Effects And Mechanism Of Rutin On Relieving Oxidative Stress In Mammary Gland Of Ruminants During The Transition Period

Oxidative stress leads to the injury in tissues through the oxidation of DNA,proteins,and lipids in cells.Oxidative stress is prevalent in the transition period in ruminants.Oxidative stress is closely related to the occurrence and development of various diseases during the transition period in ruminants.As the lactation organ of mammals,the health status of mammary gland directly affects the production performance and quality of milk.The metabolism of mammary gland is vigorous in the transition period,which is susceptible to oxidative stress.Thus,it is far-reaching significance for health of mammary gland to alleviate oxidative stress during the transition period in ruminants.Rutin,a flavonoid,which is widely distributed in plants and possess high antioxidant activity.According to the research of ruminats,rutin can improve the production performance and the energy state during the transition period.However,the protective effect and potential mechanism of rutin on mammary gland are still unclear.In the present study,we explore the effect and protective mechanism of rutin on oxidative stress of mammary gland in ruminants using H₂O₂-simulated bovine mammary gland cells（BMEC）in vitro and the transition Hu sheep in vivo,respectively.The main results of the present study are as follows:（1）The effect of rutin on H₂O₂-induced BMEC.This chapter aimed to study the effect of rutin on H₂O₂-induced the indicators of oxidative stress,inflammation,apoptosis and endoplasmic reticulum stress.After the treatment of H₂O₂,the cell viability of BMEC was decreased significantly in a dose-dependent manner.The cell viability was decreased by30%in 600μmol/L H₂O₂treatment.Thus,the concentration of 600μmol/L H₂O₂was used for the following study.After the BMEC was treated by 600μmol/L H₂O₂,the levels of ROS and MDA were increased significantly;the activity of CAT,GSH-Px and T-AOC were decreased significantly;the protein expression of p-IκBα,p-p65,Caspase3,Caspase9,Bax,GRP78 and CHOP were increased significantly;the protein expression of the Bcl-2 was decreased significantly;the relative m RNA expression of TNF-α,IL-1β,IL-6,GRP78 and ATF4 were increased significantly;the apoptosis rate was increased significantly.The BMEC treated with different concentrations of rutin had no significant change in cell viability,but it was generally higher at 12 h after treatment.Therefore,the pretreatment time of rutin was selected as 12 h.After pretreatment of rutin and 600μmol/L H₂O₂,compared to the cells treated with 600μmol/L H₂O₂,the cell viability of BMEC was increased significantly,and the level of ROS was decreased significantly,the levels of CAT,GSH-Px and T-AOC were increased significantly;the protein expression of p-IκBα,p-p65,Caspase3,Caspase9,Bax,GRP78 and CHOP were decreased significantly;the protein expression of the Bcl-2 was increased significantly;the relative m RNA expression of TNF-α,IL-1β,IL-6,GRP78 and ATF4 were decreased significantly;the apoptosis rate was decreased significantly.The most significant changes were observed in the group treated with the 50μmol/L rutin.These results indicated that rutin could relieve oxidative damage caused by H₂O₂.（2）The regulatory mechanism of AMPK/NF2L2 signaling pathway in the process of that rutin relieve oxidative damage caused by H₂O₂.After BMEC were treated with 600μmol/L H₂O₂,the protein expression of p-AMPK and the nuclear protein expression of NFE2L2were decreased significantly;the relative m RNA expression of AMPK,NFE2L2,NQO1 and HMOX1 were decreased significantly.After the BMEC was pretreated with rutin,the protein expression level of p-AMPK,the nuclear protein expression of NFE2L2,and the relative m RNA expression of AMPK,NFE2L2,NQO1 and HMOX1 were increased.The marked significant changes were observed in the group treated with the 50μmol/L rutin.After NFE2L2 was knockdown with si RNA,50μmol/L rutin failed to reduce the increase of ROS,apoptosis rate and the relative m RNA expression of TNF-α,IL-1β,IL-6,GRP78and ATF4 in BMEC induced by 600μmol/L H₂O₂.It was found that rutin activated NFE2L2 through AMPK by using AMPK inhibitor.These results indicated that the activation of NFE2L2 was a necessary condition for rutin to alleviate oxidative damage in BMEC.Rutin exert antioxidative effects by activating AMPK/NFE2L2 signaling pathway.（3）The diet was supplemented with rutin to explore the effect of rutin on blood indexes in transition Hu sheep.Thirty transition Hu sheep（-28 d relative to parturition）with similar weight and delivery date were randomly divided into 3 groups.Each group had 10 sheep.Sheep were fed with concentrate plus 0（control）,50 or 100 mg/kg BW rutin and roughage from day-28 to 28 relative to parturition.The experimental period including 7 d of adaptation period and 56 days of formal period.Blood was collected on-14 d and-7 d,+1 d,+2 d,+7 d,+14 d,+21 d and+28 d relative to parturition before morning feeding for detection and analysis.The results are as follows:compared to the control group,the supplementation of 50 mg/kg BW rutin reduced the levels of serum IL-1β,IL-6,TNF-α,H₂O₂and MDA,and increased the levels of GLB,GSH-Px,CAT,SOD and T-AOC.Compared to the control group,the supplementation of 100 mg/kg BW rutin decreased the levels of serum BHBA,IL-1β,IL-6,TNF-α,H₂O₂and MDA,and increased the levels of NEFA,GLB,GSH Px,CAT,SOD and T-AOC.（4）In order to explore the effect of rutin on the mammary gland health of Hu sheep during the transition period,single-factor and multi-level experiment was carried out.The diet was supplemented with 0（the control group）,50,100 mg/kg BW rutin to sheep during the transition period.The feeding of this test was the same as（3）.After blood collection at+28 d,the mamary gland tissue was collected for detection and analysis.The results are as follows:compared to the control group,the supplementation of 50 mg/kg BW rutin significantly reduced the inflammatory cells in the mammary gland of Hu sheep.Inflammatory cells were not observed and more papillae formed by cell bulges were observed in the mammary gland of Hu sheep in the 100 mg/kg rutin group.The supplementation of 50 mg/kg BW rutin significantly reduced the level of H₂O₂,the protein expression of Bax and GRP78,and,the relative m RNA expression of TNF-α.While,the supplementation of 50 mg/kg BW rutin significantly increased the activity of CAT,the protein expression of Bcl-2,phosphorylation level of AMPKαand the relative m RNA expression of AMPK and HMOX1 in mammary gland tissue.The supplementation of 100mg/kg BW rutin significantly reduced the level of H₂O₂,the protein expression of p-IκBα,p-p65,Bax,Caspase3,Caspase9,GRP78 and CHOP,the relative m RNA expression of TNF-α,GRP78 and CHOP.While,100 mg/kg BW rutin significantly increased the activities of CAT,GSH-Px and T-AOC,protein expression of Bcl-2,phosphorylation level of AMPKαand relative m RNA expression of AMPK,NFE2L2,HMOX1 in mammary gland tissue.In addition,the supplementation of 100 mg/kg BW rutin could significantly increase the expression of NFE2L2 protein in cells,and the majority of NFE2L2 were concentrated in the nucleus.These results indicated that rutin could improve energy metabolism,increase antioxidant capacity of blood and mammary gland,reduce oxidative damage of mammary gland,and upregulate AMPK/NFE2L2 signaling pathway.（5）Based on metabonomics,the effect of rutin supplementation on the mammary gland health status in Hu sheep during the transition period was explored.The diet was supplemented with 0（the control group）,50,100 mg/kg BW rutin to sheep during the transition period.The feeding of this test was the same as（3）.The samples of blood and mamary gland tissues were collected for detection and analysis at+28 d.Through non-specific metabonomics analysis,17 different metabolites in plasma between group C and group H were screened out.The main enrichment pathways analyzed by KEGG were secondary bile acid biosynthesis,bile secretion,steroid hormone biosynthesis,retrograde endocannabinoid signaling and arginine biosynthesis.The different metabolites involved in these pathways were ursocholic acid,cholic acid,estrone sulfate,cholesteryl sulfate,1,2-dihexadecanoyl-sn-glycero-3-phosphocholine,and N-alpha-acetyl-l-ornithine.The changes of these differential metabolites affect the energy metabolism and inflammatory response of Hu sheep.Through non-specific metabonomics analysis,22 different metabolites in mammary gland tissue between group C and group H were screened out.The main enrichment pathways analyzed by KEGG were central carbon metabolism,aminoacyl-t RNA biosynthesis,pantothenate and Co A biosynthesis,beta-alanine metabolism,biosynthesis of amino acids,ABC transporters,histidine metabolism and choline metabolism.The different metabolites involved in these pathways were aspartic acid,histidine,DL-valine and 1,5-pentanediamine.The changes of these differential metabolites affect the health of the mammary gland of Hu sheep.In conclusion,rutin protected BMEC from oxidative stress injury by activating NFE2L2 through AMPK.Rutin allivated oxidative stress in mammary gland of Hu sheep during the transition period.Rutin protects mammary gland from oxidative stress and inflammation by regulating amino acid metabolism,energy metabolism and steroid hormone synthesis of ruminant.The present study provides research basis and theoretical hints into the future application of rutin in the protection of ruminant mammary gland.