Mechanisms Of Adjuvant Action Of Platycodon D Based On Sphingomyelin Metabolisms

Radix Platycodonis,the root of Platycodon grandiflorum A.DC(Campanulaceae),is a well-known traditional Chinese medicine used as an expectorant for pulmonary diseases and a remedy for respiratory disorders such as cough,colds,sore throat,tonsillitis,and chest congestion.Platycodin D(PD),a triterpenoid saponin,is a major active ingredient in Radix Platycodonis.PD possessed multiple biological and pharmacological properties including anti-tumor,anti-obesity,anti-atherosclerosis,anti-inflammatory,antiviral,immunoregulatory and hepatoprotective activities.PD has been reported to be an ideal vaccine adjuvant,but its mechanism of action remains unclear.Meanwhile,PD is used as the quality control marker of Radix Platycodonis in the Pharmacopoeia of People's Republic of China.However,the quality of Radix Platycodonis varies with cultivation region,harvest time and processing technology.Thus,a reliable and accurate method is required for the purpose of quality control.In the study,an indirect competitive enzyme linked immunosorbent assay(icELISA)for the detection of platycodin D in Radix Platycodonis was first established.Meanwhile,to clarify the mechanism of adjuvant action of PD,among the differentially expressed mRNAs in mouse myoblast C2C12 cells induced by PD,sphingomyelin synthase 2(Sgms2)and sphingomyelin phosphodiesterase 3(Smpd3)were selected to investigate their roles in mediating adjuvant activity of PD using C2C12 cells and OVA-immunized mice.1.Establishment of indirect competitive enzyme linked immunosorbent assay for the detection of platycodin D in Radix PlatycodonisA specific polyclonal antibody against PD(PD-pAb)was developed,and PD-pAb-based indirect competitive enzyme-linked immunosorbent assay(icELISA)was established for the detection of PD in Radix Platycodonis.The coupling compounds of PD with bovine serum albumin(BSA)and human serum albumin(HSA)were prepared.The molecular weight and coupling ratio of PD-BSA and PD-HSA were determined by FT-IR and MS.New Zealand white rabbits were immunized with PD-BSA by intramuscular injection for several times.Rabbit anti PD serum was isolated and purified by column chromatography to prepare anti PD polyclonal antibody(PD-PAb).he sensitivity and specificity of PD-PAb and the recovery of PD were detected by ELISA,and the contents of PD in Platycodon grandiflorum from five producing areas were detected and compared with the results of high performance liquid chromatography(HPLC).FT-IR confirmed the coupling of PD-BSA and PD-HSA.MS showed that the coupling ratios of PD-BSA and PD-HSA were 1.5:1 and 2:1,respectively.The 50%inhibition concentration(IC50)of PD was 2.70 ?g/mL and the linearity range for PD was from 0.032 ?g/mL to 100?g/mL.No cross reactivity with PD-pAb was found in five PD analogs except for platycodin D2(PD2,0.93%).The average recovery of PD by icELISA was 97.14%(RSD=1.17%).The icELISA was used for the detection of PD in different Radix Platycodonis samples and the results were confirmed by high performance liquid chromatography(HPLC).The correlation coefficient between the two assays was 0.9654.Taken together,the established icELISA might be a simple,cheap,rapid,sensitive,reliable and high-throughput method for determining the contents of PD in Radix Platycodonis.2.Sphingomyelin synthase 2 mediates the adjuvant activity of platycodin DSgms2 was selected as objective based on the effect of PD on gene expression profile of mouse C2C12 Myoblasts.The recombinant plasmid pcDNA3.1(-)-sgms2 was constructed,and then sgms2-overexpressing C2C12 cell line was obtained by liposome transfection.The acquired function of sgms2 was studied through detecting the expression of inflammatory factors and the activity of Smpd3 in C2C12 cells by RT-qPCR,ELISA and Western blot as well as observing cell morphology of C2C12 cell by scanning electron microscope.To investigate the in vivo and in vitro"loss-of-function" of sgms2,siRNA and specific inhibitor D609 were used to detect the effects of sgms2 knockdown and inhibition on the expression of inflammatory factors in C2C12 cells induced by PD,and on the expression levels of inflammatory cytokines,the recruitment and uptake of immune cells in PD-injected mouse quadriceps muscles.The upstream regulatory factors of Sgms2 were also studied through observing the localization of PD in C2C12 cells by immunofluorescence assay and detedting the effect of Me-?-CD on the expression of Sgms2 and inflammatory factors in C2C12 cells induced PD by ELISA.Furthermore,the effects of D609 on the serum IgG and its isotype antibody titers in mice immunized with PD and OVA were determined.Sgms2 overexpression significantly up-regulated the mRNA and protein expression levels of cholesterol hydroxylase(Ch25h)and inflammatory factor COX-2 in C2C12 cells,down-regulated those of Smpd3,and inhibited the enzyme activity of Smpd3.The siRNA and inhibitor D609 of Sgms2 significantly antagonized the expression of inflammatory factor genes and proteins in C2C12 cells induced by PD,indicating that sgms2 was involved in the inflammatory response of C2Cl2 cells induced by PD.Pretreatment with D609 markedly decreased the expression levels of inflammatory cytokines,and reduced the recruitment and uptake of immune cells such as dendritic cells,monocytes,neutrophils,eosinophils,mast cells and basophils in PD-injected mouse quadriceps muscles.PD was mainly distributed in C2C12 cell membrane at the early state,and significantly reduced the content of cholesterol in C2C12 cells.The pretreatment of methyl-?-cyclodextrin(Me-?-CD)significantly inhibited the expression of sgms2 and COX-2 in C2C12 cells induced by PD.The pretreatment with D609 significantly reduced the serum OVA-specific IgG,IgGl,IgG2a and IgG2b antibody titers in the mice immunized with PD and OVA.These findings suggested that PD exerted the adjuvant activities by reducing cholesterol,activating sgms2 and then inducing inflammatory response.3.Sphingomyelin phosphodiesterase 3 mediates the adjuvant activity of platycodin DThe effect of PD on the enzyme activity of Smpd3 in C2C12 cells was first detected based on that PD significantly down-regulated the gene expression levels of Smpd3 in C2C12 cells.Meanwhile,the effects of Smpd3 specific inhibitor GW4869 on the expression levels of inflammatory factors in C2C12 cells and mouse local quadriceps muscles induced by PD as well as the serum OVA-specific IgG and its isotype antibody titers in the immunized with PD and OVA also determined by RT-qPCR,ELISA and Western blot.PD significantly inhibited the enzyme activity of Smpd3 in C2C12 cells.GW4869 pretreatment markedly enhanced the mRNA expression levels of COX-2,IL-6,IL-1?,MIP-1? and MIP-2 in C2C12 cells induced by PD,increased the levels of IL-6,MIP-1?and MIP-2 in PD-injected mouse quadriceps muscles,and promoted serum OVA-specific IgG,IgG1,IgG2a and IgG2b antibody titers in the mice immunized with PD and OVA.These results indicated that Smpd3 might be involved in mediateing the adjuvant activity of PD.