Study On Immunological Enhancement Activity Of Rehmannia Glutinosa Liposome And Its Mechanism

So far,vaccination remains one of the most effective tolls to stimulate protective immune responses against infectious diseases.Compared to traditional materials,nanomaterials can target deliver antigen,protect the antigen,minimize the systemic toxicity.So more and more nano-range adjuvants have been developed.Nanomaterials are demonstrated to provided adjuvant activity by enhancing the delivery of antigen to the immune system or by potentiating innate and/or adaptive immune responses.Liposome is the only nano drug carrier that approved by FDA.It is composed of a layer or multilayer phospholipid molecules around the core of an aqueous phase and form synthetic vesicles.One or more drugs or antigen can be encapsulated inside or conjugated on its membrance.Liposome as a novel drug delivery system and adjuvant,it holds a significant potential for the development of of novel immunomodulatory agents as easily they are taken up by antigen presenting cells.Liposome can also transport drugs for site-specific delivery to enhance efficacy and bioavailability,sustain slow-release,decrease adverse reactions and improve patient compliance.In the past few decades,researching on liposome as adjuvant is a hot spot.In this research,RGP was encapsulated in the liposome using the reverse-phase evaporation method combined with sonication.Experiments in vivo and vitro were done to determine the immunological enhancement activity of RGPL.The effects of RGPL on animal lymphocyte proliferation,antibody titers,cytokines in vivo and that on mouse peritoneal macrophages and bone marrow-derived dendritic cells in mice(BMDCs)in vitro were investigated.The details were divided into eight parts as follows:Experiment 1 Effect of RGPL on lymphocyte proliferation and differentiation Experiments in vitro showed that RGPL could successfully strengthen immunity.In a single stimulation of drugs,RGPL could significantly promote splenocyte proliferation,specifically at 200 ?g·mL-1.Moreover,in a synergistic stimulation of drugs with LPS or PHA,a significant difference was obtained between the RGPL and RGP at 100-400?g·mL-1,which indicated that the immunological enhancement of RGP was significantly improved after encapsulation with the liposome.Furthermore,RGPL significantly upregulated the ratio of CD4/CD8,which also indicated that the immunological enhancement effect of RGP was improved after being encapsulated with liposome.Experiment 2 Effects of RGPL on mouse peritoneal macrophage The purpose of the study was to investigate effects of RGPL on the activity of mouse peritoneal macrophage.Peritoneal macrophageswere obtained from ICR mice and stimulatedwithRGPL,RGP,BL and LPS respectively,and serum-free RPMI1640 medium were added as blank control.The levels of IL-6,IFN-?,IL-12,IL-1? and TNF-? production in cell culture supernatant were analyzed by ELISA.The phagocytic activity was assessed via flow cytometry.The results showed that RGPL could significantly enhance the phagocytic activity and upregulation the IL-1?,IFN-?,IL-6,IL-12 and TNF-?production.Experiment 3 Effects of RGPL on dendritic cells The purpose of the study was to investigate effects of RGPL on DCs proliferation,maturation and function.Bone marrow DCs were obtained from mouse bone marrow precursors.Effects of RGPL on primary bone marrow DCs proliferation were detected by MTT method.After 7 days of cultivation,effects of RGPL on DCs stimulating on proliferation of T cells and the ability of antigen presenting of DCs were also detected by MTT method.Besides,effects of RGPL on the expression of DC phenotype were tested by flow analysis.The results indicated that RGPL could significantly promote DCs precursor proliferation,mature,stimulating proliferation of T cells and antigen presentation of DCs.Experiment 4 Stability of rehmannia glutinosa polysaccharide liposome conjugated with different antigen The aim of this study is to investigate the size and PDI changes of liposome conjugated with different antigen both in 4? and 37?.The release profile of OVA inside the liposome was determined.Our result showed that RGPL conjugated with different antigen possess good stability,and also showed controlled-release of antigen.It provided possibility and laid a good foundation for further investigation.Experiment 5 Effects of RGPL on immune response of OVA-immuned mice The purpose of the study was to investigate effects of RGPL on immune response of OVA-immuned mice.168 between 4-week-old ICR mice were divided randomly into 7 groups.They were immunized with RGPL(4.0,2.0,1.0 mg·mL-1)+OVA,RGP(4.0 mg·mL-1)+ OVA,BL+ OVA,respectively.There were also freund's adjuvant and blank control groups.The second immunization was on 6-week-old.On the 7th,14th,21th,28th,35th and 42th day after the first vaccination,blood samples were collected from retro-orbital venous plexus in order to measure the concentration of IgG,IL-4 and IFN-? with ELISA.Four mice were sacrificed each group to separate splenic lymphocytes.The proliferation of lymphocytes was determinated by MTT method.From the results,RGPL could not only significantly increase the concentration of IgG,IL-4 and IFN-?,but also significantly promotesplenic lymphocytes proliferation.Furthermore,RGPL could effectively promote the generation of immune memory,activate DCs in LNs,and help antigen uptake to LNs.These results also indicated that RGPL could significantly improve the immunological enhancement of RGP.Experiment 6 Effects of RGPL on immune response of PCV-2-immuned mice The aim of the study was to investigate effects of RGPL on immune response of PCV-2-immuned mice.252 between 4-week-old ICR mice were divided randomly into 7 groups.They were immunized with RGPL+PCV-2,RGP+ PCV-2,BL+ PCV-2,respectively.There were also oil adjuvant and blank control groups.The second immunization was on 6-week-old.On the 1st,2nd,3rd week after the second vaccination,blood samples were collected from retro-orbital venous plexus in order to measure the concentration of IgG,IgG1,IgG2a,IgG2b,IgG3,IL-4,IL-17,TNF-? and IFN-?.From the results,RGPL could significantly induce IgG response,promote Thl and Th2 related IgG subtypes and cytokines.In all,RGPL has the protential to be developed as a effective adjuvant.Experiment 7 Effects of RGPL on DCs though TLR pathway The purpose of the study was to investigate the mechanism of RGPL's immunological function.TLR2 and TLR4 were both participated in immunological regulation,and also participate in polysaccharides on the regulation of antigen presenting cells.In the experiment,DCs were treated with RGPL,and then mRNA expression level of the molecular in the pathway were tested.According to the results,RGP was mainly depended on TLR4.While RGPL was depended on both TLR4 and TLR2.In the following experiment,TLR4 pathway was the main research object.DCs was separated from MyD88 knockout mice,and then NF-?B protein level was compared after treated with RGPL.The result indicated that,RGPL mainly depended on TLR4/MyD88/NF-?B pathway to realized its adjuvanticity.Experiment 8 Effects of RGPL on immune response of TLR4 knockout mice The purpose of the study was to investigate the effects of RGPL on TLR4 knockout mice.18 mice were divided randomly into 6 groups.They were immunized with RGPL+OVA,RGP + OVA,BL+ OVA,respectively.There were also alum adjuvant and blank control groups.The second immunization was on 6-week-old.On the 2nd week after the second vaccination,blood samples were collected from retro-orbital venous plexus in order to measure the concentration of IgG and its subtypes,IL-4 and IFN-y.The result showed no significant increase of IgG and its subtypes,and cytokines,which indicated that the immunological enhancement of RGPL could not realized in TLR4 knockout mice.