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Title: The biological activities and molecular mechanism of a herbal formula extract of Rehmanniae Radix and Astragali Radix in wound healing
Other Titles: Fu fang sheng di huang qi ying xiang shang kou yu he de fen zi sheng wu xue yan jiu
Authors: Zhang, Qi ( 張琦)
Department: Department of Biology and Chemistry
Degree: Doctor of Philosophy
Issue Date: 2010
Publisher: City University of Hong Kong
Subjects: Herbs -- Therapeutic use.
Astragalus (Plants) -- Roots -- Therapeutic use.
Wounds and injuries -- Molecular aspects.
Notes: CityU Call Number: RM666.H33 Z36 2010
xvi, 215 leaves : ill. (some col.) 30 cm.
Thesis (Ph.D.)--City University of Hong Kong, 2010.
Includes bibliographical references (leaves 197-215)
Type: thesis
Abstract: Wound healing is an intricate process that involves a variety of cell types, including fibroblast, platelet, neutrophil, macrophage, endothelial cell and keratinocyte, as well as a set of biochemical events. The classical model of wound healing is divided into four sequential but overlapping phases: hemostasis, inflammation, proliferation and remodeling. In pathological conditions, such as diabetes, an organized and orderly wound healing process is interrupted and results in chronic wounds. Fibroblast is the connective tissue cell. During the proliferation phase of wound healing, fibroblasts are stimulated by various growth factors to proliferate and migrate to the wound site, and produce extracellular matrix (ECM) including collagen and fibronectin. Fibroblast is one of the components making up granulation tissue in wound healing. It is also able to secrete growth factors associated with angiogenesis and epthelialization, such as vascular endothelial cell growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGFβ). A Chinese herbal formula, consisting of Rehmanniae Radix (RR) and Astragali Radix (AR), has been clinically demonstrated to improve the healing of diabetic foot ulcer in diabetic patients with insulin resistance. In vivo study has shown that RR extract reduced the wound area and promoted scar formation in a diabetic foot ulcer rat model. In the present study, the biological activities and molecular mechanism of the herbal formula were investigated for wound healing using the human foreskin fibroblast cell model. The formula was prepared based on the weight ratio of AR to RR at 2:1. Fibroblasts were exposed to water extract of the herbal formula (Formula AR-RR) in a serum-free medium. The results showed that Formula AR-RR at the concentration of 4mg/ml significantly increased cell viability and proliferation. Flow cytometry and Western blot analysis demonstrated that Formula AR-RR was able to affect fibroblast cell cycle, and stimulate cells to enter S phase with the expression of cyclin D1 protein. Fibroblast cell migration, adhesion and contraction were subsequently studied to further understand the effect of Formula AR-RR on wound healing. Formula AR-RR was found to stimulate fibroblast migration and to improve the adhesion of fibroblasts to the ECM protein, fibrinogen. Wound contraction occurs at a late stage of wound healing. The evaluation of fibroblast contraction revealed that Formula AR-RR did not significantly enhance cell contraction. Differentiation of fibroblasts into myofibroblasts is responsible for providing the force for wound contraction. Western blot demonstrated that the expression level of alpha-smooth muscle actin (α-SMA) protein, a cytoskeletal protein biomarker indicating the differentiation of fibroblast to myofibroblast, was not changed, suggesting that Formula AR-RR had no effect on the differentiation of fibroblast to myofibroblast and the promotion of cell contraction. Wound repair is dependent on the expression and deposition of ECM proteins, mostly collagen and fibronectin, in fibroblasts. Formula AR-RR was also found to elevate the expression of ECM proteins, including collagen I, III and fibronectin in fibroblasts. Because the deposition of ECM proteins is associated with collagenases responsible for the breakdown of the matrix protein, the cytoplasmic matrix metalloproteinase protein (MMP) and the gene expression of tissue matrix metalloproteinase inhibitor (TIMP) were measured in cells treated with Formula AR-RR. The results showed that the stimulation of fibroblasts by Formula AR-RR increased TIMP1 gene expression and decreased the synthesis of MMP3 protein. Many diverse biological events during wound healing, including cell growth, cell migration, angiogenesis, fibrogenesis, and inflammatory response, are driven by cytokines. In the present study, Formula AR-RR was found to induce fibroblasts to increase the synthesis of angiogenic and fibrogenic factors in the cytoplasm, including vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), transforming growth factor beta 1 (TGFβ1) and bone morphogenic protein 6 (BMP6). The induced production of VEGF, PDGF, TGFβ1 and BMP6 suggested that Formula AR-RR was able to affect blood vessel formation and maturation, help form granulation tissue, and promote acute wound repair. In addition, Formual RA-RR decreased the production of inflammatory factors, including tumor necrosis factor alpha (TNFα), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), interleukin-6 (IL-6) and interleukin-8 (IL-8), which are responsible for recruiting neutrophils leading to a chronic wound. The gene expression profile of fibroblasts stimulated by Formula AR-RR was studied by using a cDNA microarray containing 10,000 human genes. One hundred and twenty-six genes with significant change of expression level were identified, with 102 genes up-regulated and 24 genes down-regulated. Further analysis identified two pathways involved in wound healing - a Wnt signaling pathway and an angiogenesis signaling pathway. Eighteen genes with significant differential regulation were classified in the Wnt signaling pathway. Proteins encoded by these genes were distributed in 11 components of this pathway, including frizzled (FZD1, 2.3-fold), casein kinase 1 (CSNK1E, 0.5-fold), cadherin (PCDH1, 4.2-fold; FAT, 3.7-fold; PCDHGC3, 2.0-fold; CDH16, 2.5-fold; CELSR3, 2.9-fold and PCDH8, 2.2-fold), protein kinase C (PRKCH, 3.0-fold and PRKCZ, 2.6-fold), protein phosphatase 2A (PPP2R5A, 2.4-fold), calcineurin (PPP3CB, 2.4-fold), B-cell lymphoma 9 (Bcl9, 8.6-fold), switched/sucrose non fermentation (SMARCC1, 4.7-fold and SMARCA5, 2.2-fold), transforming growth factor beta activated kinase 1 (ACVR1B, 2.4-fold), CREB binding protein (CREBBP, 13.4-fold), and Wnt target genes (CCND1, 2.3-fold). Proteins encoded by ten genes were identified to involve in eight components of the angiogenesis signaling pathway, including protein kinase C (PRKCH, 3.0-fold; PRKCZ, 2.6-fold), sphingosin kinase (SPHK1, 2.6-fold), mitogen activated protein kinase 4 (MAPK4, 2.6-fold), platelet-derived growth factor (PDGFA, 3.7-fold), growth factor receptor-bound protein 7 (GRB7, 2.3-fold), growth factor receptor-bound protein 14 (GRB14, 20.7-fold), frizzled (FZD1, 2.3-fold), and delta/serrate (DLL1, 6.0-fold and DLL4, 3.6-fold). These two pathways are directly related to cell proliferation, angiogenesis, and ECM formation in the process of wound healing, and provide the molecular basis for the effects of Formula AR-RR. In summary, the study showed that Formula AR-RR has obvious biological effects on wound healing through promoting fibroblast proliferation, increasing cell migration and adhesion, stimulating the synthesis of cytokines involved in angiogenesis and fibrogenesis, and controlling inflammation. Recently, some chemical components, such as formononetin and genistine, were identified from Formula AR-RR. Formononetin was reported to promote angiogenesis, and genistine was found to substantially impair inflammatory response. Identification of additional bioactive chemical components from Formula AR-RR, and analysis of their functions in the future will further help understand the mechanisms of the herbal formula and develop new therapeutics for wound healing.
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