Study On The Mechanism Of Inhibitory Effect Of Acacetin On Metabolic Enteritis And Protection Of Intestinal Tight Junction In Mice

High-energy and high-protein feed and lack of movement are two major characteristics of large-scale,high-cost modern animal husbandry,long-term High-energy and high-protein Diet can cause animal metabolic diseases such as fatty liver,hyperlipemia,poultry gout and Ketosis,which have become one of the important diseases endangering animal health,and directly affect the quality and safety of animal-derived food.High-energy diet and metabolic enteritis are seldom reported in animal breeding,and the effect of acacetin on intestinal tract is not reported.In this study,the mice were fed with high-fat diet and given intragastric administration of acacetin(AC)for 8W and established a mouse model of metabolic enteritis.the expression of SREBP2,LDLR,CYP7A1 and HMGCR in the liver of mice was detected to explore the mechanism of the effect of acaacetin on lipid metabolism in mice.The expression of NF-kappa B and TLR4 in intestine was detected by Westernblot,the contents of TNF-α,IL-6 and SIg A in intestine were detected by double antibody sandwich method,and the composition of intestinal microorganisms was detected by 16 s RNA gene sequencing,To study the inhibitory effect of acacerin on metabolic enteritis in mice and its possible mechanism,as well as the effect of AC on intestinal microorganisms in mice,and to analyze the correlation between intestinal inflammation and intestinal microbial composition.The activity of serum LDH was detected by enzymatic method,the activity of serum DAO was detected by rate method,and the expression of ZO-1 and Occludin gene and protein in intestine was detected by real-time fluorescence quantitative technique and Westblot technique.The effect of acacetin on intestinal tight junction in mice with metabolic enteritis was studied,and its correlation with intestinal microorganisms was analyzed.The injury model of intestinal epithelial cells in vitro was established by stimulating mouse intestinal epithelial cells MODE-K,with LPS.The expression of tight junction proteins ZO-1,Occludin,F-action,pMLC,p MYPT1 and MLCK was detected by fluorescence quantitative PCR,immunofluorescence and Westblot techniques to study the effect of sophorin on intestinal tight junction.Furthermore,the regulatory mechanism of acacetin on intestinal tight junction was studied under the conditions of AMPK activators and inhibitors.The results were as followed:1.Acacetin inhibited metaboliceAcacetin decreased the levels of serum TC,LDL-C and HDL-C in a dose-dependent manner,decreased the contents of TNF-α and IL-6 in intestin eand increased the content of SIg A in intestine of mice fed with high-fat and high-cholesterol diet in a dose-dependent manner,TC and LDL-C were positively correlated with TNF-α and IL-6,but negatively correlated with SIg A.HDL-C was negatively correlated with TNF-α and IL-6,and positively correlated with SIg A,indicating that the level of lipid was correlated with intestinal inflammation,and the curve fitting of AC concentration with TNF-α,IL-6 and SIg A was ideal,indicating that acacetin could inhibit intestinal inflammation induced by high fat diet,that is,acacetin could inhibit metabolic enteritis.2.Effects of acacetin on metabolic enteritis and intestinal tight junction in miceThe mechanism of acacetin inhibiting metabolic enteritis in mice: acacetin inhibited the expression of TLR4 and NF-κB65,thus reducing the levels of TNF-α and IL-6 in Intestine;Effects of acacetin on intestinal tightness in mice: acacetin upregulated the expression of intestinal ZO-1,Occludin m RNA and protein,and reduced intestinal permeability.3.Effects of acacetin on intestinal microflora in miceAcacetin upregulated Brucella,Lachnospiraceae_NK4A136,Alistipes,Tyzzerella,Ruminococcaceae_UCG-009 and downregulated Bacteroides.4.Protective effect of acacetinon intestinal epithelial tight junctionThe damage model of intestinal epithelial cells in vitro: After MODE-K was stimulated for24 h by different concentrations of LPS,the fluorescence yellow transmittance of MODE-K cells increased and the cell viability decreased with the increase of LPS concentration.After incubating with different concentrations of acacetin for 2 hours,and intestinal epithelial cells were stimulated with 100μg/m L LPS.It was found that the fluorescence yellow transmittance decreased and the cell activity increased with the increase of acacetin concentration.It is determined that the optimum concentration of LPS for cell injury model in vitro is 100μg/m L,and the best concentration of acacetin 10μM.Protective effect of acacetin on intestinal epithelial tight junction: immunofluorescence results showed that ZO-1 and Occludin were reticulated in MODE-K cells in control group,ZO-1and Occludin were discontinuous in MODE-K cells in LPS group,fluorescence signals were weakened,part of Occludin transferred to intracellular,ZO-1 and Occludin in MODE-K cells in AC group showed reticular distribution,which was similar to that of the control group.The Factin of MODE-K cells in Control group and AC group showed dot distribution at the edge of cell membrane,while F-actin in LPS group showed aggregated bundle distribution at the edge of cell membrane.The results of QPCR showed that the expression of Occludin gene in LPS group was significantly lower than that in control group and AC group.The expression of ZO-1 gene in LPS group was lower than that in control group and AC group.The West blot results showed that the expression of pMLC and p MYPT1 protein in LPS group was significantly higher than that in control and AC groups(p<0.05).The expression of MLCK protein in control group was significantly higher than that in control group and AC group(p< 0.05).4.Based on AMPK/pMLC signal pathway,the protective effect of AC on intestinal epithelial tight junction was studied.The effect of acacetin on p AMPK: West blot results showed that the expression of p AMPK in MODE-K cells treated with LPS was significantly lower than that in control group and AC group(p< 0.05),indicating that LPS stimulation inhibited the expression of p AMPK in MODE-K cells,while acacetin promoted the expression of p AMPK.Effects of AMPK on cell permeability and tight junction: QPCR results: after incubation with AMPK activator AICAR,the expression of tight junction proteins ZO-1 and occludin in MODE-K cells was significantly higher than that in LPS group.Compared with AC+LPS and AICAR+LPS,AC+AICAR+LPS significantly increased the expression of ZO-1 and occludin genes.The results of immunofluorescence showed that ZO-1 and Occludin proteins were normally distributed in the cell membrane after incubation with AMPK activator AICAR,and the fluorescence staining of ZO-1 protein almost disappeared and Occludin protein distributed sporadically on the cell membrane after incubation with AMPK inhibitor Compound C.Effect of AMPK on p MYPT1 and pMLC: QPCR results showed that the expression of p MYPT1 and pMLC genes in cells incubated with AICAR,an AMPK activator,was significantly lower than that in LPS group,and the expression of p MYPT1 and pMLC genes in cells incubated with AMPK inhibitor Compound C was significantly higher than that in LPS group(p<0.05).The results of Westblot showed that the protein expression of p MYPT1 and pMLC in AICAR group was significantly lower than that in LPS group(p<0.01).The expression of p MYPT1 and pMLC genes in cells incubated with AMPK inhibitor Compound C was not significantly different from that in LPS group.After co-incubation with AC+Compound C,p MYPT1 expression decreased significantly,but pMLC expression decreased,but the difference was not significant.Conclusion:1.In this study,the metabolic enteritis model was successfully established by feeding ICR with high-fat diet,body weight,serum TC,LDL-C,HDL-C and intestine TNF-α,IL-6,SIg A as detection indexes,and the best concentration of acacetin to inhibit metabolic enteritis was30mg/kg.2.Anti-inflammatory effect of acacetin downregulated the secretion of proinflammatory cytokines TNF-α and IL-6 through TLR4/NF-κB pathway,and anti-inflammatory effect by upregulating intestinal beneficial bacteria: Blautia,Lachnospiraceae_NK4A136_group,Alistipes、Ruminococcaceae_UCG-009,Tyzzerella,and downregulating Bacteroides.3.Acacetin enhanced the tight junction between intestinal epithelium by up-regulating the expression of ZO-1 and Occludin to reduce intestinal permeability;AC Enhancement of intestinal permeability in mice was associated with AC up-regulation of Blautia,Lachnospiraceae_NK4A136_group,Alistipes,Ruminococcaceae_UCG-009,Tyzzerella,and down-regulation of Bacteroides.5.Acacetin decreased the permeability of intestinal epithelial cells by up-regulating the expression of ZO-1,Occudin,and down-regulating the expression of pMLC and promoted F-actin deaggregation.6.Acacetin upregulated the expression of ZO-1 and Occludin through AMPK/p MYPT1/pMLC signal pathway and reduced the injury of intestinal epithelial tight junction induced by LPS.