The Alleviation And Repair Mechanism Of Thiamine Supplementation On The Rumen Epithelial Barrier Damage Induced By Rumen Acidosis In Goats

Ruminants are often fed with more high-concentrate diets during the fattening and lactation stage,to meet energy demands.However,prolonged supply of easily fermentable carbohydrates in the diet can disrupt the stable environment within the rumen,resulting in damage to the rumen epithelial barrier function and imbalanced energy metabolism.Thiamine(VB1),as a water-soluble vitamin,has been shown to regulate the structure of the rumen microbiota,maintain the physiological fermentation state of the rumen,and protect the rumen epithelial barrier function.Specifically,the study focused on the ability of VB1 to alleviate inflammatory damage to the rumen epithelium and maintain energy metabolism status.Both in vitro and in vivo experiments were conducted to investigate the effects of VB1 supplementation on the maintenance of rumen epithelial homeostasis.Trail 1:Regulatory study of thiamine supplementation on high-concentrate dietinduced damage to goat rumen epithelial barrier functionIn this experiment,24 healthy Boer goats(35.62 ± 2.4 kg)were randomly divided into three groups:low-concentrate group(CON,concentrate:roughage=30:70),highconcentrate group(HC,concentrate:roughage=70:30),and high-concentrate group with 200 mg/kg DMI VB1 supplementation(HCT,concentrate:roughage=70:30).There were 8 goats in each group,and the experiment lasted for 12 weeks.After the experiment,the goats were slaughtered,and rumen fluid,blood,and rumen epithelial tissue were collected for analysis.The results showed that compared with the CON group,the HC group significantly increased the concentrations of LPS,propionate,butyrate,and total volatile fatty acids(VFA)in the rumen and decreased the pH,VB1,and acetate concentrations(P<0.05).Moreover,compared with the HC group,the HCT group showed an increase in pH,VB1,and acetate concentrations and a decrease in the concentrations of LPS,propionate,butyrate,and total VFA(P<0.05).The results of quantitative analysis of rumen microorganisms showed that exogenous VB1 supplementation reduced C.sporogenes and increased the number of Fibrobacter succinogenes,Bacteroides fragilis,and Ruminococcus flavefasciens(P<0.05).The histological examination revealed that the rumen papillae surface in the HCT groups showed normal tissue morphology,with smooth and flat rumen papillae and relatively intact mitochondrial morphology.The results of RT-PCR showed that the mRNA expression levels of THTR1,THTR2,MTPPT,Bcl-2,Claudin-4,Claudin-1,Occludin,and ZO-1 genes in the HC group were significantly downregulated,and the mRNA expression levels of Bax,Caspase 3,Caspase 8,and Caspase 9 genes were significantly upregulated(P<0.05).Exogenous VB1 supplementation reversed the expression levels of these genes.Western Blotting analysis showed that the expression levels of ZO-1,Occludin,and Claudin-1 proteins were significantly decreased after feeding the high-concentrate diet(P<0.05),and with the supplementation of VB1,the expression levels of these proteins were significantly increased(P<0.05).The results of this experiment indicate that exogenous VB1 supplementation regulates rumen microbialcommunity structure,maintains normal rumen fermentation status,and protects rumen epithelial barrier function during feeding of high-concentrate diets.Trail 2:A multi-omics integrated analysis revealed the regulatory mechanisms of thiamine supplementation on inflammation and energy status damage in goat rumen epitheliumBased on animal experiments,rumen epithelial tissues were collected and performed transcriptome analysis(four random samples per group)and tissue metabolome analysis(six random samples per group).Transcriptome analysis showed that the HCT group upregulated 112 DEGs and downregulated 1372 DEGs compared to the HC group.Gene ontology(GO)analysis showed that the effect of VB1 on tissues was mainly concentrated in biological processes(BP),such as adenosine triphosphate(ATP)metabolism,mitochondrial respiratory chain complex assembly and cellular respiration(P<0.05).In cellular components(CC),it was mainly concentrated in the composition of mitochondrial inner membrane protein complexes,mitochondrial matrix,respiratory chain complex,oxidative reductase complex and ribosome(P<0.05).In molecular functions(MF),it was mainly concentrated in electron transfer,structural molecule activity and transmembrane transporter activity(P<0.05).The KEGG analysis results indicated that thiamine supplementation mainly affected cell growth and death,cell activity,cell development and regeneration,immune regulation,signal transduction and the interaction of signal molecules(P<0.05),and the regulation of inflammation by VB1 may be related to multiple pathways such as TNF-α,MAPK and Toll-like.The analysis of epithelial tissue metabolome showed that exogenous VB1 supplementation mainly affected the content of metabolites related to VB1-coenzyme in glycolysis,TCA cycle,and pentose phosphate pathway compared to the HC group(P<0.05).Blood analysis showed that the concentration of LPS,LBP,IL-1β,and IL-6 in the HCT group was significantly decreased compared to the HC group(P<0.05),and the concentration of VB1 and IgA was significantly increased(P<0.05).In addition,exogenous VB1 supplementation also increased the content of DNA,RNA,and ATP in rumen epithelial tissues compared to the HC group(P<0.05).These results suggest that exogenous VB1 supplementation improves epithelial carbohydrate metabolism pathways and maintains tissue energy levels.At the same time,the regulation of inflammation by VB1 may be related to multiple pathways such as TNF-α,MAPK,and Toll-like.Trail 3:Cellular level reveals the regulatory mechanism of thiamine on the barrier function of rumen epithelial cellsIn this experiment,rumen epithelial cells(RECs)were isolated and cultured,stimulated with different concentrations of VB1 and LPS,and an inflammatory model was established by IL-6 stimulation.The experiment was divided into 4 groups:cells stimulated with DMEM/F12 medium without VB1 for 18 h(CON group),cells stimulated with VB1 for 18 h(THI group),cells stimulated with LPS for 6 h(LPS group),and cells pre-treated with VB1 for 18 h,washed with PBS and then stimulated with LPS for 6 h(LPTH group).Based on dose-dependent experiments,5 μg/mL LPS and 5 μg/mL VB1 were determined to be the optimal dose for cell stimulation.The results showed that the FD4 concentration and decreased TEER of the LPTH group were significantly reduced compared to the LPS group(P<0.01).Apoptosis results showed that both the lack of VB1 and LPS stimulation significantly increased the apoptosis rate of cells compared to the THI group(P<0.01).Meanwhile,compared with the CON group,the expression levels of Bax protein and mRNA in the LPS group were significantly increased(P<0.01),while Bcl-2 was significantly decreased(P<0.05).Interestingly,compared with the LPS group,VB1 pretreatment could reverse LPS-induced cell apoptosis and alter the expression levels of these proteins and mRNA(P<0.01).The cell cycle results showed that the lack of VB1 and LPS stimulation both increased the number of G0/G1 phase cells and decreased the number of S phase cells(P<0.01).After VB1 pretreatment,the number of G0/G1 phase cells in the LPTH group was significantly reduced,and the number of S phase and G2/M phase cells was significantly increased compared to the LPS group(P<0.01).To verify the effect of VB1 on the oxidative damage status of RECs cells,we detected the content of reactive oxygen species(ROS)in cells.The results showed that compared with the LPS group,VB1 pretreatment reduced the ROS content in cells(P<0.01).Tight junction protein research showed that compared with the LPS group,the pre-treatment with VB1 significantly increased the expression levels of Occludin,Claudin-1,Claudin-4,and tight junction-related protein(Zonula occludens-1,ZO-1)genes and proteins(P<0.01),and protected the barrier function of the gastric epithelium.The results of this experiment indicate that the pre-treatment with VB1 maintains normal intercellular resistance and cell permeability,reduces apoptosis,promotes cell proliferation,inhibits oxidative damage,and thus protects the barrier function of cells.Trail 4:Cellular-level elucidation of the mechanism of action of thiamine in regulating rumen epithelial energy status and inflammation through inhibition of the NFκB/AMPK/p38 MAPK pathwayBased on the experimental cell inflammation model,the regulatory effect of VB1 supplementation on cell inflammation injury and energy status and overexpressed p38 MAPK(i.e.,MAPK14 gene)was investigated to clarify its potential mechanism of action(empty vector group:LTP,pcDNA3.1(+)-MAPK14 overexpression vector group:O-LPTH).The experimental results showed that after VB1 pretreatment of cells,the mRNA expression levels of THTR1,THTR2,and MTPPT were significantly increased compared to the LPS group(P<0.01).Inflammatory studies showed that LPS stimulation induced an increase in gene expression of IL-1β,IL-6,IL-10,and TNF-α(P<0.01)and specifically triggered the expression of p-IκB in the nucleus.In contrast,compared with the LPS group,VB1 supplementation reversed the expression of the above genes(P<0.01)and reduced pIκB nuclear entry.We also determined the effect of VB1 on the p38 MAPK pathway and found that LPS stimulation induced an increase in the expression levels of p38 and p-p38(P<0.01).Interestingly,VB1 pretreatment reduced the increase in p38 protein phosphorylation induced by LPS(P<0.01).The effect of VB1 on the energy status of RECs was detected in the LPS-induced inflammation model.Compared with the LPS group,the LPTH group significantly increased ATP synthesis(P<0.01).The lack of VB1 caused an increase in the protein levels of AMPKα,PGC1α,and SIRT1 associated with energy status(P<0.01),while compared with the LPS group,the LPTH group decreased the expression of the above proteins and down-regulated the phosphorylation level of AMPKa protein(P<0.01).After overexpression of the p38 MAPK gene,we investigated the regulatory effects of VB1 on inflammation.The results showed that compared to the LPTH group,the levels of p38 protein expression and phosphorylation in O-LPTH group cells were significantly increased(P<0.05),and the phosphorylation level of p65 was also significantly increased(P<0.05),but the expression level of TLR4 protein did not change significantly(P>0.05).Studies on cellular energy status showed that compared to the LPTH group,the levels of AMPKa and PGC1α proteins and the ratio of p-AMPKa to total AMPKa were significantly increased in the O-LPTH group(P>0.05),while the expression level of SIRT1 protein was not significantly affected(P>0.05).These experimental results indicate that VB1 regulates cellular energy metabolism by participating in the regulation of mitochondrial status.In addition,the regulation of inflammation and energy status by VB1 is related to the inhibition of the p38 MAPK/NFκB/AMPK pathway,but there are still potential targets of action.In summary,the exogenous supplementation of VB1 promotes the proliferation of VB1-synthesizing bacteria and inhibits the growth of VB1 enzyme-producing bacteria in the rumen,thereby maintaining normal fermentation function and improving the rumen environment.The improvement of the rumen environment reduces the pressure of LPS,organic acids,and lactic acid on the ruminal epithelial barrier function,which is one of the mechanisms by which VB1 protects the ruminal epithelial barrier function.At the same time,exogenous supplementation of VB1 reduces ruminal epithelial cell apoptosis,promotes cell proliferation,maintains the normal morphological structure and function of mitochondria,and these comprehensive improvements protect the ruminal epithelial barrier function through exogenous supplementation of VB1.In addition,VB1 suppresses p38 MAPK/NFκB/AMPK pathways to reduce cellular inflammatory damage,maintain normal energy metabolism status,and the relief of inflammation and stable energy status are important mechanisms for VB1 to protect the ruminal epithelial barrier,but there may still be some potential sites of action.