Study On The Intervention Effect And Its Mechanism Of Rutin On Inflammatory Injury Of Rumen Epithelial Cells Induced-by LPS

Subacute ruminal acidosis(SARA)is one of the common metabolic diseases in ruminant breeding.SARA can cause the release and translocation of harmful metabolites such as lipopolysaccharide(LPS)into the blood,leading to local or systemic inflammatory response,which has many negative effects on the animal body,such as exacerbating the damage of rumen barrier function.Rutin is a natural flavonoid compound with various biological activities,such as anti-oxidant,anti-inflammatory,and anti-bacterial.However,the intervention effect of rutin on rumen inflammation has not been reported,and the intervention mechanism in rumen epithelial cell inflammatory injury has not been determined.Therefore,in this study,rumen epithelial cells of sheep were selected as the research object.LPS,the main harmful substance produced by SARA,was used as a stimulus to establish an inflammatory injury model of rumen epithelial cells The intervention effect of rutin on LPS-induced inflammatory injury of rumen epithelial cells and its molecular mechanism were explored,aiming to provide a theoretical foundation for the application of rutin as a feed additive to alleviate metabolic disorder disease such as rumen epithelial inflammation in ruminants fed with high concentrate diet in ruminant production.This paper is divided into four experiments,as follows:Experiment 1:Primary isolation and identification of sheep rumen epithelial cellsIn this experiment,fresh rumen tissues of healthy sheep were collected,and rumen epithelial cells were successfully isolated by continuous digestion of rumen epithelial mucosal layer with 0.25%-0.02%EDTA trypsin for different time(30,20,15,10,8,5 min).Under the microscope,the cells were digested and passaged after 80%of cells adherenced to the wall.CK-18 immunofluorescence was used The cytoplasmic fluorescence showed green,and the DAPI staining nucleus showed blue fluorescence,indicating that the epithelial cell was a rumen epithelial cell.In this experiment,MTT was used to detect cell viability.The results showed that the first day was the growth latency,from the second to sixth days,it began to enter the logarithmic growth phase,and followed by the growth recession phase.The optimal inoculation density of cells was 1×10~6/m L.Experiment 2:Establishment of rumen epithelial cells inflammatory damage modelThe single factor experimental design was used to stimulate the rumen epithelial cells with 0,1,5,10,50,and 100μg/m L LPS for 0,1,3,6,9,and 18 h.The cell viability,the content of inflammatory factors and their relative expression levels were determined to screen the optimal concentration and optimal action time of LPS and establish an inflammatory injury model.The results showed as follows:(1)Compared with the CON group,LPS stimulated rumen epithelial cells for 6 h,the cell viability was significantly inhibited by each concentration(P<0.05),but cell viability remained above 0.8.At 9 and 18h,the cell viability was significantly inhibited by each concentration(P<0.01),but the cell viability decreased to 0.35 at 18 h,which was not conducive to the subsequent experiments;(2)Compared with the CON group,LPS treatment with 1,5,10,50μg/m L significantly increased the content of TNF-αand the m RNA expressions of IL-1β,IL-8,IL-12 and TNF-αin the cell supernatant(P<0.01),significantly increased the content of IL-1βand the m RNA expression of IL-6(P<0.05),and significantly reduced the content of L-10 and the m RNA expressions of IL-4 and IL-10(P<0.01),indicating that the selected contents of LPS could cause inflammatory reaction in rumen epithelial cells.Based on the above experimental results,the inflammatory damage model of rumen epithelial cells was established at the LPS concentration of 1μg/m L and the action time of 9 h.Experiment 3:Intervention effect of rutin on inflammatory damage of rumen epithelial cellsIn this experiment,single factor experimental design was used to stimulate rumen epithelial cells with 0,25,50,100,150μg/m L rutin for 0,2,4,6,8 and 10 h.adding different concentrations of rutin(0,25,50,100μg/m L)on the basis of the aforementioned rumen epithelial cell inflammatory damage model,the relative proliferation rate of cells was calculated,and the optimal intervention concentration and time of rutin were screened.The results showed as follows:(1)Compared with the CON group,rutin stimulation for 8 h significantly increased the activity of rumen epithelial cells(P<0.05),and rutin stimulation for 10 h significantly inhibited the activity of rumen epithelial cells(P<0.01);(2)100μg/m L rutin could maximally alleviate LPS-induced damage to rumen epithelial cells with the relative cells proliferation rate of 70%.Therefore,the optimal stimulation conditions of rutin were determined to be a concentration of 100μg/m L for 8 h.Based on the above research,the second generation rumen epithelial cells were randomly divided into control group(CON group,without any addition),LPS damage group(LPS group,1μg/m L,9 h),rutin group(R group,100μg/m L,8 h)and rutin intervention group(R+LPS group,rutin 100μg/m L,8h+LPS 1μg/m L,9 h).The gene expression of cytokines,regulatory factors,tight junction protein and cytokine content,tight junction protein expression were measured.The results showed as follows:(1)Compared with the CON group,LPS stimulation of sheep rumen epithelial cells could significantly increase the m RNA expression of IL-1β,IL-6,IL-12,TNF-α,INF-γ,CXCL8,CXCL9,TLR-4,My D88,NF-κB,IFIT3 and TRAF6(P<0.01),significantly increase the content of IL-1βand TNF-α(P<0.05),and significantly down-regulate the m RNA expression of Claudin-4 and ZO-1 and the content of IL-10(P<0.05).The protein expression levels of Claudin1,Claudin4 and Occludin were significantly down-regulated(P<0.01);(2)Compared with the CON group,Rutin could significantly reduce the m RNA expressions of IL-1β,IL-12,TNF-α,INF-γ,CXCL9,TLR-4,My D88,NF-κB,IFIT3,IRAK1 and TRAF6(P<0.01),significantly reduce the content of TNF-α(P<0.05),significantly increase the content of IL-10(P<0.01),and significantly increase the m RNA expressions of Claudin-1,Claudin-4,Occludin and ZO-1(P<0.05).The protein expression levels of Claudin-1,Claudin-4,Occludin and ZO-1 were significantly up-regulated(P<0.01);(3)Compared with LPS group,R+LPS group could significantly reduce the m RNA expressions of IL-1β,IL-6,IL-12,TNF-α,INF-γ,CXCL8,CXCL9,My D88,NF-κB,IFIT3,IRAK1,IRF3,TRAF6 and the content of TNF-α(P<0.01),significantly up-regulate the m RNA expressions of IL-4,IL-10,ZO-1,Claudin4,andthe protein expressions of Claudin1,Claudin4,Occludin,ZO1 and the content of IL-10(P<0.01),and significantly decrease the content of IL-1β(P<0.05).The above results that rutin could alleviate the inflammation caused by LPS on rumen epithelial cells,as well as the damage of intercellular tight junctions.It could also significantly enhance the anti-inflammatory ability of rumen epithelial cells and improve the barrier function of epithelial cells.Experiment 4:Molecular mechanism of rutin intervention in inflammatory damage of rumen epithelial cellsBased on the above experiments,this study used a single factor experimental design to divide the second generation cells into 8 groups:control group(CON group,without any addition),LPS damage group(LPS group,1μg/m L,9 h),rutin group(R group,100μg/m L,8 h),rutin intervention group(R+LPS group,rutin 100μg/m L,8 h+LPS 1μg/m L,9 h),inhibitor group(PDTC group,10μM,1 h),inhibitor LPS group(PDTC+LPS group,PDTC 10μM,1 h+LPS 1μg/m L,9 h),inhibitor rutin group(PDTC+R group,PDTC 10μM,1 h+rutin 100μg/m L,8 h),inhibitor rutin intervention group(PDTC+R+LPS group,PDTC 10μM,1 h+rutin 100μg/m L,8 h+LPS LPS 1μg/m L,9 h).The results showed as follows:(1)Compared with the CON group,PDTC group significantly reduced the m RNA expressions of IL-1β,IL-6,IL-12,TNF-α,INF-γ,CXCL8,TLR-4,My D88,NF-κB,IFIT3,IRAK1,IRF3,TRAF6 and the protein expressions of P-IκBα,P-NF-κB65,P-P38MAPK,P-JNK,P-ERK1/2,P-ERK5(P<0.01);(2)Compared with LPS group,PDTC+LPS group could significantly down-regulate the m RNA expressions of IL-1β,IL-12,TNF-α,INF-γ,CXCL8,CXCL9,TLR-4,My D88,NF-κB,IFIT3,IRAK1,IRF3,TRAF6and the protein expression of P-IκBα,P-NF-κB65,IκBα,NF-κB65,P-P38 MAPK,P-JNK,P-ERK1/2,P-ERK5(P<0.01),significantly increase the m RNA expressions of IL-4 and IL-10(P<0.01);(3)Compared with LPS group,the protein expressions of P-IκBα,P-NF-κB65,IκBα,NF-κB65,P-P38MAPK,P-JNK,P-ERK1/2 and P-ERK5 were significantly decreased in R+LPS group(P<0.01).Overall,the results indicated that rutin could exert anti-inflammatory activity by regulating NF-κB/MAPK signaling pathway.In summary,rutin alleviated LPS-induced inflammatory damage of sheep rumen epithelial cells mainly by acting on the NF-κB/MAPK signaling pathway,reduced the expression and secretion of inflammatory cytokines and cell regulatory factorrelated genes,increased the expression levels of tight junction proteins,enhanced the body’s anti-inflammatory activity,and protectd the integrity of rumen epithelial cell barrier function.