Topical Application of Cleome viscosa Increases the Expression of Basic Fibroblast Growth Factor and Type III Collagen in Rat Cutaneous Wound

Cleome viscosa L. (Cleomaceae) is an important traditional medicine of the Indian-Ayurvedic and Chinese-medicine system documented for rheumatic arthritis, hypertension, malaria, neurasthenia, and wound healing. The plant is also known as Asian spider flower and is distributed throughout the greater part of India. The present study explored the wound healing property of C. viscosa methanol extract (CvME) and its related mechanism using Wistar rat cutaneous excision wound model. Wound contraction rate, hydroxyproline quantification, and histopathological examination of wound granulation tissue were performed. The healing potential was comparatively assessed with a reference gentamicin sulfate hydrogel (0.01% w/w). Western blot for COL3A1, bFGF, and Smad-2, Smad-3, Smad-4, and Smad-7 was performed with 7-day postoperative granulation tissue. Results revealed that the topical application of CvME (2.5% w/w) significantly accelerated the wound contraction rate (95.14%, 24 postoperative days), increased the hydroxyproline content (3.947 mg/100 mg tissue), and improved histopathology of wound tissue as compared to control groups. Western blot analysis revealed that CvME significantly upregulated the expression of COL3A1 and bFGF and increased the Smad-mediated collagen production in granulation tissue. These findings suggest that C. viscosa promoted the wound repair process by attenuating the Smad-mediated collagen production in wound granulation tissue.


Introduction
Cutaneous wound is a physical injury that results in structural and functional discontinuity of skin tissue, and healing is a tangled tissue restorative process that involves distinct, overlapping phases of homeostasis, inflammation, proliferation, and tissue remodeling [1,2]. Healing cascade starts from the moment of injury and involves continuous cellcell and cell-matrix interactions till tissue remodeling of the scar. Blood coagulation at injury site provides temporary fibrin scaffold matrix, which hosts the infiltrating cells and serves as a reservoir of various growth factors and cytokines. Brief initial vasoconstriction at the injury site favors homeostasis and late vasodilation prompts the extravasations of inflammatory cells (neutrophils and macrophages) to remove the nonfunctional cells and destroy the invading infections [2,3]. Proliferation phase initiated by macrophages involves the formation of granulation tissue and neovascularization. During the final remodeling phase of wound healing, the newly formed collagen fibers cross-link with other structural protein components and contribute to the strength of the scar tissue.
Cleome viscosa L. (Cleomaceae) is a sticky herb with yellow flowers, which resembles to mustard, and is distributed throughout the greater part of India. The plant is an important traditional medicine of the Indian-Ayurvedic and Chinese-medicine system for rheumatic arthritis, hypertension, malaria, neurasthenia, and snakebite [4][5][6]. Various scientific investigations revealed analgesic-antipyretic, antiinflammatory, hepatoprotective, hypoglycemic, anthelmintic, 2 BioMed Research International and antidiarrhoeal activity of C. viscosa [4,[7][8][9][10]. Panduraju et al. [11] reported the topical application of C. viscosa extract accelerated the wound healing in Wistar rat with significant wound contraction rate. Our previous experimental findings showed that methanol extract of C. viscosa (CvME) possesses potent antioxidant, antimicrobial, and fibroblast proliferation properties as compared to petroleum ether, chloroform, and water extracts [12]. However, to rationalize the wound healing activity of C. viscosa, some major supportive evidences of efficacy such as histopathological examination, estimation of growth factors, and effect on collagen production in healing tissue are yet to be investigated. Therefore, the present study aims to evaluate the C. viscosa inducedalteration in fibroblast growth factor and its effect on Smad-mediated collagen (type III) production in Wistar rat excision wound model.

Preparation of Extracts. C. viscosa leaves were collected
during August-September (2011) from the herbal garden of the Defence Research Laboratory, Tezpur, Assam (India), authenticated by the Botanical Survey of India, Shillong (Accession number 085249). About 100 g of shades dried leaf powder was successively extracted using organic solvents (petroleum ether, chloroform, and methanol) by Accelerated Solvent Extractor (ASE 1.5, Dionex, USA) as described earlier [12] and concentrated in a rotary evaporator (Rotavac, Hei-dolph2, Germany) under reduced pressure. Water extract was freeze dried by lyophilization. Preliminary phytochemical screening was performed as described earlier [13].  (20-25 g) and Wistar rats (250-300 g) were housed in the Defence Research Laboratory (DRL), Tezpur, Assam, India, and acclimatized for 3 days. Animals were given free access to feed and water ad libitum. The experiments were performed according to the Institutional Animal Ethical Committee guidelines (IAEC/DRL/05/July/2011).

Acute Skin Irritation and Toxicity
Study. The acute skin irritation and toxicity study was performed as per OECD guidelines-402 (OECD guidelines, 1987) to determine the therapeutic dose of CvME. CvME hydrogel (1 and 2.5% w/w) was applied to the shaved portion at the back of the mice and observed for 14 days for an abnormal skin response, including irritation, redness, itching, and other related symptoms [14].

Animal Grouping and Excision Wound Creation.
Rats were inflicted with excision wounds as described by Atiba et al. [15]. Animals were anesthetized and a circular 20 mm diameter wound was created on the shaved dorsal skin up to the depth of loose subcutaneous tissue. Animals were randomly divided into four groups ( = 20): group I, nontreated; group II, vehicle control (Carbopol 934 containing 5% propylene glycol); group III, CvME hydrogel treated (2.5% w/w); and group IV, gentamicin sulfate (0.01% w/w). Treatment was given once daily until complete epithelialization. For one-third of animals, wound granulation tissues (excluding any underlying muscle and extraneous tissue) were harvested on the 7th postoperative day. A portion of tissue was processed for Western blotting and another portion was fixed in Histochoice tissue fixative (Amresco, USA) for histopathological evaluations. Half of the remaining animals were euthanized on day 15 after injury; the entire remodeling tissue was used for histopathological assessment and the remaining animals were observed until complete epithelialization.

Wound Contraction Rate and Hydroxyproline Content
Estimation. The progressive changes of wounded area were photographed (Nikon Coolpix-S3000) and evaluated by using special size analysis software ImageJ (National Institutes of Health, ImageJ software, downloaded from http://rsb.info.nih.gov/ij/index.html). Wound contraction was expressed as a percentage of the original wound size (day 0).
Hydroxyproline content was analyzed on day 7 in postinjury granulation tissue as described by Neuman and Logan [16]. Acidic tissue hydrolysate was neutralized to pH 7.0 and mixed with 10 mM CuSO 4 and 2.5 N NaOH followed by 6% H 2 O 2 . The solution was mixed and incubated at 80 ∘ C for 5 min with frequent vigorous shaking. Upon cooling, 3 N H 2 SO 4 was added with agitation. Finally, 5% p-dimethylamino-benzaldehyde was added and incubated at 70 ∘ C for 15 min. Absorbance was measured at 500 nm using a UV-VIS spectrophotometer (CE7200, CECIL, USA). The standard calibration curve was plotted for pure hydroxyproline and used for estimation of the test samples.

Statistical Analysis.
The results are expressed as means ± standard deviation (S.D.). The treated groups were compared with control groups by analysis of variance following Dunnett's test. A statistical value less than 0.05 was considered significant.

Results and Discussion
3.1. Extraction of Phytochemical from C. viscosa. The powdered C. viscosa leaf (100 g) yielded 2.82 g inpetroleum ether (CvPE), 1.57 g in chloroform (CvCE), 12.03 g in methanol (CvME), and 9.11 g in water (CvWE). The preliminary phytochemical screening showed the presence of alkaloids, flavonoids, terpenes, and carbohydrates in different extracts. In our earlier study, we have reported about its potent antioxidant, antimicrobial, and human dermal fibroblast proliferation activities of CvME [12] which was similar to the previous reports of Koca et al. [18] and Gouthamchandra et al. [19], where the alcoholic extracts were reported as potent antioxidant and antimicrobial and showed significant wound healing property in comparison to other sequential extracts.
Mondal and Suresh [20] also reported the wound healing activity in other species of Cleome, where the methanol extract has shown faster wound contraction rate over other extracts. Therefore, CvME was selected for in vivo wound healing evaluation using Wistar rat.

Acute Skin Irritation and Toxicity of CvME.
Animals treated with CvME hydrogel (1 and 2.5%, w/w) did not show any symptoms of skin irritation and inflammation. Therefore, the extract was considered safe and 2.5% (w/w) of C. viscosa methanol extract hydrogel was used for in vivo wound healing study.

Effect of CvME on Wound Contraction Rate.
Wound contraction is the centripetal movement of surrounding epithelial tissues that depends on the reparative ability and general health of the tissue [21,22]. The wound appeared clean and free of exudates and distinct granulation tissues appeared at the wound edges from day 3 after injury in CvME and gentamicin sulfate treated groups. Wound areas were reduced parallel to postoperative days and CvME treated group showed 60.99, 71.13, 82.34, and 90.87% wound contraction, whereas gentamicin sulfate treated group showed 57.2, 71.58, 79.51, and 90.02% in 12, 15, 18, and 21 postoperative days, respectively (Figures 1(a) and 1(b)). On the other hand, nontreated control group showed 23.85, 28.74, 42.92, and 50.63% wound contraction and vehicle treated groups showed 33.75, 41.16, 48.54, and 57.49%, in 12, 15, 18, and 21 postoperative days, respectively (Figure 1(b)). Although the wound contraction rate by CvME was comparable to gentamicin sulfate treated groups, it showed significantly higher < 0.05 as compared to nontreated and vehicle treated control groups. These findings coincided with the  previous report on the wound healing properties of the methanol extract of Cleome species [11,12,20]. The difference in wound contraction rate and duration of healing may be due to differences in the experimental and environmental conditions. Many researchers have tried with the methanol extract of herbs/shrubs for wound healing properties to corroborate with specific ethnobotanical knowledge [23]. Similarly, methanol extract was studied and reported to possess better antioxidant, antimicrobial, anti-inflammatory, and wound healing activities than other solvent extracts [12,18,19].

Effect of CvME on Hydroxyproline Content.
Hydroxyproline is a basic constituent of collagen structure and contributes to 14% approximately. Measurement of hydroxyproline can be used as an index for collagen turnover [22]. Increase in the hydroxyproline content indicates increased collagen synthesis, which corresponds to the enhanced wound healing. In the present study, the topical application of CvME hydrogel significantly increased ( < 0.05) the hydroxyproline content as compared to nontreated and vehicle treated control groups (Figure 2). CvME treatment also showed higher hydroxyproline content as compared to gentamicin sulfate group, although the data were statistically insignificant. These results therefore reflect the potent wound healing activities of CvME in Wistar rats. Table 1 indicated that the histopathology examination of the wound granulation tissue supported the findings of CvME accelerated wound healing as an organized wound repair process (inflammation, proliferation, and remodeling). On the other hand, slow epithelialization rate and less collagen density were observed in vehicle and nontreated groups. Histopathological section of 7-postoperative-day wound tissue showed increased infiltration of macrophages/fibroblasts with less inflammatory cells and ulcer-edematous areas in both CvME and gentamicin sulfate treated groups. On the other hand, mild edema with high polymorphonuclear (inflammatory) cell density and less macrophages/fibroblasts were observed in control groups (Figure 3). Fifteen-day CvME topical application significantly accelerated the cutaneous wound repair processes as evident from the appearance of thick, well organized reepithelialized epidermis and compact dermis with abundant collagen fibers. Masson's trichrome (MT, 400×) staining for 15-day postoperative wound tissue depicted distinct mononuclear cells (macrophage and fibroblast) infiltration and faster keratinization with intraepithelial cornification in CvME (Figure 3), whereas slow reepithelialization and less collagen bundle were noticed in control groups. The results of improved histopathology of wound granulation tissue supported the faster wound contraction rate and elevation of hydroxyproline content in CvME hydrogel treated group, which corroborates the earlier reports on wound healing processes [24][25][26]. These effects of CvME in wound repair may be due to the presence of phytochemical such as tannins, triterpenoids, flavonoids [4,27], and their combined effect of antioxidant, antimicrobial, and anti-inflammatory properties [8,28,29].

Effect of CvME on Histopathology of Wound Granulation Tissue.
3.6. Effect of CvME on Granulation Tissue Protein. Proliferation phase of wound healing involves formation and vascularization of granulation tissue, collagen production, and their subsequent maturation. Collagen type III, present in remodeling wound tissues, is produced by the proliferating young fibroblasts under the influence of various growth factors like transforming growth factor-(TGF-), basic fibroblast growth factor (bFGF), and so forth. The bFGF is a potent angiogenic molecule, which promotes the neovascularization and blood supply to the granulation tissue. Increased oxygen supply facilitates the maturation ofcollagen fibers in granulation tissue [3]. Western blot analysis revealed that application of CvME significantly increased ( < 0.05) the expression of COL3A1 and bFGF protein in wound granulation tissue (Figure 4), thus further confirming the above findings of faster wound contraction rate and higher hydroxyproline content.
Various growth factors such as TGF-, bFGF, PDGF (platelet derive growth factor), and VEGF (vascular endothelial growth factor) released by the activated platelets, macrophages, and lymphocytes control the proliferation phase of wound healing [1,3]. Released TGF-bound to the fibroblast TGF-receptors in granulation tissue and initiates the TGF--Smad-mediated collagen production cascade [30]. Intracellular Smad family protein (TGF-type I receptor kinases substrate) forms heterocomplex (Smad-2/Smad-3/Smad-4) and transduced the extracellular TGFsignal to fibroblast nucleus for collagen production. Therefore, Smad family proteins were analyzed by Western blot and signal transducer protein (Smad-2, Smad-3, and Smad-4) and showed significantly ( < 0.05) higher expression in CvME hydrogel treated group, whereas the inhibitory protein (Smad-7) was observed to be equal in all treatment groups.
-Actin was used as an internal control. These findings confirmed that the Smad-mediated mechanism is involved in the increased COL3A1 expression, higher hydroxyproline content, and faster wound contraction rate that contributes to overall improved histopathology during wound repair process.
Therefore, the present study suggested that the topical application of C. viscose methanol extractenhances thewound repair process by attenuating TGF -Smad-mediated collagen production in wound granulation tissue.