| In recent years,in order to increase milk production during dairy farming,cereal starch based high-concentrate diets are fed to bovines.Although-economic benefits are achieved within the-short-term,numerous studies have shown that long-term feeding of dairy cows with high-concentrate diets causes abnormal fermentation of a huge number of carbohydrates in the rumen and yields enormous amount of volatile fatty acid(VFA).This results in reduction of rumen pH,damage to rumen epithelium,an d even the occurrence of subacute ruminal acidosis(SARA)in bovines.In SARA condition,lots of gram-negative bacteria in the rumen die and disintegrate to release harmful products such as lipopolysaccharide(LPS),lipoteichoic acid(LTA)and muramyl dipeptide(MDP),among other.So far,stud ies have shown that the products of bacterial death can an induce inflammatory response in bovines,but these studies have mostly focused on the role of LPS and LTA,rather than the role of MDP.Although,it has been reported that almost all bacteria can produce MDP in the process of proliferation,death and cleavage,however our previous studies have shown that MDP can induce an inflammatory response in bovine rumen epithelial cells(BRECs).Therefore,it is of great significance to study how MDP induces ruminal epithelial inflammatory response and its regulation mechanism for the breeding industry in C hina.Studies have shown that when MDP is absorbed into the body,it can bind to nucleotide=binding oligomerization domain-containing protein 2(NOD2),triggering a cascade of reactions that eventually induce inflammation.We hypothesized that NOD2 can mediate/moderate MDP to regulate the inflammatory response of BRECs.Therefore,the purpose of this study was to explore whether MDP can regulate the inflammatory response of BRECs through NOD2 and to provide the theoretical basis for the mechanism of rumen epithelial inflammation in SARA bovines.Experiment 1:Effects of high and low concentrate diet on MDP content in rumen fluid of bovinesThis experiment was conducted to establish a method for detecting MDP in rumen fluid of dairy cows and to study the effects of high concentrate diet on MDP content in rumen fluid of dairy cows.Bovines were divided into two groups and fed low or high-concentrate diets.Rumen fluid was collected at the same time,and rumen fermentation parameters were measured.The method for determining MDP in rumen fluid was established by thin layer chromatography(TLC)and high performance liquid chromatography tandem evaporative light scattering detector(HPLC-ELSD).The results showed that rumen pH of dairy cows was lower than 5.6 from 3h to 6 h after feeding high-concentrate diet,indicating that dairy cows were in SARA state.Compared with low-concentrate diet fed group,the contents of acetic acid,propionic acid,butyric acid,valerate acid and total volatile fatty acid in high-concentrate diet fed group were significantly increased(P<0.05).The linear regression equation of MDP content was calculated by an external standard method:Y=1.0711x+3.2201(R2=0.9943).The linear relationship was good in the concentration range of 50-1000 μg/mL,and the limits of detection and quantification of MDP were 70 ng and 210 ng,respectively.It was found through testing that,the MDP content in rumen fluid of dairy cows fed high concentrate increased significantly(P=0.005).Experiment 2:Effects of different concentrations and treatment times of MDPon BRECs inflammationIn this study,BRECs were cultured by adding MDP of different concentrations and different treatment times.The expression levels of inflammation-related genes in cells.were detected by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)to determine the relationship between the concentration of MDP and the reaction time when BRECs produced inflammatory response.The time gradient experiment was divided into 5 groups with 6 replicates in each group.Meanwhile,total RNA was extracted from cells after 10 μg/mL MDP was added to BRECs of each group and cultured for 1 h,3 h,6 h,12 h and 24 h,respectively.The concentrations gradient experiment was divided into 5 groups with 6 replicates in each group.Total RNA was extracted from cells after 0 μg/mL,1 μg/mL,5 μg/mL,10 μg/mL and 15 μg/mL MDP were added to BRECs of each group for 3 h.When compared to other time groups,the results showed that,MDP treatment for 3 h significantly increased the mRNA expression levels of pro-inflammatory cytokines IL-6,IL-8,and TNF-,chemokines CCL2,CXCL2,CXCL3,and CXCL5,and NOD1,RIPK2,and IRAK4(P<0.05).MDP treatment for 6 h significantly increased the mRNA expression levels of BRECs proinflammatory cytokines IL-1β,chemokines CCL5,CCL20,CCL28 and CXCL8;and NOD1,RIPK2 and IRAK4(P<0.05).Compared with other concentration groups,10 μg/mL MDP significantly increased the mRNA expression levels of BRECs pro-inflammatory cytokines IL-6,IL-8,TNF-α and IL-1β,chemokines CCL20,CCL28,CXCL2,CXCL3 and CXCL8,and NOD2,RIPK2,IRAK4 and IRF1(P<0.05).In conclusion,BRECs inflammatory response was the most significant when MDP concentration was 10μg/mL and treatment time was 3 h or 6 h.Experiment 3:Transcriptome analysis of MDP induced BRECs differential genes and differential signaling pathways.This study aimed to study the mRNA differential genes and differential signaling pathways between wild-type BRECs and MDP stimulated BRECs using transcriptome sequencing technology.The experiment was divided into two groups namely,wild-type BRECs and 10μg/mL MDP treated BRECs.Sequencing results showed that compared with wild-type BRECs,MDP stimulated BREC s significantly up-regulated the gene expression levels of proinflammatory cytokines IL-6,IL-8 and IL-1β,and chemokines CCL2,CCL5,CXCL1,CXCL3,CXCL5,CXCL8,and CX3CL1(P<0.05).Meanwhile,the expressions of MKK1,MKK3 and MKK4 in MKK kinase,ERK2,ERK3 and ERK5 in ERK kinase and transcription factor ATFA7 were significantly up-regulated(P<0.05).The expression levels of NF-κB inhibitory subkinases IKKE and inhibitory subkinases IKBA,IKBB,IKBD,IKBE and IKBZ in NF-κB signaling pathway were significantly up-regulated(P<0.05).In addition,the expression levels of NF-κB1 and RelB in NF-κB family were also significantly up-regulated(P<0.05).Compared with wild-type BRECs,MDP stimulated BRECs significantly up-regulated the expression levels of major kinases TRAF1,TRAF2,TRAF3,TRAF4,RIPK1 and cIAP2 in TNF signaling pathway(P<0.05).The expression levels of pattern recognition receptors NOD1 and NOD2 and downstream key kinases RIPK1,RIPK2 and RIPK4 were significantly up-regulated(P<0.05).The results showed that MDP can activate MAPK and NF-κB signaling pathways,which can trigger inflammatory responses.Experiment 4:Transcriptome analysis of NOD2 knockout BRECs-mRNA differential genes and differential signaling pathwaysThis experiment aimed to study the mRNA differential genes and differential signaling pathways of NOD2 knockout BRECs by transcriptome sequencing technology.Stable NOD2knockout BRECs cell lines were successfully obtained by CRISPR/Cas9 gene editing techniques.After monoclonal sequencing,the results showed that three effective parents were obtained.Western blot verification showed that the knockout parents almost did not express NOD2,indicating that the knockout was successful.The experiment was divided into two groups:wild-type BRECs and NOD2-knockout BRECs,Sequencing results sh owed that the expression levels of Pro-inflammatory cytokines IL-6,IL-1β,IL-1α,IL-18,IL-32 and OSMR were significantly down-regulated in NOD2 knockout BRECs compar ed with wild-type BRECs(P<0.05).The expression levels of chemokines CCL2,CXCL3,CXCL5,CXCL8,CXCL12 and CXCL16 were also significantly down-regulated(P<0.05).Meanwhile,the expression levels of TNFR2 receptor of TNF signaling pathway,key kinases TRAF5 and RIPK1,and downstream transcription factor AP-1 were significantly down-regulated(P<0.05).Compared with wild-type BRECs,the expression levels of MKK1,MKK3,MKK4,MKK6,ERK2,ERK3,ERK4 and P38,and transcription factors ATF1,ATF2,ATF3,ATF6 and ATF7 in MAPK signaling pathway were significantly reduced(P<0.05).In addition,the expression levels of pattern recognition receptors NOD1 and NOD2,and downstream key kinases RIPK1,RIPK2 and RIPK4 were significantly down-regulated(P<0.05).At the same time,the expression levels of IKKB,IKKG and IKKE in NF-κB signaling pathway were significantly increased(P<0.05).Interestingly,the expression levels of NF-κB inhibitors IKBA and IKBZ were significantly reduced(P<0.05).The expression levels of RelA,Rel and NF-κB1 in NF-κB family were significantly down-regulated(P<0.05).In conclusion,MDP can down-regulate the expressions of pro-inflammatory factors and chemokines by down-regulating the expressions of key genes in MAPK,NF-κB and TNF signaling pathways.Experiment 5:NOD2 mediates MDP to regulate BRECs inflammationThis study aimed to further investigate whether MDP can regulate the inflammatory response of BRECs through NOD2.The experiment was divided into three groups namely,wild type BRECs,wild type BRE Cs incubated with 10 μg/mL MDP and NOD2 knockout BRECs incubated with 10 μg/m L MDP.The results showed that compared with wild-type BRECs,MDP could significantly increase the mRNA expression levels of pro-inflammatory cytokines IL-6,IL-8,TNF-α and IL-1β,chemokines CCL2,CCL20 and CCL28,and CXCL2,CXCL3,CXCL5,CXCL8 and CXCL9 in BRECs(P<0.05).The mRNA expression levels of pro-inflammatory cytokines IL-6,IL-8,TNF-α and IL-1β,chemokines CCL2,CCL5 and CCL20,and CXCL2,CXCL3,CXCL8 and CXCL9 were significantly down-regulated in NOD2-knockdown BRECs stimulated by MDP compared with wild-type BRECs stimulated by MDP(P<0.05).MDP can significantly increase NOD2,RIPK2,IRAK4 and IRF1 expression levels in wild-type BRECs(P<0.05).However,NOD2,IRAK4 and IRF1 expressions were significantly down-regulated when MDP stimulated NOD2 knockout BRECs(P<0.05).Cell immunofluorescence and Western blot analysis showed that compared with the control group,MDP significantly enhanced the red fluorescence intensity of BRECs p38,p-p38,Erk1/2,p-Erk1/2,NF-κB p65 and p-NF-κB p65 and protein expression of Erk1/2,p-Erk1/2 and p-NF-κB p65(P<0.05).NOD2-knockout BRECs stimulated by MDP were compared with wild-type BRECs stimulated by MDP.It significantly decreased the redfluorescence intensity of p38,p-p38,Erk1/2,p-Erk1/2,NF-κB p65 and p-NF-κB p65 and protein expression levels of Erk1/2,p-Erk1/2,NF-κB p65 and p-NF-κB p65(P<0.05).NOD2 was significantly positive-correlated with pro-inflammatory cytokines IL-6,IL-8,TNF-α and IL-1β,chemokines CCL2,CCL20,CXCL2,CXCL3 and CXCL8,and IRAK4 and IRF1(P<0.05).However,it was negatively correlated with NOD1(P=0.07).In conclusion,MDP can regulate key genes of MAPK,NF-κB and TNF signaling pathway through NOD2,thus regulating BRECs inflammatory response.In conclusion,NOD2 mediates MDP to regulate NOD signaling pathway RIPK1,RIPK2 kinase and Toll-like receptor signaling pathway IRAK4 kinase,transcription factor IRFI expression,up-regulate MKK1,MKK3,MKK4,ERK2,ERK3 kinase and repressors IKBA,IKBZ expression.Concurrently,the protein expression of Erk1/2 and NF-κB p65 is upregulated,thereby activating the MAPK and NF-κB signaling pathways,promoting the expression of inflammatory factors,chemokines,and finally triggering an inflammatory response. |
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