Chinese herbal medicine (Tuhuai extract) exhibits topical anti-proliferative and anti-inflammatory activity in murine disease models

Abstract

While psoriasis is one of the most common skin disorders in humans, effective, safe and inexpensive treatments are still largely unavailable. Chinese herbal medicine (CHM) has been used for centuries for treating psoriasis and several reports claim that systemic administration of one such CHM, Tuhuai, mainly composed of flos sophorae, smilax glabra roxb and licorice, is effective in psoriasis. However, the mechanisms by which this CHM improves psoriasis are not yet clear. Two universal features of psoriasis are epidermal hyperplasia and inflammation. Moreover, drugs that specifically inhibit epidermal hyperplasia and/or inflammation are widely used to treat psoriasis. Here, we investigated whether topical applications of Tuhuai extract exhibit anti-proliferative and anti-inflammatory activities in two murine models of inflammatory dermatoses. To assess Tuhuai's potential anti-proliferative effect, we disrupted epidermal barrier function twice-daily for 4 days in normal hairless mice followed by topical applications of either 1% Tuhuai extract or Vehicle to both flanks immediately after each barrier perturbation. Changes in epidermal proliferation and apoptosis were evaluated by immunohistochemistry and TUNEL staining. To assess the anti-inflammatory effects of Tuhuai, both irritant (phorbol ester) and acute allergic contact dermatitis (oxazolone) models were used. Whereas topical Tuhuai extract did not alter epidermal proliferation or induce irritation in normal skin, it both reduced epidermal hyperplasia in the epidermal hyperproliferative model, and reduced inflammation in both irritant and allergic contact dermatitis models. As topical Tuhuai extract exhibits anti-proliferative and anti-inflammatory properties in a variety of human models of inflammatory dermatoses, Tuhuai could provide an effective, relatively safe and inexpensive therapeutic alternative for the treatment of inflammatory dermatoses, including psoriasis.

Introduction

Psoriasis is a common, chronic skin disorder characterized by epidermal hyperproliferation and inflammation. Although psoriasis affects up to 5% of the general population, a much smaller percentage of ethnic Chinese are affected (<0.3%). The tendency for psoriasis to frequently flare after cessation of therapy; to develop tachyphylaxis to most forms of therapy and its tenacious thick scale, represent a considerable therapeutic challenge. As a result, long-term systemic therapy or combinations of multiple agents are often used. These conventional approaches for the treatment of psoriasis include: systemic and topical anti-inflammatory, anti-proliferative, immunosuppressive and biological agents, as well as phototherapy. Yet, important side effects can occur with each of these systemic regimens, whether they are administrated systemically or topically. For example, cyclosporin A has been reported to be effective in the treatment of psoriasis (1,2), but a number of adverse effects, including kidney disease, hypertension or gingival hyperplasia, can occur (2–8). Glucocorticoids are also commonly used in the treatment of psoriasis but can produce severe side effects. Topical beta-methasone and clobetasol can induce skin atrophy, permeability barrier dysfunction, as well as increased skin infections (9–12). Liver damage can result after long-term systematic administration of methotrexate (13–15). In addition, decreased bone density has been observed with methotrexate therapy (16,17). 1,25 (OH) vitamin D₃ and its analogues have proved to be effective in treating psoriasis (18,19), however, topical irritation and hypercalciuria can occur (18,20,21). Retinoids represent another group of biological agents, which show beneficial effects in treating psoriasis (22,23). Yet, alopecia, elevated blood triglycerides, and many other side effects limit acitretin use (24,25). TNFα inhibitors, such as etancercept and infliximab, although effective, also can cause serious adverse effects including infections, exacerbation of psoriasis and increased risk of lymphomas (26–31). Finally, the considerable medical costs incurred during treatment of psoriasis with currently available medications can interfere with quality of patients’ daily life (32). Thus, substantial risks coupled with modest efficacy, limit current usage of conventional medications for psoriasis, providing rationale for an effective, relatively safe and inexpensive alternative treatment for psoriasis.

Chinese herbal medicines (CHM), which have been used for centuries in the treatment of skin diseases, including psoriasis, are both inexpensive and widely available. Moreover, some controlled clinical studies suggest that these preparations are effective, safe and inexpensive treatment for inflammatory skin disorders in general, but psoriasis in particular (33–35). Although in vivo studies suggest that CHM inhibit cell proliferation (36), which could be relevant for how CHM improve psoriasis, controlled mechanistic studies have not yet been carried out. Whereas normal mouse vaginal and rectal epithelia are often used to assess the anti-proliferative activity of CHM (37), these models do not necessarily reflect how hyperproliferative epidermis would respond. Thus, an epidermal hyperproliferative model (ideally also accompanied by dermal inflammation or by other models of inflammation) would be preferred to assess the therapeutic effects and to study the mechanisms responsible for the efficacy of CHM. Therefore, in the present studies, we employed an epidermal hyperproliferative murine model to evaluate the ability of topical CHM to reduce epidermal hyperplasia, and both TPA-induced irritant dermatitis and oxazolone-induced, allergic contact dermatitis mouse models to assess the anti-inflammatory efficacy of topical CHM.

Materials and methods

Materials

Six- to eight-week-old female hairless mice (hr/hr) and CD-1 male mice were purchased from Charles River Laboratories (Wilmington, MA, USA) and fed mouse diet (Ralston-Purina Co., St Louis, MO, USA) and water ad libitum. Phorbol 12-myristate 13-acetate (TPA), 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) and acetone were purchased from Sigma Chemical Co. (St Louis, MO, USA). Biotinylated anti-proliferating cell nuclear antigen (PCNA) antibody was from CalTag Laboratories (Burlingame, CA, USA). ABC-peroxidase kit was purchased from Vector Laboratories (Burlingame, CA, USA). The raw materials of Tuhuai mixture were purchased from Bozhou Tongrentang Pharmaceutical Company (Bozhou, China), where all herbals are collected under strict quality control conditions, according to standards set by the Chinese State Department of Drug Administration. The topical Tuhuai extract was prepared by soaking flos sophorae, smilax glabra roxb, Paeonia lactiflora, radix scutellariae, flos lonicerate and glycyrrhiza uralensis-containing mixture without further purification or analytical studies, in 100% ethanol (20%, w/v) for 3 weeks. The ethanol extract was used as the treatment agent.

Experimental protocols

All animal procedures were approved by the Animal Studies Subcommittee of the San Francisco Veterans Administration Medical Center and performed in accordance with their guidelines. An epidermal hyperproliferative model was established by repeated applications of cellophane tape on the flanks of hairless mice (hr/hr) twice daily for 4 days until transepidermal water loss reached 3–6 mg/cm²/h as measured with an electrolytic water analyzer (Meeco, Warrington, PA, USA). Either 60 μl of 1% Tuhuai extract or ethanol alone was applied to both flanks on the tape-stripped areas (4 × 4 cm) following each barrier disruption. Skin samples for immunohistochemistry were taken 12 h after the last treatment.

For anti-inflammatory studies, ear inflammation was induced by topical application of 10 μl of 0.03% TPA to both inner and outer surfaces of both ears of CD-1 mice (38). 10 μl of 1% Tuhuai in ethanol or ethanol alone was applied to both surfaces of the right or left ear at 45 min and 4 h following TPA treatment. Additionally, the oxazo-lone-induced ear inflammation model was also used to assess the anti-inflammatory effects of Tuhuai. CD-1 mice were sensitized by topical application of 3% oxazolone to the back once daily for 2 days. One week later, 10 μl of 0.5% oxazolone was applied to both inner and outer surfaces of both ears. 10 μl of 1% Tuhuai or ethanol alone was applied to both surfaces of right or left ear at 45 min and 4 h following last oxazolone treatment. Ear thickness was measured with a digital caliper (Mitutoyo, Tokyo, Japan) before and 20 h after the last oxazolone or TPA application, and samples were taken for H&E staining. The measurement of epidermal thickness of nucleated cell layers was taken at 100X on H&E sections at every 2 cm points along the epidermis.

Immunohistochemistry

For determination of epidermal proliferation, a previously described method was employed (39). Briefly, 5 μm paraffin sections were incubated with an antibody against PCNA (Ki67) (CalTag Laboratories, Burlingame, CA, USA) overnight at 4°C. Immunostaining was detected by ABC peroxidase method. Sections were visualized with a Zeiss Microscope (Jena, Germany), and digital images were taken with Axio Vision software 3.1 (Carl Zeiss Vision, Munich, Germany).

Keratinocyte apoptosis was determined with a Fluorescein-FragEL™ apoptosis detection kit (Oncogen Research Products, San Diego, CA, USA), according to manufacturer's protocol. Briefly, deparaffinized sections were incubated with proteinase K for 10 min at room temperature, followed by incubation with deoxynucleotidyl transferase for 60 min. The labelled cells were observed with a fluorescence microscope.

Epidermal thickness measurement and PCNA quantification

Thickness of the epidermal nucleated cell layers was measured on 100X micrographs taken every 2 cm along the epidermis from biopsies from normal untreated, Vehicle-treated, as well as, Tuhuai-treated skin. The data presented represent the mean of all measured points ± SEM. The number of PCNA positive cells was counted on the prints at every 1 cm segment along the epidermis, and presented as the mean of all PCNA positive cells/cm ± SEM. One-way anova test was used to determine significant differences.

Results

Topical Tuhuai does not alter epidermal proliferation in normal mice

The gross (macroscopic) appearance of mouse skin remained normal after 4 days of topical Tuhuai treatment. Notably, there were no signs of skin irritation either by visual inspection or histologically. Moreover, epidermal thickness and proliferation did not change, as assessed both by H&E and PCNA staining (Fig. S1). These results suggest that topical Tuhuai extract does not alter epidermal proliferation or produce inflammation in normal mouse skin, suggesting further that topical extract of Tuhuai is both non-toxic and non-atrophogenic.

Topical Tuhuai inhibits epidermal proliferation in an epidermal hyperproliferative model

We next assessed whether topical Tuhuai inhibits epidermal proliferation in a hyperproliferative model, produced by twice-daily tape-stripping for 4 days. As shown in Fig. 1, repeated disruption of permeability barrier function induced epidermal hyperproliferation as measured by PCNA staining (Fig. 1b versus 1d), which was accompanied by increased epidermal thickness (Fig. 1a versus 1c). Barrier disruption followed by ethanol Vehicle applications did not reduce epidermal hyperplasia. In contrast, topical Tuhuai treatment inhibited the development of epidermal hyper-plasia (Fig. 1c,d versus Fig. 1e,f,g), although it did not completely normalize the epidermal thickness (Fig. 1g,h). Thus, in contrast to normal skin, topical Tuhuai exerts antiproliferative effects in an epidermal hyperpoliferative mouse model. As no changes in keratinocyte apoptosis occurred in hyperproliferative skin following Tuhuai treatment (data not shown), it is likely that the anti-hyperplastic mechanism of the Tuhuai extract is inhibiting epidermal DNA synthesis.

Figure 1.

Topical application of Tuhuai extract inhibits epidermal proliferation in an epidermal hyperproliferative model: an epidermal hyperproliferative model was established by repeated barrier disruption. Either Vehicle or 1% Tuhuai extract was applied topically following each barrier disruption. Figure 1a, c, e are H&E staining and b, d, f are anti-PCNA staining. Figure 1a, b are normal skin. Figure 1c, d are hyperproliferative skin treated with Vehicle. And figure 1e, f are hyperproliferative skin treated with Tuhuai extract. The magnifications for all are the same. Scale bar = 20 μm. Epidermal thickness of nucleated cell layer was measured on 100X H&E sections at every 2 cm points along the epidermis (Figure 1g, n = 10 for normal; n = 30 for Vehicle treated and n = 34 for Tuhuai treated). Number of PCNA positive cells was counted on every 1 cm segment along epidermis (Figure 1h, n = 60 for normal; n = 63 for Vehicle treated, and n = 67 for Tuhuai treated). The data are expressed as mean of all measured points. The significances are shown on the figure. And *P < 0.001 is for the difference between normal and Tuhuai treated.

Topical Tuhuai exhibits anti-inflammatory activities in both irritant and acute allergic contact dermatitis models

Reduction of dermal inflammation is a major goal in the treatment of inflammatory dermatoses, including psoriasis. Hence, we next assessed whether topical applications of Tuhuai decrease inflammation in the TPA model of irritant contact dermatitis, measuring the changes in ear thickness. As shown previously (40), topical TPA treatment caused a dramatic increase in ear thickness, which was not reduced by Vehicle treatment (Fig. 2c,g). In contract, topical Tuhuai application following TPA treatment significantly reduced the development of increased ear thickness (Fig. 2e,g).

Figure 2.

Topical application of Tuhuai extract reduces the inflammatory response in both TPA and oxazolone models: ear inflammation was induced by topical TPA or oxazolone treatment. Skin samples were obtained 20 h after treatment. Significant reductions in inflammatory cell infiltration and ear thickness were seen in Tuhuai-treated ear. a and b are normal ear treated with Vehicle. c (TPA) and d (oxazolone) are inflammatory ear treated with Vehicle. e and f are inflammatory ear treated with Tuhuai. Scale bar = 100 μm. The ear thickness was measured with a digital caliper as described in Methods. Fig 2g is TPA model (P < 0.001 between all groups) and Fig 2h is oxazolone model (P < 0.01 between all groups). Data are expressed as mean + SEM. Numbers for each group are indicated in the figure.

To further evaluate the anti-inflammatory effects of topical Tuhuai, another inflammatory model was also employed. As seen in Fig. 2d,f,h, topical Tuhuai also inhibited inflammation in the oxazolone model of acute allergic contact dermatitis (38,41). The anti-inflammatory effects of topical Tuhuai were further demonstrated in H&E-stained sections, which demonstrated reductions in both ear thickness and inflammatory cell infiltration in the ears of both the TPA and oxazolone-treated mice (Fig. 2, Fig. S2). Together, these results show that topical Tuhuai exhibits anti-inflammatory effects in both irritant and allergic contact dermatitis mouse models.

Discussion

Although widely used for millennia in China, until recently CHM has attracted minimal attention outside China as potential therapy for skin diseases. CHM are given systemically to patients with psoriasis, based upon the type and extent of the patient's condition. According to traditional Chinese medical theory, psoriasis is generally classified into four types, i.e. blood heat, blood dryness, blood stasis and blood toxicity. Tuhuai is a mixture of CHM, which has shown evidence of clinical effectiveness when used systemically in the treatment of psoriasis (42,43). Most Chinese herbals used in the treatment of psoriasis must be heated for more than 1 h to extract the active ingredients prior to use, which is inconvenient for most patients, and could also inactivate some ingredients. Moreover, these herbals often leave an unpleasant taste after oral use. Furthermore, ready-to-eat herbal preparations are convenient, but usually large amounts of sugar are added to make the preparation more edible, which is not acceptable for many patients with diabetes. Thus, further development of topical CHM would be a highly desirable alternative for the treatment of psoriasis.

The pharmacological mechanisms by which CHM influence inflammatory dermatoses, including psoriasis, are not clear. It has been known that these medicinal herbs contain many ingredients, some of which are known to be active (see below). In addition, chemical reactions may occur to produce still-unknown, new chemical entities during preparation or in complex mixtures. Nevertheless, Tse et al. studied the effects of 60 CHM, previously shown to display variable effectiveness in the treatment of psoriasis, on cell proliferation in HaCaT keratinocyte cell culture (44). Significant inhibition of cell proliferation has been observed with some of ingredients in Tuhuai mixture, such as glycyrrhizin (45,46). In agreement with these in vitro finding, we demonstrated here that topical Tuhuai also inhibits epidermal proliferation in an in vivo model of hyperproliferative epidermis. Moreover, scutellarin, flavonoids and paeonol, which are chemical compounds found in the Tuihuai mixture, inhibit cell proliferation in cancer cell lines (47–50). Yet the molecular mechanism by which Tuhuai inhibits keratinocyte proliferation is not known. Furthermore, the decreased epidermal thickness induced by topical Tuhuai extract is likely due to the inhibition of keratinocyte proliferation, as keratinocyte apoptosis did not change in either normal or hyperproliferative epidermis following topical Tuhuai treatment even though some anti-psoriatic agents induce keratinocyte apoptosis (51,52). As these active chemicals are ethanol extractable, it can be assumed that they were in the Tuhuai extract used in the present study.

Cutaneous inflammation is another prominent pathological feature of certain dermatoses, including psoriasis. Accordingly, immunomodulators and steroids often are used in the treatment of psoriasis. Previous clinical observations demonstrated that systematic administration of CHM decreased cutaneous inflammation in psoriasis and eczema (53,54). Topical applications of either glycyrrhiza containing gel or Atopiclair®, a US FDA approved glycyrrhetinic acid-containing cream for atopic dermatitis treatment, improve atopic dermatitis (55–57). Moreover, CHM inhibit the production of cytokines from stimulated human monocytes (58). Smilax glabra roxb and astilbin (a chemical from smilax glabra roxb) inhibit inflammation by decreasing TNF alpha expression, lymphocyte migration, as well as matrix metalloproteinase (MMP-2, MMP-9) activities (59–64). Both blood IgE and IL-4 levels decreased in atopic dermatitis patients treated with glycyrrhizin (65). Here, we demonstrated that topical applications of Tuhuai, containing all of the above ingredients, inhibits cutaneous inflammation, as indicated by a reduction of inflammation in both irritant and acute allergic contact dermatitis models. Yet, again the molecular mechanism(s) whereby Tuhuai inhibits inflammation remain unknown. Nevertheless, while topical Tuhuai does not completely eliminate skin inflammation, it could be useful for human inflammatory dermatoses, including psoriasis.

The epidermal hyperproliferative model is easy to establish and provides a large skin area for experimental study. Previously, we have utlilized this model to successfully evaluate the anti-proliferative effects of topical PPAR and LXR activators (66). Although this model is not an exact analogue of psoriasis, the epidermal changes, i.e. hyperplasia and abnormal barrier function mimic similar changes in psoriasis, making it a useful tool for assessing potential therapies for psoriasis. Likewise, the inflammatory models employed here are also easy to establish, and highly reproducible, and therefore useful models to assess anti-inflammatory therapies (38,41). Coupling the epidermal hyperproliferative model with the inflammatory models provides a useful matrix for the testing and development of topical anti-inflammatory and proliferative medications.

In conclusion, a combination of the epidermal hyperproliferative model and the ear inflammation models provide a reliable and useful approach to assess the efficacy of anti-proliferative and inflammatory agents. In addition to its anti-inflammatory effect in both TPA and oxazolone models, topical Tuhuai extract also inhibits epidermal proliferation in the epidermal hyperproliferative model, but not in normal skin. These results explain, at least partially, the mechanism by which psoriasis reportedly is effectively treated with Tuhuai, and suggest that topical Tuhuai may be clinically useful in the treatment other inflammatory dermatoses as well. In addition to delineating the molecular mechanisms accounting for Tuhuai's activity, further clinical studies will also be required to evaluate the efficacy of topical Tuhuai on psoriasis and other inflammatory skin disorders.