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Stimulatory effect of Echinacea purpurea extract on the trafficking activity of mouse dendritic cells: revealed by genomic and proteomic analyses.

Yin SY, Wang WH, Wang BX, Aravindaram K, Hwang PI, Wu HM, Yang NS - BMC Genomics (2010)

Bottom Line: The transcriptomic and proteomic effects of this phytoextract on mouse bone marrow-derived dendritic cells (BMDCs) were analyzed using primary cultures.Treatment of BMDCs with [BF/S+L/Ep] did not significantly influence the phenotypic maturation activity of dendritic cells (DCs).Moreover, the signaling networks and molecules highlighted here are potential targets for nutritional or clinical application of Echinacea or other candidate medicinal plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Background: Several Echinacea species have been used as nutraceuticals or botanical drugs for "immunostimulation", but scientific evidence supporting their therapeutic use is still controversial. In this study, a phytocompound mixture extracted from the butanol fraction (BF) of a stem and leaf (S+L) extract of E. purpurea ([BF/S+L/Ep]) containing stringently defined bioactive phytocompounds was obtained using standardized and published procedures. The transcriptomic and proteomic effects of this phytoextract on mouse bone marrow-derived dendritic cells (BMDCs) were analyzed using primary cultures.

Results: Treatment of BMDCs with [BF/S+L/Ep] did not significantly influence the phenotypic maturation activity of dendritic cells (DCs). Affymetrix DNA microarray and bioinformatics analyses of genes differentially expressed in DCs treated with [BF/S+L/Ep] for 4 or 12 h revealed that the majority of responsive genes were related to cell adhesion or motility (Cdh10, Itga6, Cdh1, Gja1 and Mmp8), or were chemokines (Cxcl2, Cxcl7) or signaling molecules (Nrxn1, Pkce and Acss1). TRANSPATH database analyses of gene expression and related signaling pathways in treated-DCs predicted the JNK, PP2C-α, AKT, ERK1/2 or MAPKAPK pathways as the putative targets of [BF/S+L/Ep]. In parallel, proteomic analysis showed that the expressions of metabolic-, cytoskeleton- or NF-κB signaling-related proteins were regulated by treatment with [BF/S+L/Ep]. In vitro flow cytometry analysis of chemotaxis-related receptors and in vivo cell trafficking assay further showed that DCs treated with [BF/S+L/Ep] were able to migrate more effectively to peripheral lymph node and spleen tissues than DCs treated as control groups.

Conclusion: Results from this study suggest that [BF/S+L/Ep] modulates DC mobility and related cellular physiology in the mouse immune system. Moreover, the signaling networks and molecules highlighted here are potential targets for nutritional or clinical application of Echinacea or other candidate medicinal plants.

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Correlation of biological sample replicates. A, DMSO (control) replicates (Affymetrix ID: 579, 580) after 4 h treatment. Pearson correlation value: 0.99735. B, [BF/S+L/Ep] replicates (Affymetrix ID: 581, 582) after 4 h treatment. Pearson correlation value: 0.99303. C, [DMSO] (control) replicates (Affymetrix ID: 583, 584) after 12 h treatment. Pearson correlation value: 0.99628. D, [BF/S+L/Ep] replicates (Affymetrix ID: 585, 586) after 12 h treatment. Pearson correlation value: 0.99631. Data analysis used Spotfire software. Pearson correlation value and number of unchanged transcripts were calculated using GCOS software.
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Figure 2: Correlation of biological sample replicates. A, DMSO (control) replicates (Affymetrix ID: 579, 580) after 4 h treatment. Pearson correlation value: 0.99735. B, [BF/S+L/Ep] replicates (Affymetrix ID: 581, 582) after 4 h treatment. Pearson correlation value: 0.99303. C, [DMSO] (control) replicates (Affymetrix ID: 583, 584) after 12 h treatment. Pearson correlation value: 0.99628. D, [BF/S+L/Ep] replicates (Affymetrix ID: 585, 586) after 12 h treatment. Pearson correlation value: 0.99631. Data analysis used Spotfire software. Pearson correlation value and number of unchanged transcripts were calculated using GCOS software.

Mentions: We next investigated the expression of specific genes related to the cellular biochemistry and physiology of immature DCs. Affymetrix DNA microarray analyses showed that, although the gene expression of the cell surface markers tested above were not affected, a series of other genes related to DC activities were affected by 75 μg/ml [BF/S+L/Ep]. Initial experimental setups using biological sample replicates showed that our test cell culture systems and experimental protocols, e.g., RNA preparation, were highly reproducible and consistent, e.g., Pearson's Correlation values for test biological sample replicates were between 0.993 and 0.996 (Figure 2). All genes identified from the raw data of our Affymetrix gene chip analyses were subjected to a gene filtering proctocol, using established statistics software (Spotfire), to select and identify candidate differentially expressed genes for further studies. We chose genes that scored positive in at least 4 different chips and that had a coefficient of variation (CV) of over 0.4. A total of 907 and 1256 genes had changed expression significantly by 4 h and by 12 h post-treatment with [BF/S+L/Ep], respectively. Among these, 172 and 264 genes respectively were identified as immune-related genes (Table 1). For up-regulated or down-regulated genes, only those genes that showed at least a 2-fold change in RNA transcript level in two independent experiments were considered for further analysis. Differences in expression level were calculated by dividing the signal intensity values of genes from [BF/S+L/Ep]-treated cells by the intensity values of genes from the vehicle-treated (control) cells. Overall, similar numbers of genes were up-regulated and down-regulated after treatment with [BF/S+L/Ep] (Table 1). The expression of 29 genes more than doubled in [BF/S+L/Ep]-treated DCs, compared to vehicle control, at 4 h post-treatment, and a group of 31 genes were up-regulated (i.e., ≥ 2-fold change) at 12 h post-treatment. In contrast, 26 and 35 genes were down-regulated (expression more than halved) in [BF/S+L/Ep]-treated DCs at 4 h and 12 h post-treatment, respectively (Table 1). It is important to note that two different sets of 51 non-immune related genes were highly affected at 4 h and 12 h time points (Table 1). The relative changes in gene expression levels in [BF/S+L/Ep]-treated DCs are shown in Table 2 and Table 3 for 4 h and 12 h treatments, respectively. The unknown genes and sequences are not shown in these tables.


Stimulatory effect of Echinacea purpurea extract on the trafficking activity of mouse dendritic cells: revealed by genomic and proteomic analyses.

Yin SY, Wang WH, Wang BX, Aravindaram K, Hwang PI, Wu HM, Yang NS - BMC Genomics (2010)

Correlation of biological sample replicates. A, DMSO (control) replicates (Affymetrix ID: 579, 580) after 4 h treatment. Pearson correlation value: 0.99735. B, [BF/S+L/Ep] replicates (Affymetrix ID: 581, 582) after 4 h treatment. Pearson correlation value: 0.99303. C, [DMSO] (control) replicates (Affymetrix ID: 583, 584) after 12 h treatment. Pearson correlation value: 0.99628. D, [BF/S+L/Ep] replicates (Affymetrix ID: 585, 586) after 12 h treatment. Pearson correlation value: 0.99631. Data analysis used Spotfire software. Pearson correlation value and number of unchanged transcripts were calculated using GCOS software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3091753&req=5

Figure 2: Correlation of biological sample replicates. A, DMSO (control) replicates (Affymetrix ID: 579, 580) after 4 h treatment. Pearson correlation value: 0.99735. B, [BF/S+L/Ep] replicates (Affymetrix ID: 581, 582) after 4 h treatment. Pearson correlation value: 0.99303. C, [DMSO] (control) replicates (Affymetrix ID: 583, 584) after 12 h treatment. Pearson correlation value: 0.99628. D, [BF/S+L/Ep] replicates (Affymetrix ID: 585, 586) after 12 h treatment. Pearson correlation value: 0.99631. Data analysis used Spotfire software. Pearson correlation value and number of unchanged transcripts were calculated using GCOS software.
Mentions: We next investigated the expression of specific genes related to the cellular biochemistry and physiology of immature DCs. Affymetrix DNA microarray analyses showed that, although the gene expression of the cell surface markers tested above were not affected, a series of other genes related to DC activities were affected by 75 μg/ml [BF/S+L/Ep]. Initial experimental setups using biological sample replicates showed that our test cell culture systems and experimental protocols, e.g., RNA preparation, were highly reproducible and consistent, e.g., Pearson's Correlation values for test biological sample replicates were between 0.993 and 0.996 (Figure 2). All genes identified from the raw data of our Affymetrix gene chip analyses were subjected to a gene filtering proctocol, using established statistics software (Spotfire), to select and identify candidate differentially expressed genes for further studies. We chose genes that scored positive in at least 4 different chips and that had a coefficient of variation (CV) of over 0.4. A total of 907 and 1256 genes had changed expression significantly by 4 h and by 12 h post-treatment with [BF/S+L/Ep], respectively. Among these, 172 and 264 genes respectively were identified as immune-related genes (Table 1). For up-regulated or down-regulated genes, only those genes that showed at least a 2-fold change in RNA transcript level in two independent experiments were considered for further analysis. Differences in expression level were calculated by dividing the signal intensity values of genes from [BF/S+L/Ep]-treated cells by the intensity values of genes from the vehicle-treated (control) cells. Overall, similar numbers of genes were up-regulated and down-regulated after treatment with [BF/S+L/Ep] (Table 1). The expression of 29 genes more than doubled in [BF/S+L/Ep]-treated DCs, compared to vehicle control, at 4 h post-treatment, and a group of 31 genes were up-regulated (i.e., ≥ 2-fold change) at 12 h post-treatment. In contrast, 26 and 35 genes were down-regulated (expression more than halved) in [BF/S+L/Ep]-treated DCs at 4 h and 12 h post-treatment, respectively (Table 1). It is important to note that two different sets of 51 non-immune related genes were highly affected at 4 h and 12 h time points (Table 1). The relative changes in gene expression levels in [BF/S+L/Ep]-treated DCs are shown in Table 2 and Table 3 for 4 h and 12 h treatments, respectively. The unknown genes and sequences are not shown in these tables.

Bottom Line: The transcriptomic and proteomic effects of this phytoextract on mouse bone marrow-derived dendritic cells (BMDCs) were analyzed using primary cultures.Treatment of BMDCs with [BF/S+L/Ep] did not significantly influence the phenotypic maturation activity of dendritic cells (DCs).Moreover, the signaling networks and molecules highlighted here are potential targets for nutritional or clinical application of Echinacea or other candidate medicinal plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan.

ABSTRACT

Background: Several Echinacea species have been used as nutraceuticals or botanical drugs for "immunostimulation", but scientific evidence supporting their therapeutic use is still controversial. In this study, a phytocompound mixture extracted from the butanol fraction (BF) of a stem and leaf (S+L) extract of E. purpurea ([BF/S+L/Ep]) containing stringently defined bioactive phytocompounds was obtained using standardized and published procedures. The transcriptomic and proteomic effects of this phytoextract on mouse bone marrow-derived dendritic cells (BMDCs) were analyzed using primary cultures.

Results: Treatment of BMDCs with [BF/S+L/Ep] did not significantly influence the phenotypic maturation activity of dendritic cells (DCs). Affymetrix DNA microarray and bioinformatics analyses of genes differentially expressed in DCs treated with [BF/S+L/Ep] for 4 or 12 h revealed that the majority of responsive genes were related to cell adhesion or motility (Cdh10, Itga6, Cdh1, Gja1 and Mmp8), or were chemokines (Cxcl2, Cxcl7) or signaling molecules (Nrxn1, Pkce and Acss1). TRANSPATH database analyses of gene expression and related signaling pathways in treated-DCs predicted the JNK, PP2C-α, AKT, ERK1/2 or MAPKAPK pathways as the putative targets of [BF/S+L/Ep]. In parallel, proteomic analysis showed that the expressions of metabolic-, cytoskeleton- or NF-κB signaling-related proteins were regulated by treatment with [BF/S+L/Ep]. In vitro flow cytometry analysis of chemotaxis-related receptors and in vivo cell trafficking assay further showed that DCs treated with [BF/S+L/Ep] were able to migrate more effectively to peripheral lymph node and spleen tissues than DCs treated as control groups.

Conclusion: Results from this study suggest that [BF/S+L/Ep] modulates DC mobility and related cellular physiology in the mouse immune system. Moreover, the signaling networks and molecules highlighted here are potential targets for nutritional or clinical application of Echinacea or other candidate medicinal plants.

Show MeSH
Related in: MedlinePlus