A Study of the Effect of Shiunko, a Traditional Chinese Herbal Medicine, on Fibroblasts and Its Implication on Wound Healing Processes

Abstract

Significance

In China, traditional Chinese medicine (TCM) has been used for thousands of years for various acute and chronic wound care. Thus, there is a growing need to explore the possible benefits of TCM on wound healing.

Recent Advances

Nowadays, in China and some Asian countries including Korea, Japan, and Singapore, Chinese herbal therapy is used as an alternative treatment in wound care. Therefore, exploration of the possible benefits of TCM on wound healing is necessary.

Critical Issues

Development of TCM is based on the concept of Yin (negative phenomenon of nature) and Yang (positive phenomenon of nature). These opposing and complementary natural phenomena of the universe restore the normal physiological functions, consequently curing diseases and restoring health of a patient.

Future Directions

Due to lack of evidence-based research, TCM treatments are not widely accepted in the western world. Using state-of-the-art technology such as proteomics, bioinformatics, and biomolecular techniques, research studies may lead to more effective remedies for wound care in the future.

Kin-Fu Chak, PhD

Scope

The balance of two opposite natural phenomena, Yin (negative) and Yang (positive) of the body, governs everything in the world, and are the sources of growth, development, and variation of all organisms.^1,2 The concept of traditional Chinese medicine (TCM) literatures stated that poor circulation, stagnation, and poor healing are mainly due to the deficiency of Yang, whereas overheating (a terminology of TCM for inflammation and some related pathological symptoms) or having an excess of scar tissues are always associated with the deficiency of Yin. The balance between Yin and Yang is ideal for good health, and it is essential for promoting wound healing in a complementary way.

Translational Relevance

A large number of TCM literatures have documented the formulation of herbal medicines for various wound treatments. With the aim of evidence-based research, this review may explore a better formulation of TCM for various wound healing treatments of patients.

Clinical Relevance

Using the fibroblast as a cell model system, we have adapted proteomics and bioinformatics approaches to evaluate the potential efficacy of Shiunko, an herbal Chinese compound drug, on wound healing processes. Using proteomic analysis, we have identified some specific protein factors associated with the function of wound healing process. Thus, this evidence-based platform may explore the beneficial molecular basis of TCM for wound healing as a whole.

Background

The Chinesis Materia Medica

The aim of combining different herbal drugs to treat diseases is to rectify the hyperactivity or hypoactivity of Yin or Yang, resulting in restoration of normal physiological functions of the patient, which consequently cures diseases and restores health.³ In practice, the herbal formulas normally consist of four categories of herbs to rectify the balance of Yin and Yang:

• Ministerial herbs (also known as Chief herbs) address the principal pattern of the disease;

• Deputy herbs assist the ministerial herb;

• Assistant herbs are designed to reduce the side effects of the first two classes of herbs; and

• Envoy herbs detoxify and direct the therapy to a particular part of the body.

Principle of TCM for wound care

Drugs for hemostasis (to stop internal and external bleeding) and for promoting blood circulation and removing blood stasis are regarded as potent drugs of TCM for wound care.³

Hemostatics

These herbal drugs have the effects of stopping bleeding by removing heat (inflammation and related pathological symptoms) from the blood, by astringency, or by resolving blood stasis. They are indicated for the treatment of various kinds of hemorrhage and traumatic bleeding.

Notoginseng (Radix Notoginseng), for example, is the root of Panax notoginseng, which is the most powerful herbal hemostatics of TCM.⁴ This drug has the effects of arresting bleeding by resolving blood stasis and promoting blood circulation to relieve swelling and pain. It is often prescribed to treat traumatism, pain caused by ecchymoma and carbuncle, swelling, and other external skin diseases. Other herbal hemostatics³ including field thistle [Herba Cephalanoploris; Cephalanoplos segetum (Bunge) Kitam., or Cephanoplos setosum (Willd.) Kitarn.], sanguisorba root (Radix Sanguisorbae; Sanguisorba officinalis), hyacinth bletilla [Rhizoma Bletillae; Bletilla striate (Thunb.) Reichb.], and cat-tail pollen (Pollen Typhae; Typha angustifolia L. or Typha orientalis Presl) are useful for treating various skin wounds.

Drugs for promoting blood circulation and removing blood stasis

These drugs are effective for various syndromes due to obstruction of blood circulation and retention of blood stasis in traumatic injuries, arthralgia, sores, carbuncles, and other suppurative skin infections.³ The commonly used herbal drugs in this category are red sage root (Radix Salviae Miltiorrhizae; Salvia miltiorrhiza), motherwort (Herba Leonuri; Leonurus cardiaca), and safflower (Flos Carthami; Carthamus tinctorius L.).

Other drugs for assistance of wound care

For maintaining the physiological harmony of patients, properties of other drugs are usually combined with the ministerial drugs to optimize the efficacy of the drugs for wound care.³ For example, musk (Moschus) and borneol (Borneolum) with resuscitative properties are always formulated with other drugs in the TCM wound healing medicines. Moreover, drugs tonifying the deficient Qi (the most basic element for the composition and maintenance of the vital activities of the human body) and rectifying Yin and Yang of the body are often accompanied with other drugs for wound care, such as chuanxiong rhizome (Rhizoma Ligustici Chuanxiong; Ligusticum chuanxiong Hort.), astragalus root (Radix Astragali seu Hedysart; Astragalus propinquus), red sage root (Radix Salviae Miltiorrhizae), motherwort (Herba Leonuri), Chinese yam (Rhizoma Dioscoreae; Dioscorea opposita), licorice (Radix Glycyrrhizae; Glycyrrhiza glabra), Chinese angelica root (Radix Angelicae Sinensis; Angelica sinensis [AS]), fleeceflower root (Radix Polygoni Multiflori; Fallopia baldschuanica), and achyranthes and cyathula root (Radix Achyranthis Bidentatae et Radix Cyathulae; Achyranthes bidentata Bl. and Cyathula officinalis Kuan).

The TCM Strategy for Wound Care

Three golden rules of TCM⁵ for wound care, especially chronic skin ulcers (CSU), have long been proposed: (1) keeping the right amount of pus on the surface of the ulcer to stimulate the growth of granulation, (2) removing necrotic tissues to stimulate the growth of new skin, and (3) inhibition of inflammation to promote skin wound recovery. In practice, this strategy of treatment not only facilitates wound healing but also effectively decreases scarring.

Yunnan Paiyao

Yunnan Paiyao (the white herbal medicine from Yunnan province of China)⁶ is the most famous of the patent remedies in China for its ability to stop bleeding, for closure of wounds and decrease in scarring, and for the encouragement of flesh regeneration.

Yunnan Paiyao is formulated by various herbal drugs with specific characters: Radix Notoginseng (40%), Ajuga Forrestii Diels (17%), Rhizoma Dioscoreae (13%), Rhizoma Dioscoreae Nipponicae (10%), Herba Geranli & Herba Erodii (7.2%), Dioscoreae Parviflora Ting (6%), Herba Inulae Cappae (5%), Borneol (1.5%), and Musk (0.3%). Recent research demonstrated that either before or after the surgery, administration of Yunnan Paiyao can significantly reduce bleeding and swelling of the wound.⁷

Shiunko

Shiunko⁸ is commonly used in China and Japan for treating wounded skin caused by cuts, abrasions, frost, or burn.⁹ Shiunko mainly consists of sesame oil (500 mL), Lithospermi Radix (LR; Lithospermum erythrorhizon Sieb. et Zucc.) (10 g), A. sinensis (10 g), lard (3 g), and beeswax (45 g). The therapeutic effect of shiunko on granulation tissue formation, speeding wound healing, reepithelialization, and angiogenesis, in addition to anti-bacterial and anti-inflammatory effects has been proven.¹⁰

Other commonly used traditional Chinese herbal medicine for wound care

Various ancient and modern TCM literature have documented the use of herbal formulas in wound care,¹¹ such as Wuwei Xiaodu Yin (clears heat, detoxifies, cools blood, and reduces swelling), Huanglian Jiedu Tang (detoxification decoction with huanglian), Yanghe Tang (decoction for Yang harmonization, tonifies blood, expels cold, and unblocks stagnation), Tuoli Xiaodu San (power for defensive Qi enrichment and toxicant removal), and Shengji Yuhong Gao (red ointment for granulation promotion).

Current Clinical Application of TCM for Wound Care

In China, CSU patients account for 1.5%–3% of the total hospitalized patients.¹² Traditionally, TCM remedy procedure for CSU is divided into the following three steps.¹³

Clearance of heat at the early stage of CSU

The combination of the following herbal drugs is commonly used for this purpose: Lonicera japonica Thunb., Viola chinensis, Atractylodes lancea, Poria cocos, Semen coicis, Stephania tetrandra S. Moore, AS, and Platycodi.

Promoting blood circulation to dissipate blood stasis for the treatment at the middle stage of CSU

The combination of the following herbal drugs is commonly used for this purpose: Carthamus tinctorius, AS, Aucklandiae Radix, Olibanum, Comiphora Myrrha, Rhizoma Chuanxiong, Pheretima, Glycyrrhizae Radix, Salviae Miltiorrhizae Radix, Rehmanniae Radix, and Paeoniae Radix.

Supplements for deficiency to promote granulation at later stage of CSU

The combination of the following herbal drugs is commonly used for this purpose: Astragalis Raw Radix, Pseudostellariae Radix, Angelicae Sinensis Radix, Salviae Miltiorrhizae Radix, Semen Persicae, Carthami Flos, Rhizoma Chuanxiong, Rhizoma Atractylodis Macrocephalae, Poria Cocos, Cyathulae Radix, and Glycyrrhizae Radix.

Paradigm of Evidence-Based Research of TCM on Wound Healing Process

Proteomics is a powerful tool and has been widely used to elucidate protein profile changes in response to drug treatment and to identify disease-relevant biomarkers.¹⁴ In this work, proteomics and bioinformatics were employed to study the mechanism of wound healing process of a TCM wound healing drug, shiunko.

Shiunko, a Chinese herbal compound, for wound healing

AS is a basic component of many Chinese drugs for wound healing.⁹ Ferulic acid (FA) is one of the most abundant water-soluble ingredients in AS, and has been reported to be the active component of AS.^15,16 LR (also called Zicao or Gromwell) is commonly used to treat skin disorders, cuts, and burns. A recent study showed that LR has multiple activities including antimicrobial activity,¹⁷ antiviral activity,¹⁸ anti-inflammatory activity,¹⁹ and antitumor activity.²⁰ The most important components in LR are derivatives of shikonin, such as deoxyshikonin, acetylshikonin, and isobutylshikonin.²¹

To study the molecular basis of wound healing processes of this herbal medicine, Shiunko was prepared with 1:1 (w/w) of LR/AS mixture and extracted by 95% ethanol.

Proteomic approach for the analysis of proteins associated with the wound healing process

The differentially expressed proteins of fibroblasts induced by Shiunko were dominantly associated with many pathological functions (our unpublished data), including antioxidant activity (peroxiredoxin [PRDX]2, PRDX4, superoxide dismutase C [SODC], glutathione S-transferase P [GSTP1], glutaredoxin 3 [GLRX-3], and SH3 domain-binding glutamic acid–rich protein [SH3BGRL]); cell proliferation (pyruvate kinase isozyme type M2 [PKM2], spermidine synthase [SPEE], probibitin [PHB], stathmin 1 [STMN1], and eukaryotic translation initiation factor 5A-1 [IF5A-1]); anti-apoptosis (GSTP1, heat shock 27 kDa protein 1 [HSPB1], and translationally controlled tumor protein [TCTP]); collagen secretion (phosphoglycerate kinase 1 [PGK1], lactate dehydrogenase B [LDHB], ATP synthase subunit delta [ATPD], and triosephosphate-isomerase [TPIS]); and cell motility (MARE-1, vimentin [VIME], calpain small subunit 1 [CAPNS1], actin-related protein 2/3 complex subunit 5 [ARPC5], and cofilin-2 [COF2]).

Differentially expressed proteins associated with cell viability and proliferation

The relative proliferation rate of fibroblast cells induced by the optimal concentration of Shiunko 100 μg/mL (containing 50 μg/mL and AS and 50 μg/mL LR) was 150% (Fig. 1). In comparison, our previous works indicated that the proliferation rate of fibroblast induced by the optimal concentration of AS (300 μg/mL)²² and LR (50 μg/mL)²³ was only 120% to 125%. Obviously, the combination of AS (50 μg/mL) and LR (50 μg/mL), designated Shiunko 100, has a synergistic effect on cell proliferation (Fig. 1). We found that the expression level of the anti-apoptotic proteins, such as HSPB1, GSTP1, and TCTP, in cells induced by AS, LR, and Shiunko, respectively, was similar. However, the expression level of proliferation proteins was very different. Our previous data indicated that AS (300 μg/mL)²² could only induce an increase in the expression level of CAPNS1; LR (50 μg/mL)²³ could induce an increase in the expression levels of IF5A-1 and NME-1. Interestingly, proteomic analysis of the drug effect on fibroblasts indicated that more proliferation proteins were induced by Shiunko 100 μg/mL, including IF5A-1, NME-1, PKM2, SPEE, PHB, and STMN1(our unpublished data). It was known that STMN1 is a key proliferation protein,^24,25 and it can be regarded as a typical differentiation protein marker of the cell.²⁶ It is worth noting that Shiunko could induce an increase in expression level of STMN1 by up to 8.5-fold (our unpublished data), indicating that STMN1 may play an important role in the proliferation rate of fibroblasts. Further, it could not be ruled out that a higher proliferation rate of fibroblasts induced by Shiunko may be also due to more proliferation proteins induced by this treatment.

Figure 1.

Viability of fibroblast cells in response to various treatments of Shiunko. Fibroblasts and designated concentrations of Shiunko were dissolved in 0.5% dimethyl sulfoxide (DMSO). 24 h after treatment with various concentrations of Shiunko, the viability of fibroblast cells were measured when subjected to WST-1 assay. Fibroblast cells in 0.5% DMSO was used as control, and the relative viability of fibroblast cells treated with various concentrations of Shiunko was determined by comparing with the control, respectively. The Student's t-test was used to evaluate the statistical significance of the experiments, and the results were presented as mean±SD (n=3), where **p<0.01.

Differentially expressed proteins associated with antioxidant ability

With discrete drug treatments of the fibroblast cell, expression profiles of proteins associated with antioxidants are varied: AS (300 μg/mL)²²—PRDXs and PARK-7; LR (50 μg/mL)²³—PRDX-2, PRDX-4, GSTP-1, SODC, and ADI-1; Shiunko (100 μg/mL)—PRDX-2, PRDX-4, GSTP-1, SODC, GLRX-3, and SH3BRL (our unpublished data). The biochemical analysis indicated that treatment of AS (50 μg/mL) caused the reduction of ROS by 8%,²² while both LR (50 μg/mL)²³ and Shiunko (100 μg/mL) (Fig. 2) significantly reduced the level of ROS by up to 15%.

Figure 2.

Detection of reactive oxygen species (ROS) contents in fibroblast cells with various treatments of Shiunko. The contents of ROS of fibroblast cells in 0.5% DMSO was used as control, the ROS ratio of cells with various treatments of Shiunko was determined by comparing with the control. The Student's t-test was used to evaluate the statistical significance of the experiments, and the results are presented as mean±SD (n=3), where **p<0.01.

Differentially expressed proteins associated with cell mobility

Proteins involved in cell mobility, such as MARE-1, CAPNS-1, COF-2, and VIME were downregulated when cells were treated with AS,²² LR²³, and Shiunko (our unpublished data). It was known that ARPC5 is able to regulate cell mobility, probably by controlling microtubule dynamics.²⁷ In this work, we found that ARPC5 was only upregulated in cells treated with FA;²² in contrast, this protein was downregulated in cells treated with LR,²³ and with Shiunko treatment (our unpublished data). Coincidently, with FA or AS treatment, the rate of cell migration increased by up to 160%,²² while cell migration rate reduced to 40%–50% in the cells treated with either LR²³ or Shiunko (Fig. 3). Clearly, ARPC5 plays a critical role in cell migration.

Figure 3.

Mobility of fibroblast cells in response to various treatments of Shiunko. Transwell migration assay was used to evaluate the mobility of the cells treated with various concentrations of Shiunko. A total of 2×10⁴ cells were seeded onto the upper Boyden chamber, and after 6 h, number of cells that migrated down to the lower chamber of Transwell was strained by 5% Giemsa in water, and the cells that remained on the upper Boyden chamber were scraped with cotton swabs. The total number of cells was counted using an inverted microscope. Migration rate of cells in 0.1% DMSO was used as control, and the migration rate was defined as 100%. The relative migration rate of cells with various treatments of Shiunko was determined by comparing with the control. Student's t-test was used to evaluate the statistical significance of the experiments, and the results are presented as mean±SD (n=3), where *p<0.05 and **p<0.01.

Correlation between collagen secretion and expression of transforming growth factor beta

The collagen secretion rate of fibroblasts treated with either LR²³ or Shiunko 100 (Fig. 4) was increased by up to 125% to 130%, while the increase of collagen secretion rate of the AS-treated fibroblasts²⁵ was only 105%. Transforming growth factor beta (TGFβ) controls the upregulation of collagen synthesis in macrophages and fibroblasts.²⁸ Coincidentally, production of TGFβ was increased by up to 10-fold in cells treated with either LR²³ or Shiunko 100 μg/mL (Fig. 5), while there was only 1.5-fold increase in cells with AS treatment.²² It is noteworthy that when fibroblast was treated with Shiunko 300 μg/mL, collagen secretion rate and TGFβ expression level increased by up to 160% (Fig. 4) and 39.65-fold (Fig. 5), respectively. Hence, this data strongly demonstrated that Shiunko leads to increased production of TGFβ and collagen.

Figure 4.

Detection of collagen secretion from fibroblast cells treated with various concentrations of Shiunko. Fibroblast cells and designated concentrations of Shiunko were dissolved in 0.5% DMSO. 24 h after drug treatments, cells were harvested and subjected to Sircol soluble collagen assay. Collagen secretion rate of cells in 0.5% DMSO was used as control. The relative collagen secretions rate of cells with various treatments was determined by comparing with control. Student's t-test was used to evaluate the statistical significance of the experiments, and the results are presented as mean±SD (n=3), where *p<0.05 and **p<0.01.

Figure 5.

Detection of transforming growth factor beta (TGFβ) expression of fibroblast cells in response to the treatment with Shiunko. Fibroblast cells and the designated concentrations of shiunko were dissolved in 0.5% DMSO. 24 h after drug treatment, TGF-β expression was determined by immunoblotting, and the quantity of TGF-β was confirmed by using Fujifilm MultiGauge software. Expression level of TGFβ of cells in 0.5% DMSO was used as control. The relative expression of cells with various drug treatments was compared with control. This experiment was repeated thrice, and GAPDH was used as internal control.

The wound healing analysis in vivo

The effect of Shiunko on the wound healing speed was confirmed by an in vivo test. Two 8 mm biopsy punch wounds were created on the flank or back of SD rats, and then the wounds were treated with three different concentrations of Shiunko: 30, 100, and 300 μg/mL, respectively. We observed that the contraction speed of the wound treated with Shiunko 100 and 300 g/mL was similar (Fig. 6), as the opening of the wound contracted to near zero at day 11 of treatment. It was noted that the wound healing speed with Shiunko 30 μg/mL was not as good as the other two treatments. These experimental results might be due to the fact that production of both collagen (Fig. 4) and TGFβ (Fig. 5) from fibroblasts treated with Shiunko 30 μg/mL was much lower than that of the other two treatments.

Figure 6.

In vivo wound healing speed assay. Two wound circles were generated on the back of SD rats using an 8 mm biopsy punch. DMSO alone, and three discrete concentrations of Shiunko in DMSO were used to treat the wounds, and the diameter of each wound was measured twice per day. The wound healing rate was also recorded by digital camera at an interval of 4 days. Color images available online at www.liebertpub.com/wound

Discussion

Shiunko-treated fibroblasts can induce a whole range of biochemical events involved in the wound healing process, including cell proliferation and anti-apoptosis, anti-oxidant activity, secretion of collagen, and cell mobility (Fig. 7). Increase in cell viability illustrated the synergistic effect of Shiunko on the wound healing process, which is achieved by the upregulation of proteins associated with proliferation and anti-apoptosis of the treated fibroblast cells. It is worth noting that Stathmin, a differentiation marker, was highly induced by Shiunko (eightfold increase, our unpublished data); possibly, presence of this protein could be a good sign of the wound healing process.

Figure 7.

Hypothetical wound healing process revealed by proteomics and biochemical analysis of fibroblast cells treated with Shiunko. Several biological functions are involved in the wound healing processes including antioxidant activity, cell proliferation/viability, collagen secretion, and cell mobility. The proteomes detected from the fibroblast cells induced by Shiunko are grouped in places where the distinct physiological functions are located. The upward and downward arrows represent upregulation and downregulation of the distinct physiological functions of the cells. The figure illustrating the wound healing process is a combination effect of various biological functions of cells induced by the treatment of Shiunko. Color images available online at www.liebertpub.com/wound

In the early phase of the wound healing process, overexpression of reactive oxygen species (ROS) results in the formation of a secondary injury of the wound. We observed that Shiunko-treated fibroblasts can reduce the ROS concentration by up to 15%, which may alleviate the risk of scarring after the wound heals. GSTP1 and PRDX are involved in the processes of antioxidation; they are commonly expressed in cells with AS, LR, and Shiunko treatment. Interestingly, expression of SODC (3.1-fold, our unpublished data) is high only in cells with Shiunko treatment. The function of superoxide dismutase (SOD) is to catalyze the dismutation of the toxic superoxide anion O₂^– to O₂ and H₂O₂. In addition, it also has a powerful anti-inflammatory activity,²⁹ and can reverse fibrosis through reversion of myofibroblasts back to fibroblasts.³⁰ Therefore, upregulation of SOD may reveal a good sign of wound healing process.

Collagen is mainly secreted from fibroblasts, a component of skin tissue that can benefit all stages of the wound healing process. TGFβ is an upstream regulator to control expression of collagen. We found that expression of TGFβ is correlated with the secretion rate of collagen in fibroblasts with Shiunko treatment. Hence, upregulation of TGFβ should be one of the important factors for wound healing process.

Take-Home Messages.

The philosophical concept of TCM for medical care

The balance of Yin and Yang eventually invigorates the vital activities of the human body, Qi, for good health. To rectify the Qi of the patient, a combination of four categories of herbal drugs—ministerial (chief), deputy, assistant, and envoy herbs—should be prescribed.

Current practice of TCM for wound care

TCM remedy procedure for CSU is divided into three steps, namely, clearing heat, resolving stasis, and tonification methods to treat the syndromes at early, middle, and later stages of the disease, respectively.

Evidence-based research of TCM for wound care

The evidence-based research indicated that Shiunko modulates a group of proteins such as stathmin, SODC, TGFβ, and ARPC5 and acts to improve healing by doing so.

Mobility of fibroblast is restricted when the cell is treated with Shiunko. In contrast, cell migration increases by up to 150%–160% with AS and FA treatment.²² ARPC5, a positive cell mobility regulator, is only upregulated in cells with AS and FA treatment,²² while expression of this protein is suppressed in cells with Shiunko treatment (our unpublished data). In the Shiunko-treated fibroblasts (Figs. 4 and 5), increased collagen expression was found to be concomitant with overexpression of TGFβ. It is foreseeable that retention of a certain amount of fibroblasts in the wound area during the wound healing process is important. It is noteworthy that an increase of collagen production leads to downregulation of collagenase synthesis.³¹ Collagenase is a key enzyme for catalyzing the extracellular matrix; therefore, downregulation of collagenase may result in the decrease of cell mobility.³² Based on these experimental results, we propose that TGFβ may modulate the retention of an appropriate amount of fibroblasts in the wound area to improve the wound healing process.

Abbreviations and Acronyms

ARPC5

actin-related protein 2/3 complex subunit 5

AS

Angelica sinensis

ATPD

ATP synthase subunit delta

CAPNS1

calpain small subunit 1

COF2

cofilin-2

CSU

chronic skin ulcer

FA

ferulic acid

GLRX-3

glutaredoxin 3

GSTP1

glutathione S-transferase P

HSPB1

heat shock 27 kDa protein 1

IF5A-1

eukaryotic translation initiation factor 5A-1

LDHB

lactate dehydrogenase B

LR

Lithospermi Radix

PGK1

phosphoglycerate kinase 1

PHB

probibitin

PKM2

pyruvate kinase isozyme type M2

PRDX

peroxiredoxin

ROS

reactive oxygen species

SH3BGRL

SH3 domain-binding glutamic acid–rich protein

SODC

superoxide dismutase C

SPEE

spermidine synthase

STMN1

stathmin 1

TCM

traditional Chinese medicine

TCTP

translationally controlled tumor protein

TGFβ

transforming growth factor beta

TPIS

triosephosphate-isomerase

VIME

vimentin

Acknowledgments and Funding Sources

This work is supported by a research grant from the National Science Council, NSC 101-2311-B-010-004.

Author Disclosure and Ghostwriting

No competing financial interests exist. The content of this article was expressly written by the author(s) listed. No ghostwriters were used to write this article.

About the Authors

Kin-Fu Chak is the Director of the Institute of Biomedical Sciences at Mackay Medical College and a Professor of Biochemistry at National Yang Ming University, Taipei, Taiwan. Chia-Yen Hsiao contributed on the study of Angelica sinensis and Lithospermi Radix. Dr. Hsiao obtained his PhD degree in June 2012 at the Institute of Biochemistry and Molecular Biology, National Yang Ming University. Currently he is in military service. Ting-Yu Chen contributed on the study of Shiunko. Miss Chen obtained her MSc degree in June 2012 at the Institute of Biochemistry and Molecular Biology, National Yang Ming University. She is now continuing her PhD degree at the same institute of the university.