Studies On Immunoregulative Target Cells And Binding Sites By Lycium Barbarum Polysaccharides LBPF4-OL

Traditional Chinese medicine theory believed that Chinese wolfberry has "nourishing blood and promote eyesight" effects. LBPF4-OL is a homogeneous polysaccharide from glycoprotein complex 4. The purpose of this study is to identify LBPF4–OL's immunoloregulation target cells and receptors, and observes its acid decomposition products activity.Polysaccharides immunoloregulation target cells mainly belong to the innate immune antigen processing cells and humoral immune B lymphocytes, while the report of T cells is relatively rare. Toll-like receptor 4 (TLR4) is the first identified mammalian homolog of the Drosophila Toll protein. TLR4 recognizes Gram-negative bacterial lipopolysaccharide (LPS), Gram- positive bacteria eptidoglycan and also senses endogenous ligands including hyaluronic acid and so on. Japanese and Korean scholars found that TLR4 maybe the main polysaccarides receptor of Chinese herbal medicine. Current research indicates that Dectin-1 is the only specificity polysaccharide receptor, yet TLR4 and the othere polysaccarides related protain, such as TLR2, CR3, SR and CD19 et cetera, only is the non- specificity receptors. So, screening and discriminate the non-specificity receptors are our work emphasis after determining the target cells.1. Studies on the Immunogegulative target cells by LBPF4-OLFor the separation and purification of the polysaccharides, we first adopted water soak and ethanol precipitate methods to obtain the crude polysaccharide, and then used DEAE-cellulose column chromatography obtained polysaccharides components LBPF1, LBPF2, LBPF3, LBPF4 and LBPF5, and the LBPF4 component have showed absorption peaks in 490nm and 280nm, indicating that LBPF4 is a glycoprotein compound. Then using the method Pronase E to release of the polysaccharides chains, and adopt High Performance Gel Permeation Chromatography (HPGPC) observed the LBPF4 chains homogeneity. The results showed that the obtained LBPF4-OL have a single symmetrical peak; indicate it's a homogenous polysaccharides.In target cell research, we first observed lymphocytes'activation and proliferation in the condition of mixed lymphocytes and macrophages by lymphocytes proliferation experiment and flow cytometry, and then CD3+T cells and CD19+B cells were negatively selected from mouse splenocytes using mouse T and B cell magnetic bead isolation kits, and observed LBPF4-OL's effections on the three kinds of cells, at the same time, immunoregulatory characteristics of LBPF4-OL were studied.In the splenocytes research, we found that LBPF4-OL in 50 and 100μg/mL concentration can obviously increase splenocytes 3H-TdR incorporation value, and when blended with LPS can further improve incorporation values, while when blended with Con A, there was no difference in 3H-TdR incorporation values. CD3+T cells and CD19+B cells were isolated from splenocytes, and more than 90% of T and 95% of B lymphocytes were remained as determind by flow cytometry. Cell proliferation experimental results show that LBPF4-OL have no influence on T cells and B cell proliferation, but when LBPF4-OL blended with LPS, can concentration dependently promote B cells proliferations, when adding macrophages to B cells, LBPF4-OL could concentration dependently promote the 3H-TdR incorporation value too. These part of the results indicate, LBPF4–OL have no direct effect on T and B cell proliferation, but when macrophages or LPS exists, LBPF4-OL could significantly promote the 3H-TdR incorporation value in concentration dependent way.Lymphocyte activation is a prophase events of lymphocytes proliferate and transformation, and cytokines is one of the necessary conditions for lymphocyte activation. Further to determine the effect of LBPF4-OL on T, B cells activation and cytokines secretion, LBPF4-OL were cocultured with splenocytes for 24 h, the flow cytometry and Luminex were adopt. The flow cytometry results show that LBPF4-OL failed to change the percentage of CD3+CD25+T in CD3+T cells and CD19+CD86+B in CD19+B cells. Luminex results indicates that the LBPF4-OL can increase T cell type cytokine IL-2 secretion only in high concentrations of 100μg/ml, the B cell type cytokine IL-13 secretion were not significantly influenced in low, medium and high concentration. We also found a concentration dependently induction of macrophages type cytokines, including TNF- , IL-6, IL-8, and IL-10, by LBPF4-OL treatment, and promote GM-CSF, IL-12p40 secretion in 100μg/ml LBPF4-OL treatment. Combining the data indicating that LBPF4-OL can not enhance T lymphocytes proliferation and activation, can not activate B lymphocytes but can promote B lymphocytes proliferation when blending with LPS or macrophages, and can also induce macrophage type cytokines secretion indicates that macrophages and B lymphocytes are the immunoregulative target cells.We further observed the effection of LBPF4-OL on macrophage in vivo and in vitro. Mice were administrated LBPF4-OL daily for 6 days, and peritoneal macrophages were havested after the last injection. CD86 and MHC-Ⅱmolecular expression were tested by flow cytometry. The results indicating that LBPF4-OL can obviously increase CD86+CD11b+ cells and CD11b+I-A/I-E+ cells number, suggesting it can activate the macrophages and enhancing its antigen processing ability. In vitro experiments, ELISA and reverse transcription-polymerase chain reaction (PCR) were used to test the TNF- , IL-1βconcentration and IFN- , IFN–βmRNA express. To determine the affection of LBPF4-OL on proinflammatory cytokines secretion, the concentration of TNF- and IL–1βin supernatant of cultured peritoneal macrophages were test in vitro by ELISA. Our data show that glucan can not enhance TNF- and IL–1? secretion when it was cultured with macrophages in 100μg/ml concentration, and we found that LBPF4-OL can induce TNF- and IL–1βsecretion in dose dependent manner. To determine the affection of LBPF4-OL on anti-inflammatory cytokines expression, the IFN- and IFN-βmRNA expression of peritoneal macrophages was test in vitro by RT-PCR. The results showed that LBPF4-OL can time dependent induce IFN- and IFN-βmRNA expression, and both kind of mRNA expression reach the highest level at 24 h.The above results show that LBPF4-OL can both activate macrophages and enhance the antigen processing and immunoregulative ability, and indicate that macrophage is the main immunoregulative target cell.2.Studies on the target cell binding sites by LBPF4-OLIn this part of the study, we first screened TLR4, TLR2, CD19 and CR3 as four potential polysaccharide binding sites on B cells and macrophages membrane, and then taken the gene mutant mice as the research target to verify the binding site by 3H-TdR incorporation test, ELISA and biolayer interferometry.In order to observe whether CD19 or TLR4 is the LBPF4-OL binding sites on the B cells, we use the laser confocal microscope, flow cytometry and 3H-TdR incoporation method to observe the effection of LBPF4-OL on B cells function. The results showed that when B lymphocytes were pre-incubated with LBPF4-OL for varying times (9 min, 3 min and 1 min), can reduce number of B cell membrane PE-anti-CD19 fluorescence labeling antibody, and flow cytometry test results found that when B lymphocytes were pre-incubated with LBPF4-OL for 9 min, the average fluorescence intensity of PE-anti-CD19 on B cells fell 27.1%. And we found that CD19 in the concentration of 0.00625μg/ml can significantly inhibit LBPF4-OL combined with LPS induced splenocytes proliferation, the inhibition rate can achieve 58.1% when CD19 in 0.1μg/ml. TLR4/MD2 antibody in the concentration of 0.0625μg/ml can also significantly inhibit LBPF4-OL combined with LPS induced splenocytes proliferation, the inhibition rate can achieve 25.1% when TLR4/MD2 in 1μg/ml. The experimental results suggest that, both CD19 and TLR4 molecules are the binding sites of LBPF4-OL on B lymphocytes.In order to observe whether TLR4, TLR2 or CR3 is the LBPF4-OL binding sites on the macrophages, antibody block method were adopted to observe the effection of LBPF4-OL on macrophages secretion of TNF- and IL-1β. The results showed that LPS (5μg/ml) and LBPF4-OL (100μg/ml) can significantly stimulate TNF- and IL-1βsecretion, and galactan did not affect the TNF- and IL-1βsecretion. Anti-TLR4 can concentration dependent inhibit LBPF4-OL induced TNF- and IL-1βsecretion. Anti-TLR2 (0.31, 0.62, 1.25, 2.5μg/ml) only in 0.62μg/ml concentration can inhibit the TNF- secretion, and each effective concentrations of inhibition effect no obvious difference, but Anti-TLR2 can concentration dependence inhibit IL-1βsecretion.Anti-CR3 did not affect TNF- and IL-1βsecretion. Furtherer to determine whether TLR4 and CR3 are the binding sites, 3H-TdR incorporation method were used to observe LBPF4-OL induced splenocytes proliferation in the condition of anti-TLR4 and anti-CR3. The results showed that LBPF4-OL can concentration dependent induce splenocytes proliferation, anti-CR3 did not affect the splenocytes proliferation, yet anti-TLR4/MD2 can significantly inhibit the the proliferation induced. Together, the above results indicate that TLR4 maybe the main binding site on macrophages, and needs further investigation.To determine TLR4 is one of the binding sites of LBPF4-OL, we used a variety of methods to verify. First, we observe the effection of LBPF4-OL on TNF- and IL-1βsecretion of TLR4 gene mutation mice macrophages. The results showed that LBPF4-OL concentration dependent induced TNF- and IL-1βsecrete on C3H/HeJ mice abdominal macrophage, but had no influence on C3H/HeN mice. Secondly, we observed the effection of LBPF4-OL on C3H/HeN and C3H/HeJ mice splenocytes proliferation. The results showed that LBPF4-OL can obviously increase C3H/HeN mice splenocytes proliferation, but had no influence on C3H/HeJ mice splenocytes proliferation. Finally, we used biolayer interferometry to observe whether there was a direct binding of LBPF4-OL with TLR4/MD2 complex. The results showed that LBPF4-OL can directly combine with TLR4, according to its the average molecular weight calculated, its affinity KD (M) value is 4.5×10-7. The results confirmed that TLR4 is really LBPF4-OL's direct binding sites.3. The immunocompetence evaluation of fluorescence labeled LBPF4-OL and its schizolysis productsTo determine whether fluorescence labeled LBPF4-OL is still activity, the effection of fluorescamine and fluorescein isothiocyanate labeled LBPF4-OL on splenocytes proliferation were test by 3H-TdR incorporation method. The results showed that there is no difference between LBPF4-OL and FITC-LBPF4-OL in promoting splenocytes proliferation, but fluorescamine labeled LBPF4-OL lost the immunoregulative ability.Current researches basically think that the activities of polysaccharides are related with the molecular weight, the degree of polymerization, the degree of branch and the advanced conformation in solution. If molecular weight is too big or too small the polysaccharide activity will downshift, and certain branch are necessary for the polysaccharide active. In order to determine whether branch chain is necessary forpolysaccarides activity, the branch chain removed LBPF4-OL activity was measured by 3H-TdR incorporation method. The results showed that the branch chain removal LBPF4-OL in the concentration of 10, 50 and 100μg/ml can't promote splenocytes proliferation. In order to find out whether the molecular weight changes will affect the activity of LBPF4-OL, we use the same experiment methods to evaluate the activity of oligosaccharide decomposed from LBPF4-OL by acid. The results showed that the oligosaccharide completely lost its activity in promoting splenocytes proliferation. The above results showed that contain branch chain and keep up a certain degree of polymerization is necessary for LBPF4-OL's activity.Through the above studies, this paper draws the following conclusions: first of all, the immunoregulative target cells of LBPF4-OL mainly are macrophages and B lymphocytes but not T lymphocytes; Secondly, TLR4 and TLR2 mediated the activation of macrophages by LBPF4-OL; thirdly, both CD19 and TLR4 are involved in LBPF4-OL induced B lymphocytes proliferation; finally, containing some branch chain and keep up a certain degree of polymerization are necessary for LBPF4-OL's activity.