| To improve milk production and the economic benefit of lactation,dairy cows is usually fed high-concentrate diet to increase the nutrition during lactation period.However,high-concentrate diet often induces subacute rumen acidosis(SARA).During the occurrence and development of SARA,the microbial communities and abundance in rumen are disturbed,and large amounts of bacterial cell-wall components are released into rumen fluid.Lipopolysaccharide(LPS)has received broad attention due to its strong proinflammation activity.The concentration of LPS in the rumen and plasma exponentially increases during the high-concentration diet feeding mode,which in turn results in systemic inflammation and local inflammation.Like LPS,peptidoglycan(PGN)and lipoteichoic acid(LTA)are also bacterial cell-wall components and can also induce inflammatory responses.During the high-concentrate diet feeding mode,the concentration changes of PGN and LTA in rumen and plasma and their effects on inflammation and lactation of mammary gland should be studied.In addition,the effects of LPS,PGN and LPS,alone or combined stimulation on inflammation and lactation of mammary gland are deserved to be explored in depth.Sodium butyrate as a histone deacetylase(HDAC)inhibitor,can relieve SARA and the mastitis induced by LPS and D-glutamyl-meso-diaminopimelic acid(i E-DAP,the core structure of PGN)when added into the high-concentrate diet of ruminant.Butyrate absorbed by rumen is rapidly metabolized to β-hydroxybutyrate,which also is a HDAC inhibitor.β-hydroxybutyrate as a precursor substance of milk fat synthesis,can improve lactation and relieve inflammation.Whether sodium β-hydroxybutyrate addition into HC diet possesses a better effect than sodium butyrate remains unclear,and the relative regulatory mechanisms need to be explored.Summarizing the above questions to be studied,we firstly studied the effects of LPS,PGN and LPS,alone or combined stimulation on the transcriptome,inflammation,and histone acetylation of bovine mammary epithelial cells(b MECs).Next,while LPS,PGN and LTA co-stimulated b MECs,addition of HDAC inhibitor sodium butyrate and sodium β-hydroxybutyrate to research their effects on inflammation and histone acetylation of b MECs.Finally,sodium butyrate and sodium β-hydroxybutyrate were added into the high-concentrate diet of dairy cows to reveal their effects on inflammation and lactation.1.The effects of PGN and LTA,alone and combined stimulation(MIX)on transcriptome,inflammation,and histone acetylation of b MECs.This experiment explored the effects of PGN and LTA,alone and combined stimulation(MIX)on transcriptome of b MECs using RNA sequencing and bioinformation analysis and focused on the effects of PGN and LTA on inflammatory responses and histone acetylation of b MECs.The study provided a theoretical basis for interpreting the function of the cell wall components of Gram-positive bacteria(GPB)during the process of high-concentrate diet impairing mammary gland health.This study was designed as a randomized single factor trial and divided into four groups with six replicates in each group.The b MECs were seeded into 6-well plates.When the cells grew to approximately 60% confluence,the culture medium of each group was replaced with a fresh one(CON group),a fresh medium with 30 μg/m L PGN(PGN group),a fresh medium with 30 μg/m L LTA(LTA group),and a fresh medium with 30 μg/m L PGN and LTA(MIX group),respectively,and then the cells were incubated for 24 h.The results are as follows.(1)The number of high-quality(HQ)clean reads and known genes of different groups increased in the following order: PGN group < LTA group <MIX group.(2)Compared with the CON group,the number of up-and down-regulated differentially expressed genes(DEGs)of the other three treatment groups increased in the following order: PGN group < LTA group < MIX group.Meanwhile,these DEGs among the treatment groups all mainly significantly enriched in the cytokine activity GO terms and cytokine-cytokine receptor interaction pathway.(3)The relative m RNA expression of 6 inflammatory factors was determined by reverse transcription quantitative real-time polymerase chain reaction(RT-q PCR)analyses,that was basically consistent with the RNA sequencing data.(4)The expression of the proinflammatory cytokines(IL-1β,IL-6,IL-8,and TNF-α)and chemokines(CXCL1 and CXCL6)significantly increased in the following order: CON group < PGN group < LTA group <MIX group.(5)Compared with the CON group,the acetylation level of histone H3 and HAT activity in the other three groups significantly decreased.Overall,the effects of PGN on transcriptome and inflammation is weaker than those of LTA,and combined stimulation with PGN and LTA strongly changed transcriptome and increased DEG amounts and expression level,which in turn exacerbated inflammatory responses,displaying an additive effect.PGN and LTA,both alone and combined stimulation decreased the acetylation level of histone H3 by inhibiting HAT activity.2.The effects of LPS and co-stimulation with PGN,LTA and LPS(PLL)on transcriptome,inflammation,and histone acetylation of b MECs.This study explored the effects of LPS and co-stimulation with PGN,LTA and LPS(PLL)on transcriptome of b MECs also using RNA sequencing and bioinformation analysis and focused on the aspects of inflammatory responses and histone acetylation.The present study provided a theoretical basis for more comprehensively interpreting that high-concentrate diet impaired mammary gland health.This study is designed as a randomized single factor trial and divided into three groups with six replicates in each group.The b MECs were seeded into 6-well plates.When the cells grew to approximately 60% confluence,the culture medium of each group was replaced with a fresh one(CON group),a fresh medium with 0.1 μg/m L LPS(LPS group),a fresh medium with 30 μg/m L PGN,30 μg/m L LTA and 0.1 μg/m L LPS(PLL group),respectively,and then the cells were incubated for 24 h.The results are as follows.(1)The gene expression levels of different groups increased in the following order: CON group < LPS group < PLL group.(2)Compared with the CON group,the number of upand down-regulated DEGs of the LPS group was less than that of the PLL group.Meanwhile,the DEGs of the LPS group and PLL group almost significantly enriched in tumor necrosis factor(TNF)signaling pathway,cytokine–cytokine receptor interaction,nucleotide-binding oligomerization domain(NOD)-like receptor signaling pathway,chemokine signaling pathway,the Toll-like receptor(TLR)signaling pathway,and nuclear factor-kappa B(NF-κB)signaling pathway.The number of DEGs enriched in above the pathways in the LPS group was lower than that in the PLL group.The gene number and the fold change in the expression of the immune-related DEGs induced by the PLL were greater than those by the LPS.(3)The relative m RNA expression of 6inflammatory factors measured by RT-q PCR analyses was basically consistent with the RNA sequencing data.(4)The expression of the proinflammatory cytokines(IL-1β,IL-6,IL-8,TNF-α)and chemokines(CXCL1 and CXCL6)significantly increased in the following order: CON group < LPS group < PLL group.(5)Compared with the CON group,the acetylation level of histone H3 and the HAT activity significantly decreased in the LPS and PLL groups.Collectively,co-stimulation with PGN,LTA and LPS more strongly changed the transcriptome and increased DEG amounts and expression level,which in turn exacerbated inflammatory responses,displaying an additive effect.Both LPS and co-stimulation with PGN,LTA and LPS decreased acetylated histone H3 by inhibiting HAT activity.3.The effects of sodium butyrate and sodium β-hydroxybutyrate on inflammation and histone acetylation of b MECs during the PLL stimulationIn this experiment,the b MECs were treated with the HDAC inhibitors sodium butyrate and sodium β-hydroxybutyrate,respectively,for observing the function of sodium butyrate and sodium β-hydroxybutyrate on relieving the inflammatory responses and regulating histone acetylation of b MECs.The study provided a theoretical basis and regulation methods for relieving the adverse effects of bacterial cell-wall components on health of mammary gland in dairy cows fed high-concentrate diet.This study is designed as a randomized single factor trial and divided into four groups with six replicates in each group.The b MECs were seeded into 6-well plates.When the cells grew to approximately 60% confluence,the culture medium of each group was replaced with a fresh one(CON group),a fresh medium with 30 μg/m L PGN,30 μg/m L LTA and0.1 μg/m L LPS(PLL group),the fresh medium of PLL group with 1 m M sodium butyrate(PLLSB group),the fresh medium of PLL group with 1 m M sodiumβ-hydroxybutyrate(PLLHB group),respectively,and then the cells were incubated for24 h.The results are as follows.(1)The gene expression of proinflammation cytokines(IL-1β,IL-6,IL-8,TNF-α)significantly increased in the following order: CON group <PLL group < PLLSB group.Compared with the PLL and PLLSB groups,the gene expression of the four proinflammation cytokines in the PLLHB group significantly decreased.(2)Compared with the CON group,the acetylation level of histone H3 and the HAT activity in the PLL group significantly decreased.The acetylation level of histone H3 increased in the following order: PLL group < PLLHB group < PLLSB group.(3)The HDAC activity decreased in the following order: PLL group > CON group > PLLHB group > PLLSB group.Collectively,sodium butyrate improved the proinflammatory responses induced by bacterial cell-wall components,but sodiumβ-hydroxybutyrate showed opposite effect,perhaps because the effect of sodium butyrate on HDAC activity and acetylation level of histone H3 was more serious than that of sodium β-hydroxybutyrate.4.The effects of sodium butyrate and sodium β-hydroxybutyrate on inflammation and lactation in dairy cows fed high-concentrate dietIn the experiment,sodium butyrate and sodium β-hydroxybutyrate were added into the high-concentrate diet of dairy cows.Firstly,the production performance was compared.Next,the concentrations of bacterial cell-wall components in rumen and plasma were investigated.At least,the systemic and mammary inflammation was shed light on.This study was aimed at mitigating the negative effects of high-concentrate diet on health and lactation of dairy cows.This study is designed as a randomized single factor trial.Eighty healthy Chinese Holstein cows in the middle lactation were selected for the experiment,and they were divided into 4 groups with 20 replicates per group.The four groups were low-concentrate diet group(LC group,the ration of concentrate and roughage was 4:6),high-concentrate diet group(HC group,the ration of concentrate and roughage was 6:4),sodium butyrate group(HCSB group,the HC diet supplemented with 1% sodium butyrate,named the “HCSB” group),sodiumβ-hydroxybutyrate group(HCHB group,the HC diet supplemented with 1% sodiumβ-hydroxybutyrate).The feeding trial was a total of seven weeks,with a 2-week adaptation period and a 5-week measurement period.The results are as follows.(1)Diet,time and dietⅹtime interaction affected the DMI,4% fat corrected milk synthesis efficiency,and milk components.Diet tended to affect milk yield(p = 0.06).The milk yield of the HCSB group also reached a peak at the third week and remained at the peak level until the end of the trial.The milk fat contents in the HCSB and HCHB groups were significantly higher than those in the LC and HC groups.(2)The rumen p H value of the HC group was lower than the other three groups.The p H value of rumen fluid in the HCSB group was significantly higher than that in the HCHB group.Compared with the LC group,all the concentrations of LPS,PGN and LTA in rumen fluid and plasma increased in the HC.All the concentrations of LPS,PGN and LTA in rumen fluid and plasma in the HCSB group were lower than those in the HC and HCHB groups,except the PGN in rumen fluid.All the concentrations of LPS,PGN and LTA in rumen fluid and plasma were not significantly different between the HC and HCHB groups.(3)Compared with the LC group,the concentrations of proinflammatory cytokines(IL-1β,IL-6,IL-8,and TNF-α)and acute phase proteins(LBP,SAA,CRP,and Hp)significantly increased in the HC group.Compared with the HC group,the proinflammatory cytokines,CRP and Hp in the HCSB group and the IL-6 and Hp in the HCHB group significantly decreased.The concentration of IL-1β in the HCSB group was lower than that in the HCHB group.(4)Compared with the LC group,all the leucocytes(lymphocyte,monocyte,and neutrophil)significantly increased in the HC group.Compared with the HC group,both the number of white blood cells,lymphocytes,and monocytes in the HCSB group and the number of white blood cells and lymphocytes in the HCHB group significantly decreased.Both diet and time affected SCC in milk.The SCC in the HC group was significantly higher than that in the other three groups from the second week.(5)Compared with the LC group,the blood ketone bodies of the HC group significantly decreased,the blood ketone bodies of the HCSB group did not significantly change,and the blood ketone bodies of the HCHB group significantly increased.The blood ketone bodies increased in the following order:HC group < HCSB group < HCHB group.(6)Compared with the LC group,the gene expression of pattern recognition receptors(TLR4,NOD1,and PGRP2)and proinflammatory cytokines(IL-1β,IL-6,IL-8,and TNF-α)of mammary cells significantly increased in the HC group.Compared with the HC group,the gene expression of the pattern recognition receptors(TLR4,NOD1,and PGRP2)and proinflammatory cytokines of mammary cells in the HCSB group and the gene expression of NOD1,PGRP2,IL-1β,IL-6,and IL-8 of mammary cells in the HCHB group significantly decreased.Compared with the HCSB group,the gene expression of TLR4,IL-8 and TNF-α of mammary cells significantly increased in the HCHB group.Collectively,both sodium butyrate and sodium β-hydroxybutyrate mitigate the adverse effects of high-concentrate diet on health and lactation of dairy cows,and the effect of sodium butyrate was stronger than sodium β-hydroxybutyrate,might it is due to sodium butyrate inhibited the increase of bacterial cell-wall components in rumen and plasm induced by high-concentrate diet.Taken together the results above suggested the following conclusions.(1)The effects of PGN,LTA and LPS on transcriptome and inflammatory responses of b MECs displayed an additive effect.The co-stimulation with PGN,LTA and LPS induced stronger transcriptome changes and increased DEG amounts and expression level,which in turn exacerbated inflammatory responses,displaying an additive effect.(2)PGN,LTA and LPS,both alone stimulation and co-stimulation decreased HAT activity and the acetylation level of histone H3,no additive effect.(3)Sodium butyrate improved the inflammatory responses of b MECs induced by bacterial cell-wall components in vitro,but sodium β-hydroxybutyrate displayed the opposite effect,perhaps because sodium butyrate more seriously affected the HDAC activity and acetylation level of histone H3 than sodium β-hydroxybutyrate.(4)Sodium butyrate more effectively mitigated the negative effects of high-concentrate diet in dairy cows than sodium β-hydroxybutyrate via reducing free bacterial cell-wall components(LPS,PGN,and LTA)in rumen fluid and plasm. |
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