Effects Of Protocatechuic Acid On Intestinal Barrier Function Of Animal

Protocatechuic acid(PCA,3,4-dihydroxybenzoic acid)is widely distributed in a variety of plants including traditional Chinese herbal medicines like Stenoloma chusanum(L.)Ching and Ilex chinensis Sims;and it has been well studied for its potential effects on antioxidantion,antibacterial,anti-cancer,anti-ulcer,anti-diabetes,antiageing,anti-fibrosis,antiviral and anti-inflammation.Our study investigated the inhibition and the antibactericidal activity of PCA on four different bacteria in vitro.And on this basis,we explored the regulatory effect of PCA on immune barrier function in mice as well as its underlying mechanism against S.typhimurium infection.Furthermore,we investigated the effect of PCA on immune function of RAW264.7 cells and its underlying mechanism.Finally,we also investigated the effect of PCA on growth performance,meat quality and intestinal health in Chinese Yellow broilers.The main results are as follows.1.The antibacterial activity of protocatechuic acid against common pathogensIn vitro,PCA significantly inhibited the growth of S.typhimurium,Cl.perfringens,E.coli and S.aureus and high concentration of PCA(more than 2mg/mL)exerted long-term inhibition of bacteria cell growth and significant bactericidal effect on the four tested strains.In addition,PCA significantly down-regulated the expression of virulence gene such as InvA,SopD,SipB,HilA and SsrB of S.typhimurium.In conclusion,PCA excerted inhibitory and bactericidal effects on S.typhimurium,CL perfringens,E.coli and S.aureus,and mediated the expression of virulence gene in S.typhimurium in vitro.2.The effect of protocatechuic acid on modulating intestinal barrier function,and its protective role against S.Typhimurium infection in miceExcept for control group,mice were administrated with PCA(50 mg/kg weight)or GM(80 mg/kg weight)in drinking water for 28 days.We found that oral administration of PCA with 50 mg/kg/day significantly increased the body weight of mice,with nonnal structure of liver,spleen,ileum,and colon;PCA significantly increased the spleen and liver index and slightly increased NO production as well as sIgA in ileum and colon of normal mice.Furthermore,the gene expression of intestinal barrier in colon was significantly increased by PCA treatment;PCA didn't affect microbiota diversity,but significantly up-regulated the relative abundance of Firmicutes and reduced relative abundance of Bacteroidetes,with Fimicutes(phylum),Lactobacillales(from order to genus),Desulfovibronales(from order to genus),and Lachnospiraceae(order)the most frequent microbiota.In conclusion,PCA was safe for mice.It improved the intestinal flora,and enhanced the intestinal tight junction,as well as the immune function in mice.After raising for 28 days,mice were infected with 4 × 10gCFU S.typhimurium.Results showed that compared with infected-mice,PCA effectively reduced the weight loss and mortality,reduced the number of S.typhimurium in the ileum and colon,significantly relieved the ileal and colonic mucosal injury and infiltration of inflammatory cell,significantly up-regulated the expression of intestinal tight junction genes,reduced the expression and secretion of proinflammatory cytokines,such as IFN-?,IL-12,TNF-a,in serum,ileum,colon,spleen and liver,and alleviated the increase of sIgA and IgG.Furthermore,PCA reduced the colonization of S.typhimurium in the liver and spleen of mice,and alleviated S.Typhimurium-induced splenomegaly and liver lesion.It also decreased caspase-1 activity and inhibited apoptosis caused by S.typhimurium.Moreover,PCA down-regulated the relative abundance of virulence genes of S.Typhimurium in vivo(cecum),and significantly up-regulated the relative abundance of Firmicutes and reduced relative abundance of Bacteroidetes in S.typhimurium-infected mice.It indicated that PCA enhanced intestinal tight junction and immune function,as well as improved the intestinal flora of S.typhimurium-infected mice,thereby alleviating the damage caused by S.Typhimurium infection,and effectively inhibiting the invasion and proliferation of S.typhimurium in mice.3.Protocatechuic acid had regulatory effect on the function of RAW264.7 macrophage-like cellsRAW 264.7 cells were treated with PC A at the concentration of 1?500 ?g/mL.CCK-8 and LDH assays showed that PCA in doses of up to 100 ?g/mL showed no cytotoxicity to RAW 264.7 celsl,and the ACP activity increased with the concentration of PCA from 5 to 100 ?g/mL,exhibting the highest activity at the concentration of 25 ?g/mL.25 ?g/mL PCA significantly increased the intracellular LDH activity and up-regulated the percentage of CD80,CD86 and MHCII expression.PCA treatment significantly increased the NO production as well as the tNOS activity(total NOS)in RAW264.7 cells,up-regulated mRNA expression of pro-inflammatory and anti-inflammatory cytokines genes.Furthermore,PCA markedly suppressed the expression of M2-polarization genes such as MR,and Fizz,and increased that of IL-1,IL-6,IL-12 and TGF-?.In addition,PCA significantly increased the uptake of FITC-dextran as well as extracellular number of E.coli and S.typhimurium,and also enhanced the bactericidal ability of RAW 264.7 cells to E.coli.These results together suggested PCA regulated the immune function of RAW264.7 cells and induced Ml-polorization instead of M2,enhancing bactericidal capacity and protective effect against pathogen infection.4.The effect of PCA on growth performance,meat quality and intestinal barrier function of Chinese yellow feathered broilersChinese Yellow feathered broilers were treated with 300mg/kg and 600mg/kg PCA for 52 days,and non-treated and antibiotics-traeted group(enramycin)used as control groups.Results showed that 300mg/kg PCA significantly increased BW,LW,CW and decreased F:G of broilers;PCA improved meat quality indicated with the increased a*value and decreased b*value at 24h after slaughter,and significantly decreased the activity of AKP and DAO in plasma.In addition,PCA significantly increased T-AOC,the activities of T-SOD,GST-ST and GSH-Px,and decreased XOD activity.PCA also enhanced IL-10 and TGF-? levels,and reduced IL-2 and IFN-?in jejunum and plasma.PCA down-regulated the relative expression of apoptosis-related genes in liver,and up-regulated gene expression of tight junction proteins in jejunum.Furthmore,PCA significantly changed a-diversity and structure of cecal microflora,with the increased relative abundance of Firmicutes,Antinobacteria and the decreased abundance of Bacteroidetes,Protebacteria.In conclusion,PCA improved the antioxidant capacity and intestinal immune function,as well as the intestinal flora diversity and structure,which was responsible for the improved growth performance and meat quality of broilers.