| Cubosomes（Cubs）are a new type of drug carrier.Given their dual-continuous channel structure,they have a huge advantage in the process of entrapment and transportation of insoluble macromolecular drugs in the body.Eucommia ulmoides is a scarce species owing to the scarcity of resources and because of its nourishment.Since ancient times,it has been regarded as a precious Chinese medicinal plant.According to the"Shen Nong’s Materia Medica,"E.ulmoides can replenish the vital energy and strengthen the bones and muscles.Plant polysaccharides have anti-oxidative,anti-tumor and immune improvement functions.Particularly,at the level of body immunity,immune cells have multiple polysaccharide receptors;accordingly,plant polysaccharides can improve the activity of immune cells and enable them to play a better role in the immune response process.At present,there is no relevant report on the immune activity of E.ulmoides leaf polysaccharide（PsEUL）in combination with Cubs.In this study,we use ultrasonic hot water to extract PsEUL and couple PsEUL with ovalbumin（OVA）through chemical bonds via the 1-（3-Dimethylaminopropyl）-3-ethylcarbodiimide hydrochloride（EDC）condensation method.Further,we used phytantriol as the amphiphilic lipid substance to prepare PsEUL-OVA/Cubs,and conducted in vivo and in vitro tests on PsEUL-OVA and PsEUL-OVA/Cubs,respectively,to explore their immunological activity with an aim to provide a theoretical basis and data support for the development of plant polysaccharide-modified antigens and new vaccine adjuvants.The main methods,results and conclusions of this test have been reported below.1.Extraction and optimization of PsEUL.In this experiment,sonic water was used to extract PsEUL.Fourier infrared spectroscopy was used to identify extracted PsEUL,and the extracted PsEUL was decolorized and optimized using macroporous resin.The optimized The sugar content of PsEUL was determined by the phenol-sulfuric acid method.On infrared spectroscopy,the extracted PsEUL showed typical polysaccharide absorption peaks.PsEUL is identified as an acidic polysaccharide containing pyranose and furan ring;the extraction rate of PsEUL via ultrasonic water extraction was calculated by the phenol-sulfuric acid method,and the rate was 5.7%;after obtaining PsEUL,the crude polysaccharides were decolorized in two ways.When a macroporous resin was used as the decolorized material and ethanol as the eluent,the decolorization rate and polysaccharide retention rate with different concentrations of ethanol are not exhausted.The decolorization rate and polysaccharide retention rate for 5%ethanol was found to be the best.2.Preparation and characterization of PsEUL-OVA.PsEUL was reacted with OVA in EDC to obtain the coupling product of PsEUL and OVA（PsEUL-OVA）.PsEUL-OVA was further separated and purified by column chromatography.Subsequently,Fourier infrared spectroscopy,sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE）,scanning electron microscopy（SEM）,and BCA protein assay were performed for characterization.PsEUL-OVA was separated on the chromatographic column of Sephadex G-150 and UV absorption value of the eluent was recorded to further obtain purified PsEUL-OVA;PsEUL and PsEUL-OVA were then scanned by infrared spectroscopy separately.The infrared spectrum of PsEUL-OVA showed the corresponding C=O stretching vibration peak on adipic hydrazide at 1580 cm-1,indicating that PsEUL has successfully passed the covalent bond-CO-,and NH-was found to couple with OVA;OVA and PsEUL-OVA were then detected by SDS-PAGE.Compared with the band of OVA,the band of PsEUL-OVA was seen to have moved up to the compression gel portion,indicating an increase in the molecular weight and the successful coupling of OVA and PsEUL.The structure of PsEUL and PsEUL-OVA can be observed through SEM images,and it shows obvious changes.The PsEUL-OVA image shows adhesion of molecules,indicating that OVA and PsEUL have successfully combined together.As per the BCA method and phenol-sulfuric acid method,the calculated conversion rate of PsEUL-OVA（%）was 46.25%±4.2%;the ratio of polysaccharide to protein was 48.1%±6.8%.3.Immune activity of PsEUL-OVA in vivo and in vitro.To explore the immunological activity of PsEUL-OVA,we explored the activity of PsEUL-OVA in spleen cells and macrophages in vitro following the CCK-8 method,and detected the cytokine secretion level of PsEUL-OVA in spleen cells and macrophages.Using fluorescein isothiocyanate（FITC）as a fluorescent label,the phagocytosis of FITC-labeled PsEUL-OVA by macrophages was observed under a laser confocal microscope;For the in vivo experiment,90 ICR mice were divided into six groups:blank group:normal saline group;control group:PsEUL group,OVA group,and PsEUL and OVA mixed group;PsEUL-OVA group;and positive control group:Freund’s adjuvant（FCA）group.In the control group,flow cytometry was used to detect the expression of CD4+and CD8+in mouse spleen cells and the phenotypic changes in the surface maturation markers CD80,CD86,and MHC-II in dendritic cells（DCs）;the spleen,thymus,and serum were collected for evaluating these parameters.The effects of PsEUL-OVA on mouse immune organ index,including serum cytokine IFN-γ,IL-2,IL-4,and IL-6 concentration and OVA-specific IgGs and their subclasses（IgG1,IgG2a,and IgG2b）were evaluated.The results showed that when the concentration of PsEUL-OVA was 200μg/m L,the activities of mouse spleen cells and macrophages were significantly higher than those observed in the other groups（P<0.05）.Thus,PsEUL-OVA can increase the secretion levels of cytokines IFN-γ,IL-2,IL-4,and IL-6 in lymphocytes and macrophages;further,an obvious phagocytic effect of macrophages was observed in the PsEUL-OVA group by laser copolymerization microscopy compared with other groups;as per in vivo test results in mice,PsEUL-OVA can significantly improve the immune organ index of mice.PsEUL-OVA can significantly increase the levels of OVA-specific IgGs and subclasses IgG1,IgG2a,and IgG2b in mouse serum;significantly up-regulate the levels of cytokines IFN-γ,IL-2,IL-4,and IL-6 in mouse serum;increase the differentiation of CD4+T and CD8+T in splenic lymphocytes;and also enhance the level of maturation markers CD80,CD86,and MHC-II on the surface of dendritic cells.4.Preparation and characterization of PsEUL-OVA/Cubs.Phytantriol was used as an amphiphilic lipid substance into F127 aqueous solution,and PsEUL-OVA/Cubs were prepared via the ultrasonic homogenization method,and PsEUL-OVA/Cubs were measured by TEM observation,polarizing microscope observation,and Zeta potential and particle size analysis.The encapsulation efficiency and drug loading capacity were determined to characterize PsEUL-OVA/Cubs.The TEM results showed that the prepared PsEUL-OVA/Cubs present a hexahedral and cubic structure,with a relatively uniform distribution and size.Under a polarizing microscope,at room temperature,PsEUL-OVA/Cubs appear as a dark field of view,with no polarized light.On heating PsEUL-OVA/Cubs to 60℃,you can observe that the field of view becomes brighter and the structure becomes obvious.Thus,it can be confirmed that PsEUL-OVA/Cubs have a cubic phase structure,and the optical characteristic is isotropic.The average particle size（AD）of PsEUL-OVA/Cubs was 381.68±12.28 nm,the polydispersity coefficient（PDI）was0.175±0.01,and the average Zeta potential was-10.43±0.3 m V,indicating that PsEUL-OVA/Cubs have good dispersion and are relatively stable.The encapsulation rate of the prepared PsEUL-OVA/Cubs was78.12%±1.23%,and the drug load was 80.75%±2.43%as per the calculation formula of the encapsulation rate and drug loading.5.In vitro immune activity of PsEUL-OVA/Cubs.To explore the immunological activity of PsEUL-OVA/Cubs,the CCK-8 method was to explore the activity of PsEUL-OVA/Cubs in vitro in spleen cells and macrophages and to determine the PsEUL-OVA/Cub-specific cytokine secretion by spleen cells and macrophages.To determine the secretion level,FITC was used as a fluorescent label,and the phagocytosis of FITC-labeled PsEUL-OVA/Cubs by macrophages was observed under a laser confocal microscope.Finally,PsEUL-OVA/Cubs was co-cultured with macrophages and analyzed by sequencing,and the effect of PsEUL-OVA/Cubs on mouse macrophage genes was evaluated.The results showed that when the concentration of PsEUL-OVA/Cubs was 150μg/m L,the activity of mouse splenocytes and macrophages was significantly higher than that observed for other groups（P<0.05）.Thus,PsEUL-OVA/Cubs can increase lymphocyte and macrophage concentration.The secretion level of cytokines IFN-γ,IL-2,IL-4and IL-6 by macrophages was further evaluated.The phagocytic effect of macrophages in PsEUL-OVA/Cubs group was observed by laser copolymerization microscope was significantly higher than that in other groups.High throughput sequencing of the gene expression profile of mouse macrophages revealed that after 24 h of PsEUL-OVA/Cubs treatment,3385 related differentially expressed genes appeared in RAW264.7 cells,of which 1891 were significantly up-regulated.Of these,the immunological enrichment pathways of the differentially expressed genes included the tumor necrosis factor signaling pathway,Toll-like receptor signaling pathway,antigen processing and presentation signaling pathway,and phagosome and other signaling pathways.6.Immune activity of PsEUL-OVA/Cubs in vivo.In all,150 ICR mice were divided into 10 groups,the normal saline group was the blank group,and the control groups included PsEUL group,OVA group,PsEUL and OVA mixed group,PsEUL-OVA group,PsEUL/Cubs group,OVA/Cubs,PsEUL+OVA/Cubs group,and PsEUL-OVA/Cubs group.Freund’s adjuvant（FCA）was used as the positive control group.Flow cytometry was used to detect the expression of CD4+and CD8+in mouse spleen cells,and to detect the levels of surface maturation markers on dendritic cells.The phenotypic changes in the markers CD80,CD86,and MHC-II were evaluated.The spleen,thymus,and serum of mice were collected to evaluate the effect of PsEUL-OVA/Cubs on the immune organ index（including serum cytokines IFN-γ,IL-2,and IL-4）.The influence of IL-6 concentration and the level of OVA-specific IgGs and subclasses IgG1,IgG2a,and IgG2b was also determined.The results showed that PsEUL-OVA/Cubs can significantly increase the immune organ index in mice;PsEUL-OVA/Cubs can significantly increase the levels of OVA-specific IgGs and subclasses IgG1,IgG2a,and IgG2b in mouse serum,and can also significantly up-regulate the serum levels of cytokines IFN-γ,IL-2,IL-4,and IL-6.PsEUL-OVA/Cubs can improve the differentiation of CD4+T and CD8+T cells in splenic lymphocytes,and can also enhance the levels of cell surface maturation markers CD80,CD86,and MHC-II on mouse dendritic cells.In summary,the coupling of PsEUL and OVA can help enhance immune activity,and PsEUL-OVA/Cubs can enhance the in vitro activity of immune cells,promote cellular secretion of cytokines,and enhance the phagocytic ability of macrophages.Further,as per the in vivo tests,PsEUL-OVA/Cubs can enhance the antibody level of mouse serum OVA-specific IgGs and subclasses,enhance serum cytokine levels,enhance the proliferative ability of T and B lymphocytes,increase the expression of CD4+and CD8+T cells,and promote the expression of maturation markers on dendritic cells.Finally,according to the results of high-throughput sequencing,it is speculated that the effect of PsEUL-OVA/Cubs on the immune activity in the body is achieved by altering the enrichment of differentially expressed genes to express immune-related proteins. |
