cell activity to resist oxidative stress（P<0.05）,inhibited TG and milk protein content,and PPARγgene and protein expression,and reduced de novo synthesis and uptake and transport of fatty acid,and inhibited activation of m TOR and JAK-STAT pathways.It shows that silencing the Trx R1 gene weakens the protection of VA on H₂O₂-induced oxidative damage of BMECs and the alleviation of the reduction of milk fat and milk protein synthesis caused by oxidative damage.In summary,this study explored the inhibition of VA on the reduction of BMECs milk fat and milk protein synthesis caused by oxidative stress and its mechanism from the Trx R/PPARγpathway,that is,VA activates PPARγactiivity by promoting Trx R activity,promoting the de novo fatty acid synthesis,uptake and transport,promoting the activation of m TOR signaling pathway and JAK2/STAT5 pathway,thereby alleviating the decrease in the synthesis of milk fat and milk protein in BMECs caused by oxidative stress.