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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-[beta]) pathway and promoting extracellular matrix (ECM) deposition.



Astragali Radix

Rehmanniae Radix

Human skin fibroblast cells Hs27

Wound healing

Transformation growth factor


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 24 h, and in vitro scratch wound healing and quantitative cell migration assays were performed, respectively. The expression of transformation growth factor (TGF-[beta]1) 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-[beta] 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-[beta]1 and BMP-6 synthesis. qRT-PCR analysis revealed that the expression of 26 genes in Hs27 cells was changed upon NF3 induction, including TGF-[beta] superfa-mily 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 land 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-[beta]1 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.

[c] 2012 Elsevier GmbH. All rights reserved.


Chinese herbal formula Astragali Radix (AR) and Rehmanniae Radix (RR) were found to promote the formation of granulation tissue at diabetic foot ulcer bed (Wong et al. 2001; Leung et al. 2008), and stimulate the viability of primary fibroblasts in patients with insulin resistance (Liu et al. 2007). In particular, the formula used in this study, NF3 (AR:RR= 2:1, w/w), has been demonstrated to exhibit wound healing effects on diabetic foot ulcer using animal model study (Tam et al. 2011), and its effect was attributed to the synergistic interaction between its two component herbs, AR and RR (Lau etal. 2012). However, the molecular mechanisms involved in the pharmacological actions of NF3 are not very clear understood.

Wound healing comprises an ordered sequence of events including cell migration and proliferation, synthesis of extracellular matrix, angiogenesis and remodeling. Transformation growth factor (TGF-[beta]) is one of the most powerful and widely distributed profibrogenic mediators in the body and regulates many of these processes (Amento and Beck 1991). After tissue injury, TGF-[beta] can enhance fibrogenesis and remodeling in normal healing, and alter the balance between extracellular matrix synthesis and degradation by inducing an increase in synthesis of matrix components and a parallel decrease in overall ECM proteolytic activity (Wells 2000).

Once a wound occurs, a number of different types of cells are recruited to participate in the healing process (Werner and Grose 2003). Fibroblasts, the most common connective tissue cells, play a critical role in wound healing. After an injury to skin, fibroblasts are stimulated by local growth factors and cytokines, which are produced by platelets, macrophages and fibroblasts themselves, to proliferate and migrate to the wound. Fibroblasts in the wound edges begin to grow and migrate into the provisional matrix of the wound to form granulation tissue (Clark 1989; Spyrou et al. 1998). Extracellular matrix (ECM) is the largest component of the normal skin layer which forms skin connective tissue. The synthesis of ECM plays a key role in wound healing, especially in non-healing wounds with a significant loss of tissue. It also serves as a scaffold or structural support for cells and provides a transport system for nutrients and waste products (Eble and Niland 2009). Recently, a human fibroblast-derived dermal substitute (Dermagraft[R]) composed of human fibroblasts, ECM and a scaffold has been approved by FDA to repair damaged tissue in diabetic foot ulcers (DFUs), which was reported to be safe and effective in the treatment of chronic diabetic foot ulcers in a multicenter, prospective randomized study (Marston et al. 2003).

In our previous studies, NF3 has been demonstrated to be able to promote human skin fibroblast cell line Hs27 cell proliferation and cell cycle progression, and microarray analysis revealed two intracellualr signaling pathways, Wnt and angiogenesis, that may contribute to NF3 effects on fibroblasts (Tam et al. 2011; Zhang et al. 2011). In this study, we further investigated the effect of NF3 on skin fibroblast Hs27 cell migration, and elucidate the molecular mechanisms underlying the activation of TGF-f31 signaling pathway and the promoting of extracellular matrix deposition in fibroblasts.

Materials and methods

Authentication of Astragali Radix (AR) and Rehmanniae Radix (RR)

The raw herbs of AR and RR were purchased from mainland China and authenticated by morphological characterizations and thin layer chromatography in accordance with the Chinese Pharmacopoeia. Voucher specimens of RA and RR were deposited in the museum of Institute of Chinese Medicine, The Chinese University of Hong Kong, with voucher specimen numbers: 2008-3201 for AR and 2008-3200 for RR.

Extraction of NF3 and chemical profiling

Raw herbs of AR and RR were cut into small pieces and mixed in the ratio of 2:1 (w/w), the NF3 extraction were followed by previous publication (Lau et al. 2008; Or et al. 2012), which included three main steps: (1) the mixture of two dry herbs was boiled in distilled water; (2) the undissolved residue was removed by filtering; (3) the filtrate was lyophilized into dry powder. The extraction yield of NF3 was around 34% (w/w).

The chemical profile of NF3 was detected by LC-MS method and LC-MS chromatogram of melibiose, uridine, calycosin-7-0-[beta]-D-glucoside, 4H-1-benzopyran-4-one, formononetin-7-0-[beta]-p-glucoside, calycosin, and formononetin in NF3 was shown in our previous publication (Or et al. 2012).

Cell culture and NF3 preparation for cell treatment

Human skin fibroblast cell line Hs27 was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were maintained in complete DMEM with 10% FBS and 1% of penicillin-streptomycin in a humidified 5% [CO.sub.2] incubator at 37 [degrees]C.

NF3 solution was freshly prepared for each experiment by dissolving in medium and sterilized by syringe filter (0.22 [micro]m). The concentration used in the experiment was based on the dry weight of the extract (mg/m1).

In vitro scratch wound healing assay

Cells were seeded in a six-well plate and allowed to grow for 24 h. A wound was created with a p200 pipette tip. The cell debris was removed and the edge of scratch was smoothed by washing with serum free medium. The wound was exposed to NF3 at the concentration of 4 mg/m1 for 24 h at 37 C. The cells without NF3 treatment was used as the control. The closure of the scratch was observed under a microscope.

Cell migration assay

To quantitatively assess the effect of NF3 on fibroblast migration, cell suspension was placed in upper chamber (a polycarbonate membrane insert with 8 tun pore size in 24-well tissue culture plate), in accordance with the manufacture's instruction of (CytoSe-lect 24-well Migration Assay, Cell BioLabs, Inc.) The cell suspension contained 1 x [10.sup.6] cells/ml in serum free DMEM supplemented with 0.5% of Bovine serum albumin, 2.0 mM [CaCl.sub.2] and 2.0 mM [MgSO.sub.4], in accordance with the manufacture's instruction. NF3 was added directly to the cell suspension to the final concentration of 4 mg/ml. Cell suspension without NF3 treatment was used as the control. In the study, complete culture medium containing 10% FBS as a chemoattractant was added to the lower well of the 24-well plate. The cell suspension was left in the inside of each insert to migrate for 24 h at 37 [degrees]C. The insert was transferred to a new well containing Cell Detachment Solution and incubated at 37 [degrees]C for 30 min to dislodge the cells. Lysis Buffer/CyQuant GR dye solution was added to stain the dislodge cells. Fluorescence was measured with fluorescence plate reader at 480 nm/520 nm.

Western blot analysis

Hs27 fibroblasts cells were treated with or without NF3 at 4 mg/ml for 24 h. 30 flg of total protein from each sample was frac-dolled on a 4-12% gradient Bis-Tris Gel (N uPAGE Novex, lnvitrogen, USA) and transferred onto a nitrocellulose membrane. Blots were blocked with 3% of bovine serum albumin (BSA, Sigma-Aldrich, USA) in the buffer containing 10 mM Na-phosphate, pH 7.2: 0.9% NaCl and 0.05% Tween-20. Immunoblotting was performed using antibodies against transformation growth factor (TGF-[beta]) (Santa Cruz, USA), bone morphogenic protein 6 (BMP6) (Bioworld Technology, Inc., USA), and 3-actin (Beijing Biosynthesis Biotechnology Co., Ltd. China) for 2 h, respectively. After blotting with HRP-labeled secondary antibody (Beijing Biosynthesis Biotechnology Co., Ltd. China) for another 1 h, chemiluminescence was detected using an ECL kit (Amersham, Germany). The emission amount of chemiluminescence was quantified. The expression of 13-actin was used as internal control.

Quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR)

Total RNA from cells with or without NF3 treatment (4 mg/ml) for 24h was extracted using Trizol[R] Plus RNA purification kit (lnvitrogen). DNA contamination in the total RNA was removed with the addition of DNase I (Deoxyribonuclease I, Amplification Grade, Invitrogen). For measurement of type I and III collagen (COL1A1, COL3A1), matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinases (TIMP-1), cDNA was synthesized using a Transcription First Strand cDNA Synthesis Kit (Roche Diagnostics). Specific primers for target and reference genes were designed using Primer Express 3.0 software (ABI Prism; Applied Biosystems) and listed in Table 1. The expression of 18S ribosomal RNA was used as internal control. Quantitative analysis and amplification of cDNA were detected using the ABI prism 7500 sequence detection system (Applied Biosciences). Gene amplification was carried out in 10[micro]1 reaction with the following program: 45s of an initial denaturation; 45 cycles of 10 s denaturation at 95 [degrees]C and 30 s annealing and extension of at 60 [degrees]C. After amplification, a melting curve analysis from 65 to 95 [degrees]C with heating rate of 0.1 [degrees]C/s with a continuous fluorescence acquisition was carried out. The experiments were repeated four times each. Quantification of cDNA was repeated four times. Fold change of each gene expression was presented as:

Table 1

Primer sequences for genes C0L1A1. C0L3A1,
MMP9. TIMP1. and IBS for quantitative real-time PCR analysis.

Gene     Accession         Primer sequence         Product
symbol      no                                       size

C0L1A1  NM_000088.2  5'- gtgctaaaggtgccaatggt-3'   128 bp

COL3A1  NM_000090    5'-aggggagctggctacttct-3'      173 bp

MMP9    NM_004994    5'-ggcgctcatgtaccctatgt-3'     167 bp

TIM PI  NM_003254    5'-tgacatccggttcgtccaca-3'     102 bp

185     NM_022551    5'-gaggatgaggtggaacgtrt-3'     199 bp

Ct value means the number of PCR cycle

[RT.sup.2]ProfilerPCR Array System containing 84 human TGF-13 signaling pathway-focused genes was prepared according to manufacturer's protocol (SABiosciences, USA, http:// (Table 2). Reverse transcription was performed with [RT.sup.2]PCR array First Strand Kit (SABiosciences) to synthesize cDNA. Target gene expression levels were analyzed using the ABI prism 7300 sequence detection system (Applied Biosciences). Briefly, the following components were mixed in a 5 ml tube as experimental cocktail: 1275[micro]1 of 2x SABiosciences [RT.sup.2]Q-PCR Master Mix, 102[micro]1 of diluted first strand cDNA, 1173[micro]1 of dd[H.sub.2]0. Then, 25 [micro]l of the above cocktail were aliquoted to each well of the PCR array, where the gene-specific primers were immobilized, respectively. The real time PCR conditions were as follows: 1 cycle at 95[degrees]C for 10min, 40 cycles at 95[degrees]C for 15s, and 60 [degrees]C for 1 min PCR products were analyzed with PCR array data analysis web portal from the following address:

Table 2
Functional gene group in TCFp signaling targets PCR array.

Functional group           Gene symbol

Differentiation ft         ACT, BDNF. CTNNB1, EPHB2. HESl.
development                KLF10. NFKB1A (lkBa/MAD3). PPARA.
                           PTHLH, PTK2, RARA. RHOA. RUNX1
                           (AML1). SHH.
                           TGFB2, TGFBR2 BRD2, CEBPB. CREB1.
                           DNAJA1. EMP1, ENG (EVI-1). FN1,
                           1FRD1, MAPK 14 (p38 MAPK), MMP2
                           (Celatinase A).
                           MYOD1, NOTCH1, SNAI1

Proliferation & migration  ACVRI.1 (ALK1). AGT, ATF3, BCL2LI
                           (BCL-X), BDNF.CDC6. CDKN1B
                           (p27KIP1). CTNNB1, FURIN. HESl.
                           HMOX1. IL10. KLF10.
                           MSX2. MYC. NFKBIA (IKBa/MAO3).
                           PDCFA. PLC. PTCS2 (COX-2). PTHLH,
                           PTK2B, SERPINE1 (PAI-I). SHH, SOX4,
                           TGFB2, TCFBR2,
                           THBS I. TXNIP. VEGFA. ACTA2 (a-SMA).
                           ACVR1 (ALK2), AR. EMP1. FN1, FURIN.
                           IL10. PTK2

Apoptosis                  ACVR1 (ALK2). ACT, AIPL1, CDKN1B
                           (p27K1P1). CREB1, CTNNB1. FURIN.
                           CADD45B, HERPUD1, KLF10, MAP3K7
                           (TAK1). MAPK8
                           HNK11. MSX2. PLC. PTGS2 fCOX-21.
                           RAD2I. RHOA. RHOB. RYBP. S100A8.
                           SHH. SOX4. TCFB2. TNFSF10 (TRAIL).

Anti-aooptosis             BCL2L1 (BCL-X), BDNF, CEBPB. CRYAB.
                           HMOX1. IL10. NFKBIA (lkBa/MAD3).
                           PTK28, THBSI, VEGFA

Cell cycle                 ACVR1 (ALK2), RAD21,CDC6.
                           CDKNIB(p27KIPl),CADD45B. PTCS2
                           (COX-2). RHOB.TGFB2

Signal transduction        CREBBP (CBP). E2F4. EP300.1D1.
                           1D2.1D3. RBL1, SMAD1, SMAD3, SMAD5.
                           SMAD6. SP1. AR. ATF3, ATF4. BACH 1.
                           CEBPB. CREB1. CTNNB1, FOS, CTF2I,
                           HEYI. KLF10. MBDI, MSX2, MYC, MYOD1,
                           NFIB. PPARA, RARA, RUNX!
                           (AML1),S0X4, SREBF2

Enzyme-linked immunosorbent assay (ELISA)

After treatment with or without NF3 at 4 mg/ml for 24h, cells were suspended in Trysin-EDTA solution, and neutralized by Defined Trypsin Inhibitor (0.25 mg/m1) in a volume ratio of 1:1, instead of FBS, in order to remove the interference of proteins in FBS. Cell pellets were washed twice with cold D-PBS, and harvested by centrifugation. The total protein was extracted using a 1 x RayBio Cell Lysis Buffer supplemented with proteinase inhibitors (Complete Mini, Protease Inhibitor Cocktail Tablets, Roche Diagnostics). Cell lysates were spin down, and the supernatant was collected. The expression of type III collagen and fibronectin in Hs27 fibroblasts with or without NF3 treatment was quantitatively measured using specific ELISA kits (Bender MedSystems GmbH, Austria), respectively, in accordance with the manufacturer's instructions.

Statistical analysis

The results were expressed as a mean [+ or -] SD. The significant results were analyzed using a Student's t-test. The value of p <0.05 was considered as a significant statistical difference.


NF3 stimulated fibroblast migration

Migration of fibroblasts plays an essential role in wound repair. In vitro scratch wound healing assay was conducted to observe the healing process in response to NF3. After 24-h exposure of NF3, it was observed that fibroblasts were moving toward the opening to close the scratch wound (images not shown).

To further explore the activity of formula NF3 on fibroblast motility, NF3 was added to fibroblasts for quantitatively analysis of cell migration. Addition of NF3 resulted in about 29% increase in fibroblast migration compared to the control (p < 0.05, Fig. 1), showing that NF3 significantly enhanced the migration. Our study demonstrated that NF3 can act as a stimulus for fibroblast cell migration.

NF3 induced transforming growth factor-[beta]1 (TGF-[beta]l and bone morphogenic protein 6 (BMP6) expressions in fibroblasts

TGF-[beta] activation enhances fibrogenesis and remodeling in wound healing. In this study, the activation of transforming growth factor-[beta]1 (TGF-[beta]1) in fibroblast Hs27 upon NF3 treatment was investigated by western blot analysis. As a result, TGF-[beta]1 expression was up-regulated about 1.81 fold in the fibroblasts treated with NF3 compared with the control group (Fig. 2A and B).

Bone morphogenic protein (BMP), a member family within the TGF-[beta] superfamily has been well known to be associated with wound healing (Schiller et al. 2004). The regulations of BMP6 expression in fibroblast Es27 cells upon NF3 treatment was also investigated using western blot analysis, as shown in Fig. 2A and B. After fibroblasts were exposed to 4 mg/ml NF3 in serum-free DMEM for 24 h, the expression of BMP6 was up-regulated by about 1.76 fold compared to the control group.

Expression of genes involved in TGF-[beta] signaling pathway

The expression profiles of 84 TGF-13 pathway relevant genes were analyzed to further understand the molecular mechanism underlying the regulation of TGF-[beta] signaling pathway in fibroblast Hs27 cells induced by NF3. The results showed that 26 genes were differentially expressed upon NF3 treatment, where 5 genes were TGF-[beta] superfamily ligands, 5 genes were related to TGF/Smad pathway, 4 genes were related to non-Smad pathway and 12 genes were downstream effector genes (Table 3).

It was well known that transforming growth factor initiates its signaling by binding to its receptor and trigger TG93/Smad signaling pathway and/or Ras/MEK/ERK pathway (Secker et al. 2008; Stratton et al. 2002). From Table 3, in addition to TGF-131, the genes belong to TGF beta receptors, ACVR1B and TGFBR2, and co-activators, CREBBP, were up-regulated in NF3 treated fibroblast Hs27 cells. The up-regulation of SMAD1 and SMAD6 (Table 3) is consistent with the activation of TGF[beta]/Smad signaling pathway by NF3. On the other hand, several genes involved in non-Smad signaling pathway were also up-regulated, such as MAPK4 and RAB8, which belong to Ras/GTPase family and are involved in the Ras/mitogen-activated protein kinase (MAPK) pathway. A total of 12 TGF-3 pathway downstream genes were found to be differentially expressed (Table 3), which are related to regulation of cell cycle progression (CDC6, CDKN1B, GADD45B, NFKBIA), cytokine and growth factors (1110, TNFSF10, PDGFA, angiogenesis (NOTCH1, VEGFA, PDGFA) and ECM deposition (FN1).

Table 3

Differentially expressed genes for TGF-[beta]pathway
induced by NF3 (4mg/ml for 24 h) in human skin
fibroblast cell line Hs27.

Category             Symbol   Description

TCF-[beta]family     ACVR1    Activin A receptor, type 1

                     ACVRL1   Activin A receptor
                              typell-like 1

                     CREBBP   CRF.B binding protein
                              Transforming growth factor,
                              beta 2

                     TCFBR2   Transforming growth factor,
                              beta receptor II

TCF/Smad pathway     SMAD1    SMAD family member 1

                     SMAD6    SMAD family member 6

                     S0X4     SRY (sex determining region
                              Y)-box 4

                     SP1      Spl transcription factor

                     CTNNB1   CREB binding protein

Ras/MAPK(non-Smad)   RHOA     Ras homolog gene family,
pathway                       member A

                    RH08     Ras homolog gene family,
                              member B

                     MAP3K7   Mitogen-activated protein
                              kinase kinase
                              kinase 7

                     MAPK14   Mitogen-activated protein
                              kinase 14

Downstream effector  BCL2L1   BCL2-like 1

                     CDC6     Cell division cycle 6

                     CDKN1B   Cyclin-dependent kinase
                              inhibitor 1B

                     CEBPB    CCAAT/enhancer binding
                              protein (C/EBP), beta

                     FN1      Fibronectin 1

                     CADD45B  Growth arrest and

                     1L10     Interleukin 10

                     NFKBIA   Nuclear factor of kappa light
                              polypeptide gene
                              enhancer in B-cells
                              inhibitor, alpha

                     NOTCH1   Notch 1

                     PDGFA    Platelet-derived growth
                              factor alpha

                     TNFSF10  Tumor necrosis factor
                              (ligand) superfamily,
                              member 10

                     VECFA    Vascular endothelial growth
                              factor A

Category                          Function          Fold change

TCF-[beta]family            Apoptosis                     6.23

                            Proliferation &               2.86

                            Signal                       13.78

                            Apoptosis cell                5.62

                            Differentiation &             3.78

TCF/Smad pathway            proliferation ft              3.15
                            migration Signal

                            Signal                        2.76

                            Signal                        6.13

                            Signal                        3.28

                            Signal                        3.65

Ras/MAPK(non-Smad) pathway  Differentiation &             3.15
                            development apoptosis

                            Apoptosis                     5.97
                            cell cycle

                            Apoptosis                     2.60

                            Differentiation ft            3.18

Downstream effector         Proliferation ft              2.32

                            Cell cycle                    3.36

                            Cell cycle                    0.36

                            Signal                        2.10

                            Differentiation ft            5.38
                            proliferation ft

                            Apoptosis cell                0.21

                            Anti-apoptosis                0.28

                            Anti-apoptosis                3.50

                            Differentiation ft            2.60

                            Proliferation &               4.26

                            Apoptosis                     0.38

                            Anti-apoptosis                3.12

Expression of extracellular matrix (ECM)-related molecules by NF3 in skin fibroblast cells

Extracellular matrix production is one of the TGF-13 signaling targets to trigger wound healing. In this study, we analyzed the expression of several important extracellular matrix-related molecules in fibroblasts Hs27 cells upon NF3 treatment.The expression of type I and III collagens COL1A1 and COL3A1, and tissue inhibitor of metalloproteinases, TIMP-1 were up-regulated by NF3 significantly, while the expression of MMP-9 gene was down-regulated by NF3 (Fig. 3). It was also demonstrated by ELISA technique that type III collagen synthesis was enhanced in NF3 treated fibroblasts compared to the control (Fig. 4A). Moreover, the production of fibronectin, a group of glycoprotein which is important for the formation of ECM in wound healing was also found to be stimulated in Hs27 cells upon NF3 treatment compared to control group (Fig. 4B).


Previously, both in vivo and in vitro studies have demonstrated the wound healing effects of the herbal formula NF3 comprising Astragali Radix (AR) and Rehmanniae Radix (RR) (Lau et al. 2007; Tam et al. 2011). Our study also demonstrated that it could promote the migration of human skin fibroblast cell (Fig. 1).

Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes (Yarrow et al. 2004). In wound healing, TGF-[beta]1 is produced and released by various cell types, including platelets, macrophages, fibroblasts and keratinocytes (Eppley et al. 2004; Mani et al. 2002; Rolfe et al. 2007). The formation of granulation tissue at the wound site requires adjacent fibroblasts to migrate to the wound site and proliferate, which is especially important when fibroblasts migrate to wound site to produce extracellular matrix proteins in granulation tissue formation.

In this study, the activation of TGF-[beta] signaling pathway and production of extracellular matrix proteins in skin fibroblast cells upon formula NF3 treatment were investigated. Firstly, it was demonstrated that NF3 could stimulate TGF[beta]1 expression in fibroblasts promote wound healing (Fig. 2A). Our study also showed that NF3 could up-regulate BMP6 expression in fibroblasts (Fig. 2B), suggesting that the wound healing effect of NF3 is also mediated through BMP6. BMPs are members of TGF-[beta] superfamily. In vitro studies have shown that BMP6 stimulates endothelial cell proliferation, migration and tube formation. It has also been reported that BMP6 plays a role in diabetic wound healing; a deficiency of BMP6 is associated with the increase of serum glucose and diabetes risk (Vukicevic and Grgurevic 2009) and may have an adverse effect on tissue remodeling in patients with diabetes (Nguyen et al. 2006).

NF3 promoted fibroblast cells migration and ECM deposition, as shown by the analysis of the expression of 84 TGF-[beta] pathway specific genes, where 26 genes were found to be differentially expression upon NF3 treatment (Table 3). Up-regulation of the expression of TGF-[beta] and TGF-[beta] receptors type I and II (ACVR1 and TGFBR2) by NF3 suggests the initiation of the TGF-[beta] signaling pathway. Following the initiation of TGF-[beta] signaling, two signaling cascades downstream of the TG-F[beta] receptors were activated. CREBBP, a gene involved in the Sri-lad pathway, was significantly up-regulated by NF3, which has been demonstrated to be a necessary co-activator for the Smad-dependent TGF-[beta] transcriptional response (Topper et al. 1998). Studies showed that the TGF[beta]/Smad signaling pathway is crucial for the activation of several fibrillar collagen genes (Verrecchia et al. 2001). Our results showed that the expression of COL1A1 and COL3A1 were up-regulated by NF3 in fibroblast Hs27 cells (Figs. 3 and 4), suggesting that TGF[beta]/Smad signaling was activated by NF3 to trigger extracellular matrix deposition in fibroblast cells. In addition to the Smad pathway, TGF-[beta] may activate the Ras/MEK/ERK pathway (Secker et al. 2008; Stratton et at. 2002). In the study, we found that several genes related to the Ras/MAPK/ERK pathway were also up-regulated by NF3, suggesting the wound healing effect of NF3 on skin fibroblast cells may also involve via Ras/MAPK (non-Smad) pathway (Table 3).

Extracellular matrix (ECM) is the major components of granulation tissues, which provide a framework for the many processes of healing. The largest class of fibrous ECM molecules is the collagen family, including at least 16 different types (Niu et al. 2008). Among them, type I and III collagens are the primary dermal matrix. Type III collagen is the main type of collagen in granulation tissue and predominant type of collagen synthesized in the proliferation phase of wound healing. The early immature type III collagen is gradually replaced with the normal adult type I collagen, which is responsible for the tensile strength of skin and the formation of scars (Haukipuro et al. 1991). Based on our study, formula NF3 could significantly increase type III collagen production, by about 18 fold compared to the control (Fig. 4B). An increase in type I collagen gene expression was also observed (Fig. 3A) upon NF3 treatment. The result is consistent with the wound healing process that collagen type III is the predominant collagen in the proliferation phase, suggesting that NF3 could promote the formation of granulation tissue.

Fibronectins are also important components of granulation tissue in wound healing and substrate for cell migration (Repesh et al. 1982; Yamada et al. 1992). Fibronectins are the ligands of integrin receptors. They bind with cell surface integrins to connect cells with collagen fibers in the ECM, allowing cells to move through the ECM. Fibronectins also can bind to platelets during blood clotting to facilitate cell movement to the site of tissue injury. Using ELISA, our study revealed that NF3 could stimulate fibronectin synthesis significantly (Fig. 4B).

Catabolism is the work of catabolic enzymes known as collagenases or matrix metalloproteinases (MMPs), which cleave the fibrous proteins of the ECM (Parks and Mecham 2008). Our study showed that the gene expression of MMP9 in Hs27 fibroblast cells was down-regulated by NF3, while the expression of TIMP1 was up-regulated by NF3 (Fig. 3). TIMP1 plays a role in inhibiting MMPs deposition and preventing ECM degradation. In addition to MMP-inhibiting activity, TIMP1 has been known to promote fibroblast proliferation and enhance new blood vessel formation (Lovelock et at. 2005; Liu et at. 2008).

In summary, the study showed that NF3, an herb formula com-pring Astragali Radix (AR) and Rehmannia Radix (RR) in the ratio of 2:1, could enhance skin fibroblast cell migration and promote wound healing effect. A tentative scheme describing how NF3 activates TGF-[beta]1 signaling pathway to trigger ECM deposition and wound healing in skin fibroblast cells was proposed (Fig. 5), where NF3 regulates gene transcription for ECM synthesis via the TGF/Smad pathway and co-activator activation, and mediates gene transcription for cell-matrix adhesion and cell motility via the Ras/MAPK pathway.


This study is supported by a grant from UGC Area of Excellence project "Chinese Medicine Research and Further Development" (Project No. AoE/B-10/01) and the Shenzhen Double Hundred Project.

Abbreviations: BMP6, bone morphogenetic protein 6; COL1A1, collagen, type I, alpha 1; COL3A1, collagen, type Ill, alpha 1: MAPK, mitogen-activated protein kinases; MMP-9, matrix metalloproteinase 9; TIMP-1, a tissue inhibitor of metalloproteinases 1; TGF-[beta]1, transformation growth factor pl.


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Qi Zhang (a), Chi Chun Fong (a), (b), Wai Kin Yu (a), Yao Chen (a), (b), Fan Wei (a), Chi Man Koon (c), (d), Kit Man Lau (c), (d) Ping Chung Leung (c), (d), Clara Bik San Lau (c), (d), Kwok Pui Fung (c), (d), (e), Mengsu Yang (a) (b) (*)

(a.) Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong

(b.) Shenzhen Key Laboratory of Biochip Research, City University of Hong Kong, Shenzhen 518057, PR China

(c.) Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

(d.) State Key Laboratory of Phytochemistry & Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

(e.) School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

* Corresponding author. Tel.: +852 34427797; fax: +852 34420552.

E-mail address: (M. Yang).

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Author:Zhang, Qi; Fong, Chi Chun; Yu, Wai Kin; Chen, Yao; Wei, Fan; Koon, Chi Man; Lau, Kit Man; Leung, Pin
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Dec 15, 2012
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