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

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. 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. 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. 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. 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.

Lau KM ，Lai KK ，Liu CL ，Tam JC ，To MH ，Kwok HF ，Lau CP ，Ko CH ，Leung PC ，Fung KP ，Poon SK ，Lau CB ... -  《-》

Transcriptional profiling of human skin fibroblast cell line Hs27 induced by herbal formula Astragali Radix and Rehmanniae Radix.

The herbs Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer. Chinese herbal formulus comprising Astragali Radix (AR) and Rehmanniae Radix (RR) have been shown to improve the healing of diabetic foot ulcer through enhancing the viability of primary fibroblasts in diabetic patients suffering insulin resistance. Our previous study demonstrated that the herbal formula NF3 comprising of AR and RR in the ratio of 2:1 was effective in promoting wound healing in diabetic rats, and in vitro data indicated that the wound healing effects of NF3 might be due to the regulation and coordination of inflammation, angiogenesis and tissue regeneration. However, the underlying molecular mechanism has not been well investigated. In this study, we investigated the cellular and molecular effects of the herbal formula NF3 on human skin fibroblast cells. Human skin fibroblast cells Hs27 were treated with NF3 ranging from 0 to 8 mg/ml for 24h, and the cells without NF3 treatment were used as control. Cell proliferation assay and cell cycle analysis were performed. Transcriptional profiles of Hs27 cells upon NF3 treatment were acquired by using a human cDNA microarray containing 10,000 genes, and the signaling pathways differentially regulated by NF3 were identified and analyzed. NF3 promoted Hs27 cell proliferation and cell cycle progression. Microarray analysis revealed that 116 genes were differentially expressed upon NF3 treatment. Functional analysis of the genes indicated that NF3 mainly activated Wnt and angiogenesis related pathways, which are directly related to cell proliferation, angiogenesis, extracellular matrix (ECM) formation and inflammation during the process of wound healing. This study provides insight into the molecular mechanism of how the herbal formula Astragali Radix and Rehmanniae Radix may serve as potential therapeutics for wound healing.

Zhang Q ，Wei F ，Fong CC ，Yu WK ，Chen Y ，Koon CM ，Lau KM ，Leung PC ，Lau CB ，Fung KP ，Yang M ... -  《-》

Herbal formula Astragali Radix and Rehmanniae Radix exerted wound healing effect on human skin fibroblast cell line Hs27 via the activation of transformation growth factor (TGF-β) pathway and promoting extracellular matrix (ECM) deposition.

Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and as the principal herbs in treating diabetic foot ulcer. In this study, we investigated the effect of NF3, which comprises of AR and RR in the ratio of 2:1(w/w), on skin fibroblast cell migration and the activation of selected genes and proteins related to wound healing. Human skin fibroblast cell line Hs27 was treated with NF3 at 4 mg/ml for 24h, and in vitro scratch wound healing and quantitative cell migration assays were performed, respectively. The expression of transformation growth factor (TGF-β1) and bone morphogenetic protein 6 (BMP6) in Hs27 cells with or without NF3 treatment was analyzed by western blot analysis. In addition, the expression of a panel of genes involved in human TGF-β signaling pathway was analyzed in Hs27 cells upon NF3 treatment (4 mg/ml, 24 h) by quantitative real-time PCR (qRT-PCR). Furthermore, the expression of several genes and proteins associated with ECM synthesis was investigated by qRT-PCR analysis or/and ELISA techniques. The results suggested that NF3 promoted the migration of human skin fibroblast cells. Western blot analysis demonstrated that NF3 up-regulated TGF-β1 and BMP-6 synthesis. qRT-PCR analysis revealed that the expression of 26 genes in Hs27 cells was changed upon NF3 induction, including TGF-β superfamily ligands and down stream effectors genes, and genes involved in TGF/Smad pathway, and Ras/MAPK (non-Smad) pathway. Among the extracellular matrix (ECM)-related molecules, it was found that NF3 up-regulated the expression of type I and III collagens, fibronectin as well as TIMP-1, and down-regulated the MMP-9 expression in skin fibroblast cells. This study demonstrated that herb formula NF3 could enhance skin fibroblast cell migration and activated genes involved in TGF-β1 pathway. NF3 could regulate gene transcription for extracellular matrix synthesis via the Smad pathway, and gene transcription for cell motility via the Ras/MAPK (non-Smad) pathway.

Zhang Q ，Fong CC ，Yu WK ，Chen Y ，Wei F ，Koon CM ，Lau KM ，Leung PC ，Lau CB ，Fung KP ，Yang M ... -  《-》

Angiogenic efficacy of simplified 2-herb formula (NF3) in zebrafish embryos in vivo and rat aortic ring in vitro.

Diabetic foot ulceration results in high risk of lower extremity amputation, and represents a significant health care expenditure worldwide. Radix Astragali (RA) and Radix Rehmanniae (RR) are widely used Chinese medicinal herbs in treating diabetes, and have shown positive effects in enhancing wound healing in diabetic foot ulcer animal model. The angiogenic efficacy of NF3, a simplified 2-herb formula consisting of RA and RR in 2:1 ratio, was investigated. Median lethal concentration (LC50) and median effective concentration (EC50) were determined by treating zebrafish embryos with different concentrations of NF3 from 20 hpf to 72 hpf. The angiogenic activity of NF3 was examined in zebrafish embryos in vivo and by rat aortic ring assay in vitro. Cell cycle analysis of endothelial cells induced by NF3 was analyzed by flow cytometry using transgenic zebrafish Tg(fli1:EGFP). Real-time PCR was used to analyze mRNA expression profiles of selected genes involved in VEGF, FGF and MAPK pathways. NF3 enhanced blood vessel formation as indicated by extra growth of intersegmental vessels in zebrafish embryos, and increased microvessels formation in rat aortic ring. NF3 also enhanced endothelial cells proliferation as shown by increased percentage of cells accumulating in S phase and G2/M phase of the cell cycle. NF3 exposure significantly induced up-regulation of VEGF-A, Flk-1, fgf1 and bRaf expression in zebrafish embryos. Our results demonstrated that NF3 was effective in promoting angiogenesis in zebrafish embryos and by rat aortic ring assay, which provided scientific basis to support the use of NF3 as potential therapeutics in treating diabetic foot ulceration.

Tse HY ，Hui MN ，Li L ，Lee SM ，Leung AY ，Cheng SH ... -  《-》

Healing effect of a two-herb recipe (NF3) on foot ulcers in Chinese patients with diabetes: a randomized double-blind placebo-controlled study.

In the present study, we examined the effect of a two-herb traditional Chinese medicine (NF3), comprised of Astragali Radix and Radix Rehmanniae, on the healing of diabetic foot ulcer and the possible molecular mechanisms involved. This was a prospective randomized double-blind placebo-controlled study. Sixteen diabetic patients were randomized to receive either placebo or NF3 for 6 months. Ulcer healing and sensory changes were examined. Molecular studies included measurement of serum tumor necrosis factor (TNF)-α and RNA microarray investigation. The daily rate of reduction in ulcer area was 3.55% in the NF3 group and 1.52% in the placebo group (P = 0.062). In the index limb, the number of negative tests for sensory neuropathy using monofilament was reduced from 27% to 7% in the NF3 group and from 37% to 35% in the placebo group (P < 0.001). In addition, NF3 significantly decreased serum TNF-α levels (P = 0.034). Microarray studies revealed concerted changes following NF3 treatment in the expression of genes implicated in fibroblast regeneration, angiogenesis, and anti-inflammation. In this proof-of-concept study, 6-month treatment with NF3 was associated with improved wound healing and sensation accompanied by concerted changes in gene expression.

Ko CH ，Yi S ，Ozaki R ，Cochrane H ，Chung H ，Lau W ，Koon CM ，Hoi SW ，Lo W ，Cheng KF ，Lau CB ，Chan WY ，Leung PC ，Chan JC ... -  《-》