| In intensive dairy farms,in order to efficiently increase milk production,lactating dairy cows are often fed diets with high concentrate and low forage.Although feeding high-concentrate diet can increase milk production in a short time,it can cause subacute rumen acidosis（SARA）in dairy cows,with excessive LPS produced in the rumen to be released into the blood circulation system,which in turn causes a series of metabolic disorders for dairy cows.This article used high-concentrate diet to feed lactating dairy cows for a short time.It was found that high-concentrate diet can cause inflammatory reaction,oxidative stress,and milk fat anabolic reduction in mammary gland of dairy cows.Stimulating bovine mammary epithelial cells（BMECs）with LPS can also induce these phenomena,indicating that LPS is one of the reasons for mammary gland damage and MFD caused by high-concentrate diet feeding.Conjugated linoleic acid（CLA）is mainly consist of cis-9,trans-11-CLA and trans-10,cis-12-CLA.It is an important component of fatty acids in milk and has many biologically active functions.Among them,trans-10,cis-12-CLA is considered to be the main reason for inducing MFD.However,this study found that cis-9,trans-11-CLA can play anti-inflammatory response and anti-oxidative stress roles and increase milk fat synthesis in BMECs.The application of cis-9,trans-11-CLA in dairy cows needs further exploration.1.Effect of high concentrate diet on inflammatory response,oxidative stress and milk fat synthesis metabolism in the mammary gland of dairy cowsAlthough high-concentrate diet feeding can temporarily increase milk production,it can cause a series of metabolic diseases,such as SARA and MFD.The main purpose of this experiment was to study the effects of high-concentrate diets on inflammatory response,oxidative stress and milk fat synthesis in mammary gland of dairy cows.12 mid-lactating Holstein cows equipped with rumen fistulas were randomly divided into 2 groups,each with 6 cows,fed with low concentrate diet（low concentrate,LC）and high concentrate diet group（high concentrate,HC）,the test period was 21 days.On the 20th and 21st days of the experiment,rumen fluid was collected to measure the pH value and milk samples were collected for milk component analysis and LPS concentration test.On the 21st day,mammary vein blood was collected to detect the LPS concentration.After the experiment,mammary gland tissue was collected for HE staining to observe morphological changes,and the expression of inflammatory response,oxidative stress,and milk fat synthesis-related genes and proteins in mammary gland were analyzed by real-time quantitative PCR,WB and immunohistochemistry in the laboratory.The pH of rumen fluid in the HC group was significantly lower than that in the LC group,and the period of pH lower than 5.6 was more than 3 hours,indicating that short-term feeding of high-concentrate diet induced SARA.The concentration of LPS in the mammary vein blood increased significantly after high-concentrate feeding,resulting in the destruction of the mammary tissue structure and a large number of neutrophil infiltrations.For the inflammatory response,the pro-inflammatory cytokines（IL-6 and IL-1α）and innate immune factors（LAP and TAP）in mammary gland of the HC group were significantly increased,and the TLR4-NF-κB signaling pathway was activated.For oxidative stress,after high-concentrate diet feeding,the content of malondialdehyde（MDA）in mammary vein blood and mammary gland tissue increased,the content of glutathione（GSH）in mammary vein blood decreased,the activity of SOD and the total antioxidant capacity（T-AOC）in mammary gland tissue and mammary vein decreased,and the expression of antioxidant enzymes and antioxidant transcription factor NFE2L2 in mammary gland decreased.For milk fat metabolism,high-concentrate diet feeding reduced the milk fat content in milk samples and the triglyceride content in mammary gland,and also inhibiting de novo synthase（ACACA and FASN）,long-chain fatty acid converting enzymes（ACSL1 and SCD）,fatty acids transporters（CD36,FATP,FABP3 and LPL）,triglyceride synthase（AGPAT6,DGAT1 and LPIN1）,lipid droplet releasing enzyme（PLIN1）and transcription factors SREBP1 and PPARG.In summary,high concentrate diet can induce SARA with increased concentration of LPS in dairy cows,stimulate inflammatory reaction and oxidative stress,and inhibit milk fat synthesis in mammary gland of dairy cows.2.Effect of cis-9,trans-11-conjugated linoleic acid on inflammatory response and signaling pathway in bovine mammary epithelial cells induced by heat-inactivated E.coliThe anti-inflammatory effects of cis-9,trans-11-conjugated linoleic acid（cis-9,trans-11-CLA）in diverse cells have been demonstrated in recent studies.The present study was conducted to observe the anti-inflammatory effects and involved mechanisms of CLA in bovine mammary epithelial cells（BMECs）exposed to Escherichia coli.According to the gene expression of IL-6,to optimize the treatment period and dose of CLA,50μM and 100μM CLA were chosen to pretreat the cells for a period of 48h.BMECs were exposed to1×107/mL E.coli for 6 h（ECO group）,and cells were pretreated with 50μM and 100μM CLA for 48h followed by E.coli challenge（C50 and C100 groups）.After E.coli challenge,compared with that in the CON group（control group）,the gene expression of pro-inflammatory cytokines（IL-1βand IL-6）,chemokines（IL-8 and CCL-20）and antimicrobial peptide BNBD5 were increased while the gene expression of the anti-inflammatory cytokine IL-10 was decreased significantly;CLA reversed this inflammation effect.Pretreatment with CLA also repressed the secretion of IL-6,IL-8 and TNF-αfrom BMECs in the culture medium following E.coli challenge.Therefore,cis-9,trans-11-CLA exerted anti-inflammatory effects in BMECs.The cells that were pretreated with CLA expressed remarkably lower levels of phospho-p65,phospho-IκB,TLR4 and higher level of PPARγafter E.coli challenge at the gene and protein levels.Compared to that in the ECO group,the nuclear translocation of phospho-p65 was suppressed when CLA was added.Combined with the above results,50μM CLA showed a better anti-inflammatory effect.In conclusion,CLA can reduce inflammation caused by E.coli in bovine mammary epithelial cells,and this effect is mediated through the TLR4-NF-κB pathway and PPARγparticipation.3.Effect of cis-9,trans-11-conjugated linoleic acid on oxidative stress caused by LPS in bovine mammary epithelial cellsFeeding dairy cows with high concentrate diets induces subacute rumen acidosis（SARA）,which is accompanied by an increase in the concentration of LPS in the blood of the mammary vein,resulting in damage to the mammary gland tissue.The accumulation of reactive oxygen species（ROS）in the body is the cause of many metabolic disorders,and it is reported that cis-9,trans-11-conjugated linoleic acid（CLA）has anti-oxidative stress activity.The purpose of this experiment is to explore whether LPS can cause oxidative stress in bovine mammary epithelial cells and whether cis-9,trans 11-CLA can alleviate oxidative stress and its mechanism.In this experiment,followed by LPS with different treatment time to stimulate cells and CLA with different pretreatment time to protect cells,the mRNA expression of transcription factor NFE2L2 and anti-oxidative stress-related enzymes were detected to determine the optimal action time of LPS and CLA.Then flow cytometry and laser confocal were used to detect the ROS content in the cells.Finally,the cells were pretreated with 50μM CLA for 24h and 8μg/mL LPS stimulated for 12h.The effects of LPS and CLA on the transcription factor NFE2L2-dependent anti-oxidative stress system were analyzed by assay kits,real-time quantitative PCR,WB and immunofluorescence.Results showed that,after LPS stimulated BMECs,the mRNA and protein expression of NFE2L2 decreased,indicating that LPS inhibited the expression of NFE2L2,thereby inhibiting the expression of its downstream target genes.At the same time,LPS induced cells to produce more ROS and MDA,and reduced the content of non-enzymatic antioxidant GSH.Cis-9,trans-11-CLA can reduce the production of ROS and MDA by LPS stimulation,and increase the expression of the transcription factor NFE2L2 and its regulated antioxidant enzymes.Treatment with CLA alone can also increase CAT activity and the expression of antioxidant enzymes.In summary,LPS stimulated BMECs to produce more ROS and MDA,reduced non-enzymatic antioxidant GSH and the transcription factor NFE2L2-related antioxidant enzyme system,which in turn caused oxidative stress;however,cis-9,trans-11-CLA can alleviate oxidative stress caused by LPS.This experiment further determined the mechanism of oxidative stress-induced mastitis caused by feeding high-concentrate diet,and provided a new method for the treatment of clinical mastitis,but the antioxidant capacity of cis-9,trans-11-CLA needs to be further confirmed by in vivo test.4.Regulation of cis-9,trans-11-conjugated linoleic acid on milk fat depression caused by LPS in bovine mammary epithelial cellsFeeding dairy cows with high-concentrate diets can cause milk fat suppression（MFD）,which is often accompanied by an increase in the LPS concentration in the blood circulation system.Previous studies have shown that trans-10,cis-12-conjugated linoleic acid（CLA）is the main cause of MFD,but its isomers,cis-9,trans-11-CLA,does not have a clear effect on milk fat metabolism.Therefore,the purpose of this experiment was to study the effect of LPS on milk fat metabolism and the regulation of cis-9,trans-11-CLA on milk fat metabolism in BMECs.First,cells were treated by LPS and CLA for different time,and the best treatment time was decided.Then,oil red O staining,real-time quantitative PCR,WB and cell immunofluorescence were used to detect the influence of CLA pretreatment followed by LPS stimulation on triglyceride synthesis,milk fat synthesis related transcription factors,fatty acid de novo synthase,long chain of fatty acid invertase,fatty acid transport protein,triglyceride synthase and lipid droplet releasing protein.Results showed that 8μg/mL LPS can reduce the synthesis of triglycerides and the expression of milk fat metabolism synthase in BMECs.Cells treated with 50μM and 100μM H2O2 for 6h also inhibited the mRNA expression of transcription factors SREBP1 and PPARG and milk fat synthase ACACA and FASN.Cells treated with 50μM CLA alone had an unstable effect on milk fat synthesis-related enzymes,but treatment for 24 hours can significantly increase the expression of milk fat synthesis-related enzymes,which was consistent with the protein results of SREBP1,PPARG and SCD.With the extension of CLA treatment time,the content of triglycerides in BMECs was also increased;and CLA can alleviate the inhibitory effect of LPS stimulation on the mRNA expression of SREBP1,ACC and ACSL1 and the protein expression of SCD.In summary,LPS and its induced oxidative stress is one reason of MFD caused by high-concentrate diet,and cis-9,trans-11-CLA can increase the expression of milk fat synthesis-related enzymes and relieve the inhibitory effect of LPS on milk fat metabolism,but the application of cis-9,trans-11-CLA in dairy cows feeding needs further exploration. |
