Gene Expression Profiling on the Molecular Action of Danshen-Gegen Formula in a Randomized Placebo-Controlled Trial of Postmenopausal Women with Hypercholesterolemia

The Danshen-Gegen formula (DG) is a traditional Chinese herbal formula which has long been used to treat cardiovascular disease. DG was found to be a cardiovascular tonic in our recent research. However, a comprehensive investigation of the molecular mechanism of DG in cardiovascular disease has not been performed. The aim of this study was to clarify the transcriptional profiling of genes modulated by DG on postmenopausal women by using DNAmicroarray technology. We obtained 29 whole blood samples both from DG-treated and placebo-treated subjects. Blood lipid profile and intima-media thickness (IMT) were measured. Affymetrix GeneChip was used to identify differentially expressed genes (DEGs), followed by validation by the real-time PCR method. The results showed that DG-treated group has a significant improvement in IMT and lipid profile as compared to placebo-treated group. For the genomic study, the DG-treated group has a higher number of DEGs identified as compared to the placebo-treated group. Two important biological processes of “regulation of systemic arterial blood pressure by hormone” and “regulation of smooth muscle proliferation” have been identified by GePS in the DG-treated group. No significant biological process and cellular components were identified in the placebo-treated group. This genomic study on the molecular action of DG in postmenopausal women gathered sufficient molecular targets and pathways to reveal that DG could improve neointima thickening and hypertension

In Vivo Study on the Pharmacological Interactions between a Chinese Herbal Formula ELP and Antiresorptive Drugs to Counteract Osteoporosis

Antiresorptive drugs, alendronate and raloxifene, are effective in lowering bone mineral density (BMD) loss in postmenopausal women. However, long-term treatment may be associated with serious side effects. Our research group has recently discovered that a Chinese herbal formula, ELP, could significantly reduce BMD loss in animal and human studies. Therefore, the present study aimed to investigate the potential synergistic bone-protective effects of different herb-drug combinations using ovariectomized rats. To assess the efficacy of different combinations, the total BMD was monitored biweekly in the 8-week course of daily oral treatment. Bone microarchitecture, bone strength, and deoxypyridinoline level were also determined after 8 weeks. From our results, coadministration of ELP and raloxifene increased the total tibial BMD by 5.26% (2.5 mg/kg/day of raloxifene; P=0.014) and 5.94% (0.25 mg/kg/day of raloxifene; P=0.026) when compared with the respective dosage groups with raloxifene alone. Similar synergistic effects were also observed in BMD increase at distal femur (0.25 mg/kg/day; P=0.001) and reduction in urinary deoxypyridinoline crosslink excretion (2.5 and 0.25 mg/kg/day; both P=0.02). However, such interactions could not be observed in all alendronate-treated groups. Our data provide first evidence that ELP could synergistically enhance the therapeutic effects of raloxifene, so that the clinical dosage of raloxifene could be reduced

Identification of Target Genes Involved in Wound Healing Angiogenesis of Endothelial Cells with the Treatment of a Chinese 2-Herb Formula

<div><p>Angiogenesis is vitally important in diabetic wound healing. We had previously demonstrated that a Chinese 2-herb formula (NF3) significantly stimulated angiogenesis of HUVEC in wound healing. However, the molecular mechanism has not yet been elucidated. In line with this, global expression profiling of NF3-treated HUVEC was performed so as to assess the regulatory role of NF3 involved in the underlying signaling pathways in wound healing angiogenesis. The microarray results illustrated that different panels of differentially expressed genes were strictly governed in NF3-treated HUVEC in a time-regulated manner. The microarray analysis followed by qRT-PCR and western blotting verification of NF3-treated HUVEC at 6 h revealed the involvement of various genes in diverse biological process, e.g., MAP3K14 in anti-inflammation; SLC5A8 in anti-tumorogenesis; DNAJB7 in protein translation; BIRC5, EPCAM, INSL4, MMP8 and NPR3 in cell proliferation; CXCR7, EPCAM, HAND1 and MMP8 in migration; CXCR7, EPCAM and MMP8 in tubular formation; and BIRC5, CXCR7, EPCAM, HAND1, MMP8 and UBD in angiogenesis. After 16 h incubation of NF3, other sets of genes were shown with differential expression in HUVEC, e.g., IL1RAPL2 and NR1H4 in anti-inflammation; miR28 in anti-tumorogenesis; GRIN1 and LCN1 in anti-oxidation; EPB41 in intracellular signal transduction; PRL and TFAP2A in cell proliferation; miR28, PRL and SCG2 in cell migration; PRL in tubular formation; and miR28, NR1H4 and PRL in angiogenesis. This study provided concrete scientific evidence in support of the regulatory role of NF3 on endothelial cells involved in wound healing angiogenesis.</p></div

Western blotting of NF3 on HUVEC for 6 and 16 h.

<p>Immunobloting was performed three times using independent cell lysates and representative blots of (A) 6 h and (B) 16 h were shown.</p

Primer list of NF3-treated HUVEC versus control at 16 h.

<p>Primer list of NF3-treated HUVEC versus control at 16 h.</p

Hypothetical diagram of mechanistic actions of NF3 on HUVEC at 16 h.

<p>The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 16 h. Yellow molecule represented differentially expressed genes identified from microarray analysis. Blue box represented the suggested signaling pathway. Purple box represented the molecular mediators. Green box represented the biological responses. + indicated as positive regulation and–indicated negative regulation.</p

Network connectivity of identified genes in NF3-treated HUVEC by GePS.

<p>(A) Differentially expressed genes with frequent co-citations and interactions in NF3 treated HUVEC at 6 h. (B) Differentially expressed genes with frequent co-citations and interactions in NF3 treated HUVEC at 16 h. Genes were identified by their gene symbols (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139342#pone.0139342.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139342#pone.0139342.s002" target="_blank">S2</a> Tables). Dashed lines indicated association by co-citation. Up arrow and down arrow corresponded to up- or down-regulation, respectively.</p

Gene ontology classification of the identified genes in NF3-treated HUVEC at 16 h by UniProt.

<p>Gene ontology classification of the identified genes in NF3-treated HUVEC at 16 h by UniProt.</p

Hypothetical diagram of mechanistic actions of NF3 on HUVEC at 6 h.

<p>The molecular gene targets, respective signaling pathways and molecule mediators associated with the biological responses was demonstrated in NF3-treated HUVEC at 6 h. Yellow molecules represented differentially expressed genes identified from microarray analysis. Blue box represented the suggested signaling pathway. Purple box represented the molecular mediators. Green box represented the biological responses. + indicated as positive regulation and–indicated negative regulation.</p

Primer list of NF3-treated HUVEC versus control at 6 h.

<p>Primer list of NF3-treated HUVEC versus control at 6 h.</p