Distinct Transcriptional Immunomodulating Signatures Of COLD-FX In Murine Spleen Cells Of Different Functional Status

Objective：The exploration of using plants as a method of preventing andtreating diseases has been lasting for centuries in the eastern countries in thetraditional medical system. With the development of immunology and thetechnical methods, the research of American ginseng and its pharmacologicalmechanism became increasingly popular. In the past two decades someachievements of the research have been reported. According to the report themainly two components of American ginseng are ginsenosides andpolysaccharides. Many studies have demonstrated that the polysaccharidescomponents may have stronger immunomodulating capacity than ginsenosides.A standardized polysaccharide-rich product of American ginseng namedCOLD-FX was shown to be able to stimulate immunoglobulin production byB lymphocytes and activate peritoneal exudate macrophages. However, thecontradictory results on the research about the mechanism COLD-FX havebeen reported. Likewise, the unstable results of clinical trails of the Americanginseng product have also reported. These results have demonstrated that thereare some unclear but distinct bias caused the conflicting results. This studyaims to investigate the gene expression level in mice spleen cells in differentfunctional status and the relationship with the COLD-FX treatment. This studywill provide the clues for future study of the mechanism American ginseng.Methods：1COLD-FX was purchased commercially and the powder was dilutedwith Phosphate buffered saline (PBS) to the concentration used in theexperiment.2The establishment of immune deficiency mouse modelSeven weeks adult C57BL/6mice were used. The immune suppressionmice model was made by using Dexamethasone in the cell culture media after the cell was isolated from the spleen. At first use different concentration ofDexamethasone to determine the target concentration by analyzing the data.The splenocytes was cultured with Dexamethasone about2hours. After that,collect the cell media and centrifuge for the pellet, then wash the pellet withPBS to get rid of the Dexamethasone.3Treatment in different groupsSpleen cells from adult male C57BL/6mice were divided into4groups:1)normal na ve, treated with saline;2) normal activated, treated with Con A (1μg/well);3) deficient na ve, treated with Dexamethasone (DEX,20ng/ml for2hours); and4) deficient activated, treated with DEX(20ng/ml for2hours)and Con A (1μg/well). Then, COLD-FX was added to all groups (500ng/well)for24hours.4MTT assaysMTT assays were conducted to determine the effects of COLD-FX on theproliferation of lymphocytes. Splenocytes were cultured in RPMI-1640containing10%fetal bovine serum. After24h and48h incubation MTT testwas processed. The Cell proliferation rate (CPR) was calculated to determinethe lymphocytes proliferation.5Flow cytometry assaysFlow cytometry assays were performed to evaluate the expression ofbiomarkers CD3, CD4, CD8, and CD25in the treated spleen cells. The spleencells were labeled with anti-mouse antibodies markers respectively andwashed using PBS, then analyzed with BD LSR II flow cytometer. The finalanalysis was performed by using FlowJo software.6Real-time PCR validate the expression level of Ifng and some othergeneProbes were obtained from Life Technologies. The PCR reactions werecarried out with ABI7900Real Time PCR System using ABI’s standardprotocol. Relative gene expression change was calculated with ddCt method.7Microarray gene evaluationIllumina gene expression system was used to identify transcriptional diff- erences in different mice spleen cell groups treated with COLD-FX.Results:1The proliferation status of different groups of splenocytesThe MTT results show the absorbance value (OD value) of the deficientna ve group (0.142±0.002) is lower than that of normal na ve group (0.203±0.006), the difference has statics significance (P<0.05). The OD value ofdeficient activated group (0.532±0.008) is significantly lower than normalactivated group (0.717±0.015) the difference has statics significance(P<0.05).2The effects of COLD-FX on the cell proliferation rate in differentgroupsEffects of Cell Proliferation Rate by COLD-FX in deficient na ve (17.31±0.03) is higher than that in normal na ve group (10.18±0.06) the differencehas statics significance (P<0.05). With the treatment of COLD-FX the CellProliferation Rate in deficient na ve group (17.31±0.03) and deficientactivated (24.91±0.3) is higher than that in normal na ve group (10.18±0.06)and normal activated group (11.92±0.07) respectively and the difference hasstatics significance (P<0.05).3Effect of COLD-FX on the expression of T cell marker in splenic cellsThe results showed that there were no common expression changes ofthese genes among these4different groups. However in deficient na ve groupthe CD25increased from (30.8±0.45) to (82.3±0.2) and the ratio ofCD4/CD8increased from1.59to2.39with the treatment COLD-FX. Indeficient activated group the CD3decreased from (52.1±0.05) to (33.2±0.4)and CD25decreased from (72.6±0.25) to (27.8±1.75).4Global gene expression prolife of different groups in response to thetreatment of COLD-FXThe results showed that only na ve groups were affected by COLD-FXtreatment and many more genes were regulated in the deficient na ve group.Among the top ten gene expression changes in the normal na ve group and the deficient na ve group only three were in common for both groups, i.e. theupregulation of Slpi and Wnt10a as well as the downregulation of Hbb-b1.Gene functional classification analysis was conducted with DAVIDBioinformatics Resource and found that in the normal na ve group, a cluster ofup-regulated genes was enriched.5clusters of genes were enriched in thedeficient na ve group, among them1cluster of up-regulated genes and3clusters of down-regulated genes were enriched.5Ifng gene expression validated by real-time PCRDual effects of COLD-FX on the expression of Ifng were observed indifferent groups and significant down regulation was identified in the normalna ve group by microarray assays. Using real-time PCR, the expressionpattern of Ifng was validated.Conclusions:Different proliferation and biomarker expression were observed in4different functional groups of murine spleen cells. Microarray analysisresults demonstrated that many more genes have been modulated byCOLD-FX in the deficient na ve group in comparing with other groups; andgene functional classification analysis indicated that cell cycle and IFN-γpathway activities were suppressed in the deficient na ve group as well. Onlyone gene (i.e. Hbb-b1) was found down-regulated in all three groups exceptthe na ve activated group. Two genes, Slpi and Wnt10a, were up-regulated inboth normal na ve and deficient na ve groups. Pharmacological response ofspleen cells to COLD-FX treatment depends on the functional status of thetreated cells. The study indicates that the physiological state plays animportant role in the phenotypic and transcriptional variations in response toCOLD-FX treatment.