Protective Effects And The Underlying Mechanisms Of Viola Yedoensis Makino On Intestinal Injury Induced By Lipopolysaccharide In Broiler

Background: The immune and digestive systems are immature in the early stage of hatching,and how to improve the immune function of the body and to maintain the intestinal health in the chickens,is extremely important to resist the invasion of external pathogens.Viola Yedoensis Makino(Vy),a traditional Chinese herbal medicine,exerts immunomodulatory,antioxidant and anti-inflammatory effects as well as regulation of microbial flora in animal models,which indicates that Vy may have a promising clinical application in the animal farming.However,to date,no studies have assessed the effects of Vy and possible mechanisms involved in the regulation of intestinal health in broiler chickens.Objective: The model of intestinal damage in broiler chickens was induced by intraperitoneal injection of lipopolysaccharide(LPS)and Vy was supplemented in broilers’ diet.The effects of Vy on the intestinal damage as well as the potential mechanisms was explored in broiler chickens.This research is expected to provide a new intervention method for maintaining intestinal health,and lay a scientific and theoretical basis for Vy as a feed additive.Methods: A total of 50 1-day-old AA male broilers were divided into 5 groups.The control group and the intestinal injury group were fed the basal diet,and the other three groups were fed the diet supplemented with 0.5%,1.5%,and 4.5% of Vy.Intestinal injury was induced by intraperitoneal injection of LPS(1 mg/kg.BW)on the day 17,day 19 and day 21,and the control group was injected with an equal dose of normal saline(1 mg/kg.BW).The growth performance of broilers was monitored throughout the whole process,and the jejunum,ileum,and cecum contents were collected after slaughtering of broilers on the day 21.In order to explore the protective effect of Vy on LPS-induced intestinal injury,the morphological changes of jejunum and ileum were observed by H&E staining,the ultrastructure of jejunum was observed by electron transmission microscope,and the apoptosis rate of jejunum was detected by TUNEL assay.Moreover,the redox system was assayed by kits,and the m RNAs expression of nuclear factor E2-related factor 2(Nrf2)/mitogen-activated protein kinase(MAPK)signaling pathways were detected by RT-q PCR,which to investigate the antioxidative effect and regulatory mechanism of Vy on LPS-induced intestinal injury.Meanwhile,the levels of inflammatory cytokines and inflammatory mediators as well as the nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3(NOD-like receptor protein 3,NLRP3)signaling pathway-related m RNA expression were detected by RT-q PCR,which to explore the anti-inflammatory effect and the regulatory mechanism of Vy on LPS-induced intestinal injury.Furthermore,the effect of Vy on the microflora of broilers was clarified by detecting the microbial diversity of cecal contents.Results:(1)Supplement with Vy alleviated the reduction in growth performance,shortening of villus length,deepening of crypt depth,mitochondrial injury and cell apoptosis in jejunum of broilers caused by LPS,indicating that Vy had a protective effect on LPS-induced intestinal injury.(2)Supplement with Vy exerted antioxidative property by down-regulating malondialdehyde(MDA)content in jejunum and ileum,up-regulating superoxide dismutase(SOD)activity in ileum,and compensatively decreasing m RNA expression of antioxidant enzymes such as superoxide dismutase 1(SOD1),glutathione peroxidase 7(GSH-PX7),catalase(CAT)and NAD(P)H:quinone oxidoreductase 1(NQO1)in jejunum and ileum.In addition,Vy inhibited LPS-induced elevation of Nrf2 and Heme Oxygenase-1(HO-1)m RNA expression in jejunum,and enhanced m RNA expression of mitogen-activated protein kinase 9(MAPK9),c-Jun N-terminal kinase(JNK)and proto-oncogene adaptor protein(CRK).But Vy didn’t change the expression of Nrf2 protein by immunohistochemical assay,suggesting that Vy might relieve LPS-induced oxidative stress by regulating Nrf2/MAPK signaling pathways.(3)Supplement with 1.5% Vy significantly down-regulated m RNA levels of Interleukin-1β(IL-1β),IL-8 and IL-18 in jejunum,while supplement with Vy could inhibit the m RNA expression IL-1β,IL-8 and IL-18 in ileum and upregulate IL-10 m RNA expression in jejunum and ileum of LPS-treated broilers.These results suggested that Vy alleviated LPS-induced intestinal inflammation.Besides,RT-PCR results indicated that Vy supplement up-regulated m RNA expressions of Toll-like receptor 4(TLR4)and NF-κB in jejunum and ileum.At the same time,immunohistochemical results showed that Vy pretreatment reduced NLRP3 protein expression in jejunum.Theses finding implied that Vy prevented LPS-induced intestinal inflammation in broilers through regulating NF-κB/NLRP3 signaling pathways.(4)Vy pretreatment reduced the Chao index of alpha diversity in cecal microbe,and the composition of groups supplemented with Vy has a higher similarity by analyzing the β diversity.It was found that Vy increased the intestinal beneficial bacteria(Lactobacillus,Lachnospiraceae and Romboutsia)and decreased the pathogenic bacteria().The results demonstrated that Vy alleviates LPS-induced intestinal injury by improving the intestinal microbiota.Conclusion: Vy has a protective effect on LPS-induced broiler intestinal injury by restoring the balance of the intestinal redox system,regulating the secretion of inflammatory cytokines,inhibiting excessive apoptosis through regulation of the Nrf2/MAPK and NF-κB/NLRP3 signaling pathways.Meanwhile,Vy improved the intestinal microbiota by increasing beneficial bacteria(Lactobacillus,Lachnospiraceae and Romboutsia),and reducing the pathogenic bacteria.