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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)
|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
|Online Catalog Link: ||http://lib.cityu.edu.hk/record=b4086565|
|Appears in Collections:||BCH - Doctor of Philosophy |
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