Cheuk-Lun Liu

Synergistic interaction between Astragali Radix and Rehmanniae Radix in a Chinese herbal formula to promote diabetic wound healing

ETHNOPHARMACOLOGICAL RELEVANCE Astragali Radix (AR) and Rehmanniae Radix (RR) are two traditional Chinese medicines widely used in China for treating diabetes mellitus and its complications, such as diabetic foot ulcer. AIM OF STUDY In our previous study, a herbal formula NF3 comprising AR and RR in the ratio of 2:1 was found effective in enhancing diabetic wound healing in rats through the actions of tissue regeneration, angiogenesis promotion and inflammation inhibition. The aims of the present study were to investigate the herb-herb interaction (or the possible synergistic effect) between AR and RR in NF3 to promote diabetic wound healing and to identify the principal herb in the formula by evaluating the potencies of individual AR and RR in different mechanistic studies. MATERIALS AND METHODS A chemically induced diabetic foot ulcer rat model was used to examine the wound healing effect of NF3 and its individual herbs AR and RR. For mechanistic studies, murine macrophage cell (RAW 264.7) inflammation, human fibroblast (Hs27) proliferation and human endothelial cell (HMEC-1) migration assays were adopted to investigate the anti-inflammatory, granulation formation and angiogenesis-promoting activities of the herbal extracts, respectively. RESULTS In the foot ulcer animal model, neither AR nor RR at clinical relevant dose (0.98g/kg) promoted diabetic wound healing. However, when they were used in combination as NF3, synergistic interaction was demonstrated, of which NF3 could significantly reduce the wound area of rats when compared to water group (p<0.01). For anti-inflammation and granulation formation, AR was more effective than RR in inhibiting lipopolysaccharide (LPS)-induced nitric oxide production from RAW 264.7 cells and promoting Hs27 fibroblast proliferation. In the aspect of angiogenesis promotion, only NF3 promoted cell migration of HMEC-1 cells. CONCLUSIONS AR plays a preeminent role in the anti-inflammatory and fibroblast-proliferating activities of NF3. The inclusion of RR, however, is crucial for NF3 to exert its overall wound-healing as well as the underlying angiogenesis-promoting effects. The results of present study justified the combined usage of AR and RR in the ratio of 2:1 as NF3 to treat diabetic foot ulcer and illustrated that AR is the principal herb in this herbal formula.

Molecular mechanisms of angiogenesis effect of active sub-fraction from root of Rehmannia glutinosa by zebrafish sprout angiogenesis-guided fractionation

ETHNOPHARMACOLOGICAL IMPORTANCE The root of Rehmannia glutinosa (Rehmanniae Radix (RR)) is clinically used as a wound-healing agent in traditional Chinese medicine. Angiogenesis acts crucially in the pathogenesis of chronic wound healing. The present study investigated the angiogenesis effect and its underlying mechanism of RR through zebrafish sprout angiogenesis guided-fractionation. MATERIALS AND METHODS The in vivo angiogenesis effect was studied by analyzing the number of ectopic sprouts formed upon sub-intestinal vessel of transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish embryos by fluorescence microscopy. Quantitative real-time PCR gene expression of the zebrafish embryos was further performed using a panel of 30 angiogenesis-associated genes designed for zebrafish sprout angiogenesis. Classical in vitro angiogenesis assays using human microvascular endothelial cells (HMEC-1) was accompanied. RESULTS We demonstrated that among all RR sub-fractions tested, C1-1 treated-zebrafish embryos possessed the most potent angiogenesis activities (from 190 to 780 ng/ml, p<0.001) in sprout formation in the zebrafish model. Quantitative gene expression of the treated embryos demonstrated significant up-regulation in MMP-9 (p<0.05), ANGPT1 (p<0.05), EGFR (p<0.05), EPHB4 (p<0.01), and significant down-regulation in Ephrin B2 (p<0.05), Flt-1 (p<0.05) and Ets-1 (p<0.05). C1-1 treatment could also significantly (p<0.001-0.05) stimulate HMEC-1 cell migration in scratch assay. Significant increase (p<0.05) in mean tubule length was observed in the C1-1-treated HMEC-1 cells in the tubule formation assay. CONCLUSIONS Our zebrafish sprout angiogenesis model-guided fractionation revealed that C1-1 possessed the most potent angiogenesis effect in RR. The design of the panel with 30 tailor-made angiogenesis-associated genes exhibited in zebrafish gene expression analysis showed that C1-1 could trigger differential expression of various angiogenesis-associated genes, such as VEGFR3 and MMP9, which played key role in angiogenesis. The pro-angiogenic activity of C1-1 was further confirmed in the translated study in motogenic and tubule-inducing effect using HMEC-1.

Molecular angiogenic events of a two-herb wound healing formula involving MAPK and Akt signaling pathways in human vascular endothelial cells

The emergence of electric cell‐substrate impedance sensing (ECIS) technology has provided new insight in advanced cell behavioral study by its nanometer sensitivity, precise electrical wounds generation, and high reproducibility that can be monitored in real time in a noninvasive way. However, little is known regarding pro‐angiogenic agents in wound healing studies using endothelial cells evaluated with ECIS technology. Our previous studies showed a prominent wound healing effect of a two‐herb formula (NF3) comprising of Astragali Radix and Rehmanniae Radix in a rat chronic wound model through actions including angiogenesis. Here we further investigated the angiogenic effect and its underlying molecular mechanism through proliferation, motility, and tubule formation of human vascular endothelial cells (HECV) using ECIS technology. It was first shown that HECV treated with NF3 had a higher resistance than that of control using ECIS cell attachment and cell migration model (p < 0.01). We further validated in a scratch assay that NF3 treatment significantly stimulated HECV cell migration (p < 0.01–0.05). Also, NF3‐treated HECV were observed to develop into a significantly more branched tubular structure when compared with control (p < 0.05–0.01). Meanwhile, Western blot analysis of NF3‐treated HECV revealed the activated expression of p‐Akt, and mitogen‐activated protein (MAP) kinases for p‐ERK, p‐p38, and p‐JNK. We propose that the effect of NF3 in the promotion of endothelial cell migration and tubule formation could be mediated through pathways involving p‐Akt and activated MAP kinases. Hence, we demonstrated the complexity of the angiogenic effect activated by NF3 molecularly and functionally. NF3 treatment could offer therapeutic value to chronic wound healing for its pro‐angiogenic efficacy.

Pro-angiogenic effects of Carthami Flos whole extract in human microvascular endothelial cells in vitro and in zebrafish in vivo

AIM Carthami Flos (CF) is a Chinese herb traditionally used for cardiovascular disease and bone injury in China with pharmacological effects on improving blood circulation. The aim of this study was to investigate the angiogenic potential of CF whole extract (extracted by boiling with water, followed by ethanol) and the underlying mechanisms in human microvascular endothelial cells (HMEC-1) in vitro and in transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish with transgenic endothelial cells expressing EGFP (Enhanced Green Fluorescent Protein) in vivo. METHODS Effects of CF whole extract on cell proliferation, migration and tube formation in HMEC-1 cells in vitro were detected by MTT assay, wound healing assay and tube formation assay. Its angiogenic effect in zebrafish was investigated by monitoring the sprout number in the sub-intestinal vessel (SIV), and the underlying mechanisms were tested by quantitative real-time PCR. RESULTS CF whole extract increased cell proliferation, migration and tube formation in vitro in HMEC-1 cells. Its angiogenic effect was also confirmed in vivo in zebrafish by increasing the sprout number in the SIV. As determined by quantitative real-time PCR, CF whole extract up-regulated the expression of angiogenesis-related genes in zebrafish, including angiogenic and its associated growth factors and receptors (e.g. IGF1, CTGF, NRP2, and VEGFR3), transcription factor (e.g. HIF1A), matrix degradation and endothelial cell migration-related factors (e.g. MMP2, MMP9, TIMP2, PLG and PLAU), cell adhesion molecules (e.g. ITGAV, ITGB3, beta-catenin and PECAM1), tubule formation factors (e.g. ANGPT1, TIE-2, PDGFR-B, CDH5, S1PR1, FGF2, Shh, and TGFRB1), and blood vessel maturation/formation factor (e.g. Ephrin B2). CONCLUSIONS CF whole extract increased angiogenesis in HMEC-1 cells in vitro and in zebrafish in vivo with multiple mechanisms.

In vitro and in vivo mechanistic study of a novel proanthocyanidin, GC-(4→8)-GCG from cocoa tea (Camellia ptilophylla) in antiangiogenesis

Angiogenesis, the process of blood vessel formation, is critical to tumor growth. Ant-angiogenic strategies demonstrated importance in cancer therapy. Cocoa tea (Camellia ptilophylla), a naturally decaffeinated tea commonly consumed as a healthy drink in southern China, had recently been found to be a potential candidate for antiangiogenesis. A novel proanthocyanidin, GC-(4→8)-GCG, which consisted of gallocatechin and gallocatechin 3-O gallate moieties, was discovered and thought to be one of the effective candidates for antiangiogenesis. Hence, the present study aimed to evaluate the antiangiogenesis activities of GC-(4→8)-GCG in vitro and in vivo, and SU5416 was applied as a positive control. The inhibitory effects of GC-(4→8)-GCG on three important processes involved in angiogenesis, i.e., proliferation, migration and differentiation, were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using transgenic zebrafish embryos TG(fli1:EGFP)y1/+(AB) as an animal model of angiogenesis, the antiangiogenic effect of GC-(4→8)-GCG was further verified in vivo. Our results demonstrated that GC-(4→8)-GCG significantly inhibited migration (P<.001) and tubule formation (P<.001-.05) of HMEC-1 in dose-dependent manner. Regarding intracellular signal transduction, GC-(4→8)-GCG attenuated the phosphorylation of ERK, Akt and p38 dose-dependently in HMEC-1. In zebrafish embryo, the formation of new blood vessels was effectively inhibited by GC-(4→8)-GCG in a dose-dependent manner after 3 days of treatment (P<.001-.05). In conclusion, these results revealed that our novel proanthocyanidin, GC-(4→8)-GCG might be a potential and promising agent of natural resource to be further developed as an antiangiogenic agent.

Stachyose: One of the Active Fibroblast-proliferating Components in the Root of Rehmanniae Radix (地黃 dì huáng)

This study aimed to investigate and compare the fibroblast-proliferating activities of different Rehmanniae Radix (RR) samples and its chemical components using human normal fibroblast cells Hs27. Those active components were quantified in differently treated RR samples using UPLC so as to correlate activity with component content. Our results showed that dried RR aqueous extract exhibited the most potent fibroblast-proliferating activity. Stronger effect was observed when ethanol with heating was applied in the extraction process. Stachyose and verbascoside were demonstrated for their first time to exhibit significant stimulatory effects on fibroblast proliferation. However, the proliferating effect of dried RR extract did not correlate with the stachyose content, and verbascoside was not responsible for the fibroblast proliferative effect of RR since it was undetectable in all samples. In conclusion, stachyose only contributed in part to the activity of RR, suggesting that other active components might be present and yet to be found.

Preparative separation of gallocatechin gallate from Camellia ptilophylla using macroporous resins followed by sephadex LH-20 column chromatography

Gallocatechin gallate (GCG) possesses multiple potential biological activities. However, the content of GCG in traditional green tea is too low which limits its in-depth pharmacological research and application. In the present study, a simple, efficient and environment-friendly chromatographic separation method was developed for preparative enrichment and separation of GCG from cocoa tea (Camellia ptilophylla) which contains high content of GCG. In the first step, the adsorption properties of selected resins were evaluated, and XAD-7HP resin was chosen by its adsorption and desorption properties for GCG. In order to maximize column efficiency for GCG collection, the operating parameters (e.g., flow rate, ethanol concentration, and bed height) were optimized. We found that the best combination was the feed concentration at 20mg/mL, flow rate at 0.75 BV/h and the ratio of diameter to bed heights as 1:12. Under these conditions, the purity of GCG was 45% with a recovery of 89%. In order to obtain pure target, a second step was established using column chromatography with sephadex LH-20 gel and 55% ethanol-water solution as eluent. After this step, the purity of the GCG was 91% with a recovery of 68% finally.