Ginsenoside Rg1 Enhanced Immune Responses To Infectious Bursal Disease Vaccine In Chickens With Oxidative Stress

Ginseng stem and leaf total saponins(GSLS)are saponins extracted from the stems and leaves of Panax ginseng C.A.Mey.Previous studies have shown that oral GSLS can enhance the humoral and cellular immune responses to Newcastle disease virus(NDV)and infectious bursal disease virus(IBDV)vaccines,as well as alleviate oxidative stress induced by cyclophosphamide(Cy).Ginseng stem and leaf saponins have been approved by the Ministry of Agriculture and Rural Areas as new veterinary drugs((2017)New Veterinary Medicine Certificate No.20)on the market.GSLS contains more than 30 monomers,of which Rgl,Re,Rc,Rd,Rbl and Rb2 are listed in the Veterinary Pharmacopoeia of the People's Republic of China in 2015 as components for quality control.In order to effectively control the quality of veterinary drugs with GSLS and provide basis for the clinical application of GSLS,the effects of the six monomers on intestinal mucosal immunity of chickens were studied in this paper.It was found that Rgl could significantly enhance mucosal immunity.On this basis,the effects of oral Rgl on immune response to vaccine in chickens were studied.The results can provide a basis for the clinical application of GSLS,and is helpful for exploring its mechanism of action.In this study we attempted to explore the effect of ginsenosides on intestinal mucosal immunity of chickens.192 specific-pathogen-free chickens were randomly divided into 8 groups according to body weight gradient,each consisting of 24 female animals.After 2 weeks of acclimatization,ginsenosides of same concentration was added into drinking water respectively.Before administration and 1,2,3 weeks after administration,the intestinal lavage solution was collected for detecting the total sIgA.Duodenum tissues were sampled and fixed immediately in 4%(v/v)neutral buffered formalin.Tissue sections were prepared and the interepithelial lymphocytes(IELs)in duodenum were observed and counted under a microscope.Results showed that oral administration of 1 mg/kg BW of Rgl,Rd or 5 mg/kg BW of GSLS could significantly enhance the total sIgA level in intestinal contents and increase the number of IELs in chicken duodenum.As one of the main components in GSLS,Rgl was used to evaluate its effect on immune responses to infectious bursal disease virus(IBDV)vaccine in chickens of oxidative stress.96 chickens were randomly divided into 4 groups,each consisting of 24 birds.Groups 2 and 3 received intramuscular injection of Cy at 100 mg/kg body weight for 3 d to induce oxidative stress and immune suppression.Groups 1 and 4 were injected with saline in the same way as groups 2 and 3.Then chickens in group 3 were orally administrated Rgl of 1 mg/kg BW in drinking water for 7 d.After that,groups 1 to 3 were orally vaccinated with attenuated infectious bursal disease(IBD)vaccine(Strain B87).Blood samples were collected from chickens before Cy treatment and 7,14,21 d after immunization for determination of IBDV specific antibodies,cytokines and antioxidative parameters.Splenocytes were prepared for lymphocyte proliferative assay.The results showed that Rgl significantly enhanced serum specific antibody,IFN-y and IL-6 responses,and lymphocyte proliferation induced by concanavalin A and lipopolysaccharides compared with the Cy group.Antioxidant activity of Rgl was also observed in chickens by increasing total antioxidant capacity,total superoxide dismutase,catalase,glutathione peroxidase,glutathione,ascorbic acid,and a-tocopherol,as well as decreasing malondialdehyde and protein carbonyl.We further observed the effects of ginsenoside Rgl on intestinal mucosal immunity and immune-related mRNA expression in the duodenum of chickens.After sacrifying,duodenal lavage fluid of chickens was collected to detect total sIgA and specific slgA content.Duodenal tissue was collected to observe the number of intraepithelial lymphocytes(IELs)and IgA+ cells in the lamina propria.The total RNA of duodenum was extracted first week after immunization,and the expression of mRNA in intestinal tissue was detected by RT-qPCR.The results showed that oral administration of 1 mg/kg ginsenoside Rgl could significantly increase intestinal total sIgA and specific sIgA content compared with the Cy group.In addition,oral administration of Rgl significantly increased the number of IELs and IgA+ cells in the chicken duodenum.Results from RT-qPCR showed that oral administration of Rg1 up-regulated the mRNA expression of TLR4,p65,TGF-beta,pIgR and CCR9 genes in the duodenum of chickens.The present study was designed to investigate the protective effects and potential mechanisms of Rgl on H2O2-induced oxidative stress in chicken splenic lymphocytes.ROS,MDA and SOD in cells were used to evaluate the protective effect of Rg1 on oxidative stress.Cell viability,cell apoptosis,mitochondrial transmembrane potential and[Ca2+]i were detected in this experiment.Transcriptome analysis and quantitative real-time polymerase chain reaction were used to estimate the effect of Rg1 on gene expression of the cells.The results showed that pre-treatment with Rg1 dramatically enhanced cell viability,reduced apoptotic cells,increased SOD activity and decreased ROS and MDA content,when compared with the Model.In addition,treatment ofRg1 reversed[Ca2+]i overload and restored mitochondrial transmembrane potential.Transcriptome analysis showed that 323 genes were down-regulated and 105 genes were up-regulated in Rgl-treated cells.The differentially expressed genes were involved in GO terms such as regulation of protein serine/threonine kinase activity,protein kinase binding,tumor necrosis factor receptor binding and KEGG pathways such as Toll-like receptors,PPARs signaling pathway and cytokine-cytokine receptor interaction.In summary,Rg1 could enhance intestinal mucosal immunity in chickens.Rg1 increased the immune responses of chickens to IBDV vaccine under oxidative stress,displayed by enhanced specific antibody titers and cytokines concentration in serum,and increased the stimulation index of lymphocyte to ConA and LPS.Meanwhile,the antioxidant parameters of serum in Rgl group is better than that in Cy group,which is manifested by the elevated antioxidant enzymes and decreased oxidative stress.Oral administration of Rg1 enhanced intestinal mucosal immunity.Rg1 increased sIgA concentration,number of IgA+ cells and IELs in duodenum.Compared with Cy group,TLR4,NF-?B,pIgR,TGF-? and CCR9 in duodenum tissue of Rg1 group were up-regulated.Rg1 could effectively protect chicken spleen lymphocytes against oxidative stress,indicated by reduced cell apoptosis,restored mitochondrial membrane potential and alleviated calcium overload.By transcriptome sequencing,we identified 428 differentially expressed genes,which were involved in Toll-like receptors,PPARs signaling pathway and cytokine-cytokine receptor interaction.It is speculated that the effect of Rgl on enhancing the immune response of chickens under oxidative stress may be by alleviating oxidative damage and reduceing apoptosis of lymphocyte.