Adjuvant Activity Of Tubeimoside Ⅰ From The Tuber Of Bolbostemma Paniculatum And Its Mechanisms

Vaccination is the most economical and effective strategy for preventing and controlling infectious diseases.Adjuvants are essential components for both traditional and novel vaccines,not only affecting the strength of the body’s adaptive immune response to vaccines,but also inducing the most effective type of immune response against specific pathogens.There are many types of immunoadjuvants reported in the current research,but few have been practically applied due to inevitable defects such as toxic side effects or safety hazards.Saponin is an important class of immunological active substances widely found in natural medicines.Since the discovery of saponins to enhance the body’s immune response to foot-and-mouth disease vaccines in 1951,their adjuvant effects have received widespread attention.Bolbostemmatis Rhizoma is the dry tuber of Bobostemma paniculatum(Maxim.)Franquet.As a traditional Chinese medicine,Bolbostemmatis Rhizoma has curative effects on various diseases such as inflammation,wart and tumor.The bioactive substances in Bolbostemmatis Rhizoma include triterpenoid saponins,alkaloids,polyphenols,flavonols,and sterols.The triterpenoid saponins,tubeimosides(TBMS),were regarded as the most active constituents in Bolbostemmatis Rhizoma.Two sugar chains at C-3 and C-28 of aglycons combined to form a unique structural cyclic bisdesmosides via 3-hydroxy-3-Hydroxy-3-methyl glutaric acid.Given its unique chemical structure,TBMS is assumed to have potential adjuvant activity.To provide scientific basis for its development as a new adjuvant,in this study,tubeimoside I(TBMS I)isolated from Bolbostemmatis Rhizoma was investigated for its adjuvant activities for Ovalbumin(OVA)in mice,and explored for its innate immune mechanism of action,so as.Part I: Adjuvant activity of tubeimoside I on the immune responses to ovalbumin in miceICR mice were immunized subcutaneously with OVA 25μg alone or with OVA 25 μg dissolved in saline containing Quil A(10 μg)or tubeimoside I(25,50 or 100 μg)on days 1 and 15.Animals injected with 200 μl of PBS were included as a negative control.Sera and splenocytes were collected 2 weeks after the boosting immunization.The effects of TBMS I on serum OVA-specific antibody titers,splenocyte proliferation,natural killer(NK)cell activity,mRNA expression of cytokines and transcription factors,secretion of cytokines,as well as delayed type hypersensitivity(DTH)in the mice immunized OVA were determined by ELISA,MTT assay,and quantitative real-time PCR(qRT-PCR).TBMS I not only significantly enhanced the serum OVA-specific IgG,IgG1,IgG2 a and IgG2 b antibody titers(P<0.05,P<0.01 or P<0.001),but also promoted the concanavalin A(Con A)-,lipopolysaccharide(LPS)-and OVA-stimulated splenocyte proliferation(P<0.05,P<0.01 or P<0.001),the activities of NK cells(P<0.01 or P<0.001)and delayed type hypersensitivity(DTH)in the mice immunized with OVA.TBMS I also remarkably induced the production of both Th1(IFN-γ)and Th2(IL-10)cytokines,and up-regulated the mRNA expression levels of Th1 cytokines(IFN-γ and IL-2)and transcription factors(T-bet and STAT4)as well as Th2 cytokines(IL-10)and transcription factors(GATA-3 and STAT6)in splenocytes from the OVA-immunized mice.These results demonstrated that TBMS I had a potential to enhance and improve immune responses and elicit both Th1/Th2 response to OVA,and that TBMS I would be a promising adjuvant candidate for vaccine.Part II: Innate immune mechanism of adjuvant action of tubeimoside IMice were immunized intramuscularly(i.m.)with 50 μl/quadriceps of PBS containing Alexa Flour 488 conjugated OVA(OVA-AF488)alone or combined with various doses of TBMS I.Quadriceps and draining lymph nodes(dLNs)were collected at 12,24 and 48 h after injection for preparation of single cell suspension,respectively.Then the cells were stained with combination fluorescence labeling specific antibody and analysed by flow cytometry to evaluate the dynamics of immune cell recruitment and identify the main antigen uptake cell populations induced by TBMS I.For cytokine and chemokine mRNA and protein expression assay,mice were injected intramuscularly(i.m.)with 50 μl/quadriceps muscle of TBMS I or PBS alone.Quadricepses were collected at 2,4 and 6 h after injection.The total RNA was isolated using Trizol reagent and the reverse transcription was conducted.The qRT-PCR was performed using FastStart Universal SYBR Green Master.Quadricepses were collected at 3,6 and 12 h after injection.After PBS grinding,the supernatant was collected after centrifugation,and the levels of cytokines and chemokines were detected by ELISA kit or Western-Blot assay.The results showed that compared with mice immunized with OVA alone,TBMS I could dramatically induced the recruitment of neutrophils,monocytes,macrophages,dendritic cells,eosinophils and basophils at the injection site,as well as enhance the uptake of OVA-AF488 by these innate immune cells both at injection site and in dLNs.Meanwhile,TBMS I intramuscular injection significantly up-regulated the mRNA expression levels of cytokines and chemokines in local tissues and induced the secretion of cytokines and chemokines at injection site.These results suggested that TBMS I could improve the immune microenvironment by inducing local cytokine and chemokine secretion,contributing to the recruitment and activation of immune cells,thus promoting the antigen presenting and adaptive immune responses.In conclusion,the present study demonstrated that TBMS I could improve both OVA-specific antibody and cellular immune responses in mice.TBMS I enhanced the magnitude of the immune responses,and,most importantly,it modulated the quality of the immune responses resulting in concurrent Th1/Th2 immunity.Meanwhile,the results that TBMS I up-regulated the mRNA expression and induced strong local production of cytokines and chemokines at the injection site,recruited various immune cells to the injection site,as well as enhanced antigen uptake by immune cells both at injection site and in dLNs suggested that the adjuvant action of TBMS I could be achieved by inducing local immunostimulatory environment.