| Croci Stigma,the dried stigmas of the Crocus sativus L.(Iridaceae),is the most expensive traditional Chinese medicine.It was recorded to activate blood circulation and remove blood stasis,cool blood and detoxify,relieve depression and tranquilize mind,and used for the treatment of amenorrhea,postpartum blood stasis,mild poison,depression,palpitation and madness in traditional Chinese medicine.Modern pharmacological studies showed that Croci Stigma possessed neuroprotective,anti-tumor,antidepressant,anticonvulsant,anti-schizophrenia and antioxidant activities.C.sativus petals(CSP)are usually discarded as a waste in the processing of crude drug Croci Stigma in production place.CSP has been reported to contain large amounts of flavonoid,anthocyanin and terpenoid components similar to crude drug Croci Stigma.CSP were reported to possess neuroprotective,hepatoprotective,antioxidants,hypotensive and antitussive activities.In this study,the water extract of CSP was subjected to ethanol precipitation,dialysis,and gel column chromatography affording two homogeneous heteropolysaccharides CSPPA and CSPPB.The structural characteristics and immunoregulatory effects of these two novel polysaccharides were systematically investigated.1.Isolation,purification and structural characterization of C.sativus petal polysaccharidesThe crude polysaccharide(CSPP)was obtained by water extraction and ethanol precipitation from C.sativus petals.The crude polysaccharide CSPP was isolated by DEAE-Sephadex A-50 column chromatogram into the three main fractions F1,F2,and F3.The fractions F2 and F3 were further purified using Sephadex G-200 column to afford two homogeneous heteropolysaccharides(C.sativus petal polysaccharides,CSPPs),namely CSPPA and CSPPB.The homogeneity of CSPPA and CSPPB was identified by HPGPC-RID as a single peak with the dispersion coefficient being 1.21 and 1.14,respectively.The molecular weights were determined to be 1.98×106 and 2.53 × 106 Da for CSPPA and CSPPB.Infrared spectrum analysis revealed that CSPPA and CSPPB have polysaccharide characteristic absorption signal,contain acetyl and sulfonic groups.The content of total sugar,sulfonic group and acetyl group in CSPPA were 95.33±0.55%,7.95±0.07%and 5.10±0.06%,and those in CSPPB was 93.22±0.42%,13.02±0.77%and 3.33±0.07%,respectively.CSPPA consisted of Gal,Rha,Ara,and Xyl in the molar ratio of 16:5:7:3,while CSPPB were composed of Gal,Glc,Man,Rha,Ara,and Xyl with the molar ratio of 16:2:7:19:15:16.The monosaccharide residues of CSPPA consisted of?4)-Me-?-D-GalpA-(1?,?-L-Rhap-(1?,?3,6)-?-D-Galp-(1?,?4)-?-L-Arap-(1?,?4)-?-D-Xylp-(1?,and?3,4)-?-D-Xylp-(1?.The acetylated or sulfonated monosaccharide residues were identified as?3,6)-2-OAc-?-D-Galp-(1?,?3,6)-4-OAc-?-D-Galp-(1?,and ?4)-3-SO3-?-D-Xylp-(1?.The monosaccharide residues of CSPPB consisted of ?-L-Araf-(1?,?2)-?-L-Arap-(1?,?3)-?-D-Galp-(1?,Me-?-D-GalpA-(1?,?4)-?-L-Rhap-(1?,?4)-3-SO3-?-L-Rhap-(1?,?3,4)-?-L-Rhap-(1?,?6)-?-D-Manp-(1?,?6)-2-OAc-?-D-Manp-(1?,?4)-3-SO3-?-Xylp-(1?,?4)-?-D-Xylp-(1?,?3,4)-?-D-Xylp-(1?,?6)-?-D-Glcp-(1?,?6)-3-OAc-?-D-Glcp-1(?,and?3,6)-?-D-Glcp-(1?.Congo red experiment showed the three-helix structure in aqueous solution of CSPPA,and the flake structure and rod structure in CSPPA and CSPPB were observed under scanning electron microscope.The island structure formed by spontaneous curl of polysaccharide chain in CSPPA was found with its surface fluctuation being more obvious than CSPPB under atomic force microscope.In total,there are obvious differences in the composition of monosaccharide residues,advanced structure and microstructurebetween the two polysaccharides.2.Adjuvant activity of C.sativus petal polysaccharides and its mechanismsThe adjuvant activity of CSPPA and CSPPB were investigated in mice immunized with OVA and FMDV 146S.Serum and splenocytes were collected 2 weeks after the second immunization for measurement of serum antigen-specific antibody,splenocyte proliferation,NK cell activity,and production and mRNA expression of cytokines from splenocytes.The OVA-and FMDV 146S-specific serum IgG,IgGl,IgG2a,IgG2b antibody titer level was significantly enhanced by CSPPA and CSPPB(P<0.05,P<0.01 or P<0.001).CSPPA and CSPPB remarkably increased the proliferation of Con A-,LPS-,and antigen-induced splenocyte and NK cells activity in the immunized mice(P<0.05,P<0.01 or P<0.001).CSPPA and CSPPB also significantly promoted the production of Th1(IFN-y)and Th2(IL-10)cytokines as well as up-regulated the mRNA expression levels of cytokines(IL-2,IFN-y,IL-4 and IL-10)and transcription factors(T-bet,STAT4,GATA-3 and STAT6)in splenocytes from the immunized mice(P<0.05,P<0.01 or P<0.001).The results indicated that both CSPPA and CSPPB were ideal adjuvant candidates that increased both cellular and humoral immune responses and elicited a balanced Thl/Th2 response.Accumulating evidence suggested that most adjuvants induced early activation of innate immunity which then translates into higher antibody and cellular responses to vaccines.Key features of the innate immune response induced by CSPPA and CSPPB were explored at the intramuscular injection site in mice.H&E stain observation revealed the infiltration of inflammatory cells in quadriceps muscle tissues induced by both CSPPA and CSPPB.CSPPA and CSPPB significantly up-regulated the mRNA and protein expression levels of inflammatory factors(IL-1(3 and IL-6)and chemokines(CCL3,CXCL1 and CXCL2)in the quadriceps muscles(P<0.05,P<0.01 or P<0.001).CSPPA and CSPPB also significantly induced the recruitment and promoted antigen uptake of neutrophils,dendritic cells(DCs)and macrophages into the quadriceps muscles in the OVA-treated mice(P<0.05).Furthermore,CSPPA and CSPPB enhanced the number of DCs,B cells,macrophages,mast cells,neutrophils and T cells in the draining lymph nodes(P<0.05).These results suggest that CSPPA and CSPPB enhanced the adaptive immune responses by inducing the robust early recruitment of innate immune cells at the injection site and the dLNs.3.Activation of RAW264.7 macrophages by C.sativus petal polysaccharides and its molecular mechanismsRAW264.7 macrophages were used to investigate the immunomodulatory effects of CSPPA and CSPPB on macrophages in vivro.CSPPA and CSPPB were no cytotoxic to RAW264.7 cells up to the concentrationof 5.0 ?g/mL(P>0.05).CSPPA and CSPPB significantly induced the mRNA expression and secretion of cytokines(IL-1?,IL-10,IL-12 p40 and TNF-?)and chemokines(CCL5 and CCL22)in RAW264.7 cells in a concentration-and time-dependent manner(P<0.05,P<0.01 or P<0.001).Both polysaccharides also significantly concentration-dependently promoted the expression of CD40,CD80,CD86,MHC ? and MHC ? in RAW264.7 cells(P<0.05,P<0.01 or P<0.001).To further insight into the molecular mechanism on the immunomodulatory action of CSPPA and CSPPB,the phosphorylation levels of MAPK members(ERK,JNK and p38 MAPK)and NF-?B p65 in cells were detected using Western blotting.CSPPA and CSPPB significantly induced the phosphorylation of ERK,JNK,p38,and NF-?B p65 in RAW264.7 cells(P<0.05,P<0.01 or P<0.001).Furthermore,pretreatment of SB203580(p38 MAPK inhibitor)and Bay 11-7082(NF-?B p65 inhibitor)could result in significant suppression of the production of IL-10 and IL-12 p40 in RAW264.7 cells induced by CSPPA(P<0.001),whereas CSPPB-induced IL-10 and IL-12 p40 secretion could be inhibited by SP600125(JNK inhibitor),and PD98059(ERK inhibitor).These results further confirmed the involvement of MAPK and NF-?B in the activation of RAW264.7 cells by PCSPA and PCSPB.In conclusion,two homogeneous polysaccharides from petal of C.sativus were first isolated and purified,and their physicochemical properties and primary structure were analyzed by various analytical methods to determine the monosaccharide residues,the possible linkage of monosaccharide residues,and the repeated structural units.CSPPA and CSPPB could activate APCs via MAPK-NF-?B signaling and rapidly induce innate immune response resulting in their strong immunological adjuvant activity,and thus may be ideal adjuvant candidates suitable for a wide spectrum of prophylactic and therapeutic vaccines. |
PDF Full Text Request