Inhibitory Effect And Underlying Molecular Mechanism Of Epicatechin On LPS Induced Bovine Mastitis

Mastitis,a most common disease affecting dairy cows worldwide,causes huge economic losses to the dairy industry.Mastitis is mainly caused by infection with pathogenic bacteria such as Escherichia coli.In canonical signaling pathway of inflammation,toll-like receptor 4(TLR4)recognizes lipopolysaccharide(LPS),and promotes the activation of mitogen-activated protein kinases(MAPK)and nuclear factor kappa B(NF-κB)pathways by myeloid differentiation factor 88(My D88),leading to the production of pro-inflammatory factors such as interleukin 1 beta(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin 6(IL-6)and other inflammatory mediators,i.e.cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(i NOS).At present,antibiotics are widely used for treatment of mastitis.However,antibiotic treatment of mastitis has a low cure rate and easily leads to bacteria resistant to drugs and antibiotic residues in food chain,which have detriment to consumers’ health.Plant active substances have the advantages of non-drug resistance,no residue,and multi-functionality.As a substitute for antibiotic drugs,they have great potential for application in the prevention and treatment of mastitis.Epicatechin(EC),belonging to natural polyphenols,is abundant in cocoa,grapes,tea,and many other fruits and vegetables,and is one of the monomers of flavanol compounds.EC shows high activities of anti-inflammation and anti-oxidation,however its role in mastitis and the underlying molecular mechanism have not been reported.In this study,LPS-induced in vivo and in vitro models,respectively.The inhibitory effects of epicatechin on inflammatory factors and oxidative stress mediators were tested by ELISA.The effect of EC on T lymphocyte infiltration in mouse mammary tissue was tested with immunohistochemical methods.Western blot,ELISA and immunofluorescence methods were applied to detect the effects of EC on NF-κB and MAPK signaling pathways.Transcriptomic and proteomic profiling was performed with RNA-seq and TMT technology,and the protein interaction was analyzed with co-immunoprecipitation(Co IP)to reveal the upstream target genes and the molecular mechanism underlying the anti-inflammatory effects of EC.The results obtained in this study are shown as fellows:1.MTT experiments showed that 1.5,7.5,15,30 mg/m L of EC treatment had no effect on MAC-T cell viability(P>0.05).LPS stimulation at 1 μg/m L for 24 h significantly increased the levels in MAC-T cells of proinflammatory factors,including IL-1β(interleukin-1),IL-6(interleukin-6),TNF-α(tumor necrosis factor-α),and other inflammatory mediators,i.e.COX-2(cyclooxygenase-2)and i NOS(inducible nitric oxide synthase)(P<0.05).2.The expression of inflammatory cytokines in MAC-T cells was detected by ELISA.Compared with the LPS treatment group,EC at 1.5,7.5,15 and 30 mg/m L in combination with LPS(1 μg/ml)treatment group significantly reduced:(1)IL-1β(P<0.01),IL-6(P<0.01),TNF-α,COX-2(P<0.05)and i NOS(P<0.01)protein levels;(2)phosphorylation levels of IκB and p65(P<0.05)and p65 nuclear transfer immunofluorescence signal(P<0.05)in NF-κB pathway;(3)phosphorylation levels of p38,ERK1/2 and JUN1/2 in MAPK pathway(P<0.05).3.Oligomer proanthocyanidins(OPC)consisted of EC was chosen.ELISA assays indicated that OPC treatment at 1,3 and 5 μg/ml significantly reduced the protein of COX-2(P<0.05),i NOS(P<0.01),IL-6(P<0.01),IL-1β(P<0.01)and TNF-α(P<0.05)in LPS-induced MAC-T cells.Moreover,OPC downregulated LPS-induced phosphorylation of p65 and IκB in the NF-κB signaling pathway(P<0.01),and they inhibited p65 translocation from the cytoplasm to the nucleus as revealed by immunofluorescence test and western blot.Additionally,OPC decreased phosphorylation of p38,EREK1/2 and JUN1/2 in the MAPK signaling pathway(P<0.01).4.To further verify the results of the in vitro study,lactating female mice on the 7th day were selected,and 50 μL LPS at 0.2mg/m L was injected into the fourth pair of nipples,and then 10,20 and 30 mg/kg were injected intraperitoneally.After 24 h,compared with the LPS treatment group,the group co-treated various concentrations of EC and LPS was significantly reduced:(1)the levels of T cell molecular marker CD3(P<0.05);(2)the expression of inflammatory factors(IL-1β,IL-6 and TNF-α)and other inflammatory mediators(COX-2 and i NOS)(P<0.05);(3)phosphorylation levels of NF-κB pathway(IκB and p65)(P<0.05)and MAPK pathway(p38,ERK1/2,JNK1/2)(P<0.05)and p65 nuclear transfer immunofluorescence signal.5.To screen the upstream targets of EC,the transcriptomic and proteomic changes of MAC-T cells co-treated with EC and LPS were detected.Compared with the LPS group,RNA-seq analysis revealed that 156 genes were differentially expressed(FDR<0.05),mainly enriched in KEGG pathways,such as cytokine-cytokine receptor interaction,MAPK signaling pathway and inflammatory mediator regulation of TRP channels,and GO category including extracellular space,external side of plasma membrane,inflammatory response,immune response,etc.TMT analysis disclosed 30 proteins that were differentially expressed(FDR<0.05),which were mainly enriched in GO category including endoplasmic reticulum unfolded protein response and ER to Golgi transport vesicle membrane.6.TMEM35A(transmembrane protein 35A)and TMPO(thymopoietin)were chosen from the differential proteins,and CRISPR-Cas9 constructs for their corresponding genes were designed.The results showed that the phosphorylation level of p65 and the protein levels of TNF-α and i NOS were significantly increased after knock-down of TMPO and TMEM35 A,respectively(P<0.01).7.To verify whether TMEM35 A regulates NF-κB signaling pathway through TMPO,TMEM35 A,overexpression vector with His tags was further designed.When only transfection with Cas-TMEM35 A,TMPO levels in MAC-T cells was significantly reduced(P<0.01).However,in MAC-T cells co-transfected with Cas9-TMEM35 A and p CMV-TMEM35A-His,TMPO levels did not differ from those in the control group(P>0.05)and protein levels of TLR4,p-p65,TNF-α and i NOS were significantly increased(P<0.01).8.To investigate whether TMEM35 A interacts with TMPO to block LPS-stimulated inflammatory responses,Western blot analyses were performed using antibodies against TMEM35(anti-His)and TMPO in MAC-T cells co-transfected with p CMV-TMEM35A-His and co-treated with EC and LPS for 24 h.Co IP analysis indicated interaction between TMEM35 A and TMPO.Taken together,the above results indicated that EC inhibited inflammatory response in MAC-T cells and mouse mammary gland via MAPK and NF-κB pathways.TMEM35 A mediates the transmembrane transport of EC,and the interaction between TMEM35 A and TMPO inhibits the NF-κB signaling pathway.The results of this study provide new data for comprehensively revealing the anti-inflammatory mechanisms of EC and its application in the prevention and treatment of mastitis.