Study On Preparation And Immunity Enhancement Mechanism Of Acanthopanax Senticosus Polysaccharide Nanoparticles

Animal immunosuppressive diseases seriously affect the development of modern livestock and poultry breeding industry,and have a great impact on social development and human health.How to enhance the immunity of animals and improve the immune effect of vaccines has become an urgent problem to be solved.Acanthopanax senticousus polysaccharide(ASPS)is a polysaccharide extracted from the dry rhizome or bark of Acanthopanax senticosus by water extraction and alcohol precipitation.It has many biological activities,such as enhancing body resistance,anti-tumor,anti-oxidation,regulating blood lipid and so on.It can be used as an immunopotentiator.In veterinary clinic,Acanthopanax senticosus polysaccharide has the problems of fast metabolism and low bioavailability,which seriously limits its clinical application.1.Preparation and Characterization of Acanthopanax Senticosus Polysaccharide NanoemulsionAcanthopanax senticousus polysaccharide-nanoemulsion(ASPS-NE)was prepared in order to solve the problems of rapid metabolism,low bioavailability and difficult dissolution of some alkali-soluble polysaccharides in vivo.And were characterized.K-Car-ASPS was prepared as aqueous phase,ethanol as cosurfactant,Tween-80 and span-80 as surfactant,liquid paraffin and IPM as oil phase according to the nanoemulsion region in the pseudo-ternary phase diagram.The final ASPS-NE formulation was composed of 46.2%Tween-80,15.4%Span 80,7.6%ethanol,15.4%liquid paraffin,11.5%IPM and 3.9%ASPS aqueous solution.The results showed that ASPS-NE was a W/O nanoemulsion with good dispersion.The particle size of ASPS-NE was 35.82±11 nm,and the particle size distribution was uniform.The average content of ASPS in ASPS-NE was 25.06 mg/mL.The results of stability test showed that ASPS-NE had good stability.2.Safety Evaluation of Acanthopanax Senticosus Polysaccharide NanoemulsionIn order to evaluate the safety of ASPS-NE in animal clinical use,LD50 in mice and toxicity test in L929 cells were carried out in this study.The results showed that the LD50 of ASPS-NE was 1157 mg/kg while the LD50 of blank nanoemulsion was 1008.2 mg/kg.According to the international acute toxicity classification standard,ASPS-NE belonged to low toxicity drug;In addition,ASPS-NE was of grade 0 toxicity when the drug concentration was less than or equal to 31.25 μg/ml,indicating that ASPS-NE had low cytotoxicity and was safe for clinical use in veterinary medicine.3.Study on the Mechanism of Immune Enhancement of Acanthopanax Senticosus Polysaccharide NanoemulsionIn Order to study the immunopotentiating effect of ASPS-NE,cyclophosphamide was used to establish the immunosuppressive animal model in mice.The effects of ASPS-NE on the immune function of splenocytes and macrophages in immunosuppressed mice,the expression of cytokines in serum,the ratio of CD4+/CD8+in peripheral blood and the expression of P65/JNK signaling pathway related proteins in macrophages were investigated.The results showed that ASPS-NE reversed immunosuppression by enhancing the immune function of spleen and macrophages.In addition,ASPS-NE induces the secretion of various cytokines(IL-2,IL-6,TNF-α and IFN-γ)and participates in the immune regulation of TLR4-NF-κB signal transduction pathway.It was preliminarily revealed that ASPS-NE could activate P65/JNK/ikkα signaling pathway and regulate the ratio of Th1/Th2 to regulate the immune activity of macrophages in immunosuppressed mice4.Preparation and Quality Evaluation of Calcium Carbonate Nanoparticles Loaded with Acanthopanax Senticosus PolysaccharidesIn order to develop a new type of immune adjuvant,CaCO3,CaCO3-ASPS,CaCO3-ASPS-PEI and CaCO3-ASPS-PPV(PPV,Porcine Parvovirus)nanoparticles were prepared and characterized.The results showed that the nanoparticles were spherical,and the average particle size of CaCO3,CaCO3-ASPS and CaCO3-ASPS-PPV were 1381±39 nm,2286±54 nm and 2777±49 nm,respectively.PDI<0.3.The increase of Zeta potential can improve the stability of the solution.The Zeta potentials of CaCO3-ASPS nanoparticles and CaCO3-ASPS-PEI nanoparticles were-18.12±1.24 mV and 23.81±0.38 mV,respectively.The Zeta potential of CaCO3-ASPS-PPV nanoparticles was-5.12±0.69 mV,indicating that PPV was adsorbed on the surface of ASPS-CaCO3.Compared with CaCO3 and CaCO3-ASPS,there were visible particles on the surface of CaCO3-ASPS-PPV,indicating the successful adsorption of PPV.The surface of nanoparticles is rough,which is more conducive to the loading of ASPS.The stability test showed that the PDI and Zeta potential of CaCO3,CaCO3-ASPS and CaCO3-ASPS-PPV nanoparticles did not change significantly within 28 days,and the PDI was less than 0.3,indicating that the nanoparticles had uniform size distribution within 28 days.The AE(%)of CaCO3-ASPS-PPV was lower than that of CaCO3-ASPS,and the AE of PPV antigen in CaCO3-ASPS-PPV did not change significantly within 28 days.5.Regulation of autophagy pathway by calcium carbonate nanoparticles loaded with Acanthopanax senticosus polysaccharidesIn order to study the regulation of CaCO3 nanoparticles loaded with Acanthopanax senticosus polysaccharides on autophagy pathway,By stud that effect of CaCO3-ASPS-PEI on the protein expression of autophagy factors(LC3-Ⅰ/Ⅱ,ATG3,ATG5,ATG6/Beclin1,ATG7,ATG9,ATG12 and ATG14)in Raw264.7 cell,the effect of CaCO3-ASPS-PEI on the mRNA expression of the autophagy factors,The expressions of ATG6 and LC3-Ⅱ were detected by immunofluorescence,and the effects of ATG6 and LC3-Ⅱ on Raw264.7 autophagic flow were analyzed.The results showed that the expression of LC3-Ⅰ and LC3-Ⅱ was dose-dependent and time-dependent,and CaCO3-ASPS-PEI could up-regulate the expression of Atg5-Atg12,Atg6/Beclin1,Atg7 and Atg14.The data of real-time PCR showed that CaCO3-ASPS-PEI could up-regulate the expression levels of LC3,ATG3,ATG6/Beclin1,ATG9 and ATG12 in a dose-dependent manner.Immunofluorescence results showed that CaCO3-ASPS-PEI could significantly up-regulate the protein expression levels of ATG6/Beclin1 and LC3-Ⅱ,and CaCO3-ASPS-PEI could up-regulate the degradation rate of LC3.The results showed that CaCO3-ASPS-PEI could positively regulate the cellular immune response by promoting the autophagy level of Raw264.7 cells to enhance the immune response.6.Enhancement of Immune Response of PPV Vaccine by Calcium Carbonate Nano-adjuvant Loaded with Acanthopanax Senticosus PolysaccharideThe immune response of CaCO3-ASPS-PEI to PPV vaccine was investigated by measuring the activity of CaCO3-ASPS-PEI on macrophages,the expression of MHCⅡ and CD86,cytokines secreted by macrophages,PPV antigen-specific IgG antibodies,and cytokines in serum.The results showed that CaCO3-ASPS-PEI could significantly upregulate CD86 and MHCII,activate macrophages,and promote the production of TNF-α and IL-1β by macrophages.CaCO3-ASPS-PEI has a good activation effect on macrophages,suggesting that CaCO3-ASPS-PEI may be an effective way for macrophages to adsorb antigens and promote antigen uptake.In order to study the effect of CaCO3-ASPS-PEI adjuvant,mice were injected with CaCO3-ASPS-PPV adsorbed with PPV antigen.The results showed that CaCO3-ASPS-PPV could produce PPV-specific IgG and induce Th1/Th2 mixed immune response(dominated by Th1).Therefore,the developed CaCO3-ASPS-PEI drug delivery system can be better used to induce antiviral and anti-infectious disease-related immune responses.In conclusion,ASPS-NE and CaCO3-ASPS-PEI can be used as new immune adjuvants to enhance the immune function of animals.