Effect And Mechanism Of Butyric Acid On Relieving Oxidative Stress In High-Yielding Cows

Redox reaction in the organism is one of the important reactions to promote the normal and orderly life activities.When the balance of redox reaction is broken and the oxidation reaction in the organism is far more than the reduction reaction,the organism will be in oxidative stress.Compared with the normal lactating cows,the milk production and metabolism rate of the high-yield cows increased significantly.The high metabolism rate led to the greatly enhanced aerobic respiration of the Bovine mammary epithelial cells(BMECs),so it will produce too much free radicals to make the mammary gland in the state of oxidative stress.Oxidative stress can not only damage the mammary gland of dairy cows,but also damage the blood milk barrier,and cause bacteria to invade the mammary gland.Serious oxidative stress can also damage the lactation life of dairy cows,even lead to other lactation diseases such as mastitis,and cause huge economic losses to dairy farms.Therefore,it is of great significance to effectively control the oxidative stress of mammary gland in high-yield dairy cows Butyric acid is a kind of short chain fatty acids,which has been studied to have antioxidant function,but its mechanism is not clear,and its antioxidant effect in mammary gland has not been reported.In addition,some studies have shown that butyric acid,through its receptor GPR109A,plays many roles such as anti-inflammatory and repairing intestinal barrier.Therefore,we speculated that butyric acid may activate antioxidant pathways through GPR109A,so as to alleviate oxidative stress in high-yield dairy cows.Therefore,in this study,the effects of butyric acid on oxidative stress in high-yield dairy cows and their BMECs were studiedFirst,in order to analyze the correlation between GPR109A and oxidative stress in high-yield dairy cows,we screened high-yield dairy cows(n=10,45±5 kg/d)and normal yield dairy cows(n=10,25±5 kg/d),collected serum and breast tissue,and detected the expression of GPR109A in serum and mammary gland.The results showed that the levels of MDA,H2O2,CAT,FRAP,GSH,SOD and GPR109A in high-yield dairy cows were significantly higher than those of normal dairy cows.These results indicate that high-yield dairy cows are in a state of high oxidative stress,and the significant up regulation of GPR109A may indicate that GPR109A plays a regulatory role in oxidative stressSecondly,in order to further study whether butyric acid can alleviate oxidative stress through GPR109A,we used H2O2-induced oxidative stress response model of BMECs.In this model,we studied the role and mechanism of butyric acid in antioxidant stress through GPR109A.The results showed that sodium butyrate(NaB)could significantly promote the expression of FRAP,GSH and SOD,and inhibit the expression of MDA and ROS,but this effect was inhibited by adding GPR109A-shRNA,AA or RA.Further results showed that NaB promoted Nrf2 nuclear accumulation and H3K9/14 acetylation through GPR109A/AMPK Signaling pathway in BMECs,and the interaction between Nrf2 and H3K9/14 acetylation could promote the expression of antioxidant molecules.Oxidative stress often leads to cell apoptosis In order to further clarify the antioxidant stress effect of butyric acid,we tested the effect of butyric acid on the apoptosis in BMECs.The results showed that NaB or TSA could significantly reduce the apoptosis induced by H2O2,while GPR109A-shRNA or RA could inhibit this effect.These results showed that butyric acid promotes Nrf2 nuclear accumulation and H3K9/14 acetylation in BMECs through GPR109A/AMPK,and promotes the interaction between Nrf2 and H3K9/14 acetylation,thus promoting the synthesis of antioxidant molecules and plays an antioxidant roleThirdly,we sequenced the mRNA of the two groups by high-throughput sequencing.We found 639 up-regulated mRNA and 91 down-regulated mRNA,41 of which were related to Nrf2 and oxidative stress.Through the analysis of GO and KEGG,we found that the difference gene regulated by butyric acid is mainly enriched in cAMP signaling pathway,which is closely related to G protein coupled receptor.The results of interaction network analysis showed that these proteins regulate various functions of BMECs in the form of interaction network,including antioxidant functionFinally,in order to further study the mechanism of butyric acid in antioxidant stress of dairy cows.We collected the serum,whey and peripheral lymphocytes of two groups of dairy cows(one group was fed with tributyrin)on day 0,7,14 and 21.The results showed that tributyrin could significantly increase the content of FRAP,GSH,CAT and SOD,and inhibit the content of H2O2 and MDA.Western blot and qRT-PCR results showed that the expression of GCLC,GCLM,NQO1,Nrf2 and HO-1 in lymphocytes of tributyrin group was significantly up-regulated.The results showed that tributyrin could also relieve oxidative stress in dairy cowsThrough the above study,the mechanism of butyric acid in anti-oxidative stress was systematically elucidated for the first time,and the important biological functions of butyric acid in BMECs of dairy cows were elucidated by high-throughput sequencing This study provides a new target for anti-oxidant stress in dairy cows,and provides a theoretical basis for butyric acid as an antioxidant.