Psoriasis is a common immune-mediated chronic inflammatory skin disease characterized by thick and erythema raised plaques with adherent silvery scales. T-cells are activated via the IL-23/Th17 axis which is involved in psoriasis pathogenesis. Conventional treatments of psoriasis have adverse events that influence patients’ adherence. Wannachawee Recipe (WCR) is Thai traditional medicine that is known to be effective for psoriasis patients; however, preclinical evidence is still lacking. This study investigated the therapeutic potential of WCR on antiproliferant activity using imiquimod- (IMQ-) induced psoriasis-like dermatitis in a mouse model. Psoriasis-like dermatitis was induced on the shaved dorsal skin and right ear pinna of BALB/c mice by topical application of IMQ for 15 consecutive days after which WCR was administered to the mice by oral gavage for 10 days. Phenotypical observations, histopathological examinations, and ELISA of skin and blood samples were conducted. WCR significantly ameliorated development of IMQ-induced psoriasis-like dermatitis and reduced levels of Th17 cytokines (IL-17A, IL-22, and IL-23) in both serum and dorsal skin. Histopathological findings showed a decrease in epidermal thickness and inflammatory T-cell infiltration in the WCR-treated groups. The WCR has pharmacological actions which regulate Th17 related cytokines suggesting that it is a potential alternative therapeutic strategy for psoriasis.
Psoriasis, a noncommunicable and immune-mediated inflammatory skin disorder, is characterized by sharply demarcated, red, scaly plaques most often on the elbows, knees, scalp, and lumbar area [
Conventional therapies such as corticosteroids, vitamin D3 analogues, and calcineurin inhibitors are currently used as topical therapies in mild psoriasis. Severe psoriasis often necessitates treatment with phototherapy or systemic agents including methotrexate, cyclosporine, and acitretin [
Traditional systems of medicine in Thailand employ many different plants for the treatment of dermatological conditions [
For the experimental design, we used imiquimod (IMQ) for induction of psoriasis-like dermatitis. IMQ activates the toll-like receptor-7/8 (TLR-7/8), which is used to treat genital warts in patients [
Unless stated, all chemicals and reagents were purchased from Sigma Aldrich Co. (Merck KGaA, Darmstadt, Germany). Acitretin 25 mg was purchased from Silom Medical Co., Ltd. (Thailand), and IMQ cream (Aldara) from 3M Pharmaceuticals. The rodent diet CPF 082 was obtained from CP MICE FEED, SWT Co., Ltd., Samut Prakan, Thailand, and sterile disposable plastic needles were obtained from Strategic Applications, Inc., USA.
WCR is composed of 8 Thai herbs as shown in Table
Plants used in WCR and their original sources.
Thai name | Scientific name | Family name | Part used | Original source | |
---|---|---|---|---|---|
(1) | Kha |
|
Zingiberaceae | Rhizomes | Chanthaburi Province, Thailand |
(2) | Khao Yen Tai |
|
Smilacaceae | Rhizomes | |
(3) | Khao Yen Nuea |
|
Smilacaceae | Rhizomes | |
(4) | Khao Yen Jeen |
|
Smilacaceae | Rhizomes | |
|
|||||
(5) | Hua Ta Pead |
|
Stemonaceae | Roots | Chachoengsao Province, Thailand |
(6) | Non Tai Yak |
|
Stemonaceae | Roots | |
|
|||||
(7) | Thong Pan Chang |
|
Acanthaceae | Aerial part | Chanthaburi Province, Thailand |
(8) | Ngueak plaamo |
|
Acanthaceae | Aerial part |
WCR was prepared as previously described [
Eight- to eleven-week-old BALB/cMlac male mice
Histological and clinical features of dermatitis in mice induced by IMQ are similar to psoriasis manifestations, including erythema, scaling, and inflammation [ Group I: normal (negative control) group received only a Vaseline® on shaved dorsal skin surface and right ear pinna. Group II: positive control group received a daily topical dose of 5% IMQ cream on shaved back and right ear pinna (left ear pinna was untreated). Group III: standard group received IMQ plus acitretin at 5.14 mg/kg once daily by oral gavage. Group IV: sample group received IMQ plus WCR 800 mg/kg once daily by oral gavage. Group V: sample group received IMQ plus WCR 1,600 mg/kg once daily by oral gavage. Group VI: sample group received IMQ plus WCR 3,200 mg/kg once daily by oral gavage.
Experimental procedure to induce psoriasis-like dermatitis in mice group II to group VI. IMQ, imiquimod; WCR, Wannachawee Recipe.
All the mice in groups I to VI were sacrificed at the end of the experiment by an intraperitoneal injected overdose (50–90 mg/kg) of pentobarbital (Nembutal®; Ceva Santé Animale, France). Pentobarbital was used in this study because its profile on the sleeping time and loss of righting reflex has been well established after pentobarbital administration [
Psoriasis area and severity index (PASI) scores were determined by evaluating the degree of erythema, thickening, and scaling on the affected dorsal skin surface and ear pinna. PASI for each was measured on a four-point scale (0 = none; 1 = slight; 2 = moderate; 3 = marked; 4 = very marked). The severity of skin inflammation was measured by the combined scores (erythema plus scaling plus thickening) giving a range of scores of 0–12. Ear thickness was measured twice every other day using digital calipers (BEC, China). An increase in ear thickness was used to indicate the extent of epidermal proliferation and inflammation.
Selected tissue samples were fixed in 10% neutral-buffered formalin before processing and embedding in paraffin blocks. Sections of the samples were prepared at 4
The spleen from each mouse was isolated and a photograph was taken before being weighed. Splenomegaly was evaluated by calculating the ratio of the weight of the spleen to the bodyweight [
To measure cytokine levels in serum, mouse blood was collected at 24 h after the final treatment using the cardiac puncture method; serum was stored at −70°C until analysis. To measure cytokine levels in skin tissue, the central dorsal skins of the mice were removed and stored at −80°C. The skins were later homogenized in tissue protein lysis buffer (Bio Basic Inc., Canada) at 4°C, and the supernatants were stored at −80°C until analysis. The concentrations of IL-17A, IL-22, and IL-23 in the mouse serum and skin tissue were measured using mouse IL-17A, IL-22, and IL-23 ELISA MAX™ Deluxe (BioLegend, USA). ELISA was performed in accordance with the manufacturer’s instructions.
Differences between treatment groups were analyzed by one-way analysis of variance (one-way ANOVA), followed by post hoc Fisher’s least significant difference (LSD) test using the SPSS for Windows (version 20.0). Differences were considered significant at
Characteristics and health status such as food and water consumption, bodyweight, behavior signs, respiratory patterns, and cardiovascular signs of all mice were normal throughout the experimental period. Two or three days after starting IMQ application, it was observed that both the dorsal skin and the right ear pinna of the mice exhibited signs of erythema, scaling, and thickening (Figures
Phenotypical observations of dorsal skin and ear pinna in treated mice groups. (a) Control mice (group I) with daily topical application of Vaseline on the shaved dorsal skin and ear pinna. (b) Test mice with daily IMQ-treated (5%) dorsal skin (group II) on day 7 after IMQ treatment showing psoriasis-like inflammation and erythema lesions on treated dorsal skin. (c) Ear pinna of the control mice. (d) IMQ-treated inflamed right ear pinna (marked with
Analysis of phenotypical presentations of WCR-treated IMQ-induced inflammation accompanied by structural feature characteristic of psoriasis found that dorsal skin started to display erythema, thickening, and scaling beginning 2 to 3 days after the first IMQ application, with maximum inflammatory severity occurring at days 7 and 8. Intensity of the psoriasis-like symptoms in the IMQ-only treated group (group II) was observed to have steadily increased from day 1 through day 16 (Figure
Phenotypical presentation of the effect of WCR treatment on the dorsal skin of IMQ-induced psoriasis-like dermatitis. (A) Phenotypical presentation of dorsal skin of the five dermatitis groups; group II (IMQ, (a–d)), group III (acitretin 5.14 mg/kg + IMQ, (e–h)), group IV (WCR 800 mg/kg + IMQ, (i–l)), group V (WCR 1,600 mg/kg + IMQ, (m–p)), and group VI (WCR 3,200 mg/kg + IMQ, (q–t)) on day 1 (no treatment), day 7 (after IMQ treatment, psoriasis-like dermatitis-induced mice), day 12 (acitretin- or WCR-treated psoriasis-like dermatitis-induced mice), and day 16 (end of acitretin or WCR treatment). (B–F) PASI intensity scores (erythema, thickness, and scales) recorded on days 1–16 for mice groups II–VI. (G) PASI cumulative scores recorded for 10 days after administration of acitretin or WCR.
Analysis of H&E stained sections from the IMQ-treated dorsal skin was found to be in line with the phenotypical observations and PASI score results. The dorsal skin and right ear pinna sections of the IMQ-treated mice showed significantly increased acanthosis, hyperkeratosis of the epidermis, and inflammatory infiltration (Figure
Histological examinations of the six different treatment groups, stained with hematoxylin and eosin (H&E). (a) H&E stained dorsal skin and right ear pinna of control and IMQ-treated mice. Images magnified 100x (A, C, E, and G) and 200x (B, D, F, and H). (A, B) Control dorsal skin tissue section treated with Vaseline showing normal epidermis, dermis, sebaceous glands, and hair follicles. (C, D) Dorsal skin tissue section of IMQ-treated mice showing flaky crust, acanthosis, and hyperkeratosis of the epidermis. Abundant inflammatory infiltration and elongated rete ridges were also shown with IMQ treatment. (E, F) Control group ear pinna section showing normal epidermis, dermis, and cartilage. (G, H) Right ear pinna of IMQ-treated mice showing epidermal hyperplasia and disturbance in the normal pattern of the dermal layer. (b) Histopathology of acitretin- and WCR-treated dorsal skin sections (A, C, E, and G) and right ear pinna sections (B, D, F, and H) of IMQ-induced psoriasis-like dermatitis mice in different treatment groups. (A–H) After treatment for 10 days. (A, B) sections of IMQ-induced psoriasis-like dermatitis dorsal skin and right ear pinna treated with acitretin showed greatly decreased inflammatory infiltration and hyperplasia of the epidermis. (C, D) WCR 800 mg/kg treated mice showed slightly reduced inflammatory infiltrates relative to IMQ-treated mice. (E, F) WCR 1,600 mg/kg treated group had significant decreases in thickened epidermis, hyperplasia, and inflammatory cell infiltration. (G, H) WCR 3,200 mg/kg treated group showed maximum efficacy of treatment with recovered tissue having normal epidermis, dermis, sebaceous glands, and hair follicles. (c) H&E stained tissue sections from the spleens of mice in different treatment groups. (A) A sectional view of a control group spleen showing normal white pulp and normal red pulp. (B) A sectional view of an IMQ-treated mouse spleen showing depletion of white pulp. (C–F) Sectional views of acitretin, WCR 800, 1,600, and 3,200 mg/kg treated mice, respectively, showing dose-dependent depletion of white pulp. Ctrl, control; IMQ, imiquimod; Gr, group; DS, dorsal skin; and EP, ear pinna.
The size and weight of the spleen were markedly enlarged in IMQ-induced psoriasis mice [
Effect of WCR treatment on the ratio of spleen weight to bodyweight. (a) Representative photographs of IMQ-induced mice: normal, untreated (IMQ alone), treated with acitretin (5.14 mg/kg), and WCR 800 mg/kg, WCR 1,600 mg/kg, or WCR 3,200 mg/kg. (b) At 24 h after the final administration, mice were sacrificed and the ratio of spleen weight to bodyweight was determined. Data presented are mean ± SD (
Inflammatory cytokines responsible for inflammation in psoriasis were measured in the serum and skin samples of normal, IMQ-treated mice and WCR-treated psoriasis-like dermatitis-induced mice by ELISA assay. Compared to the normal group, the levels of Th17-mediated cytokines, particularly IL-17A, IL-22, and IL-23, were significantly higher in both serum and dorsal skin samples from IMQ-treated mice. However, a marked dose-dependent reduction in the expression of IL-17A, IL-22, and IL-23 was noted in the WCR-treated groups compared to the IMQ-only treated group. There was no significant difference of IL-22 in skin samples between the treatment groups and the normal group (Figure
Effect of WCR treatment on serum and skin cytokine profiles. The concentrations of IL-17A, IL-22, and IL-23 in (a) the serum and (b) skin were measured using ELISA 24 h after the final treatment. Data presented as mean ± SD of 7 mice.
A combination of plant species (called a formulae) based on Thai traditional medicine is often prescribed by qualified Thai traditional doctors to enhance therapeutic efficacy and reduce side effects [
IMQ is an agonist of the toll-like receptor-7/8 (TLR-7/8) which has been approved for the treatment of actinic keratosis, external genital warts, and superficial basal cell carcinoma [
In this study, all the IMQ-treated mice developed prominent characteristics of psoriatic plaques such as erythema, skin thickening, scaling, parakeratosis, and acanthosis. Although these characteristics were clearly observable, our preliminary study found that the psoriasis-like symptoms tended to spontaneously decline after 6 days of IMQ treatment. These findings are consistent with recent research which has reported instability in the IMQ-induced psoriasis-like model due to the adaptive reaction of the mouse skin to IMQ stimulation [
We demonstrated that the IMQ-induced psoriasis-like dermatitis was noticeably improved after the treatment with WCR. Furthermore, a decrease in production of Th17-related cytokines including IL-17A, IL-22, and IL-23 in serum and skin tissue occurred in the WCR-treated mice. It has been well documented that Th17 is involved in many other processes with inflammatory and autoimmune diseases [
The spleen is the largest secondary lymphoid organ in the immune system [
Systemic treatment of psoriasis is amenable to immunosuppressants; however, they produce undesirable side effects and have serious toxicity profile. Acitretin, a retinoid derivative, offers the advantage of being a nonimmunosuppressive drug with a better safety profile. It exerts its effects on gene expression by activating two families of receptors—retinoic acid receptors (RARs) and retinoid X receptors (RXRs)—that are members of the steroid receptor superfamily [
The present study demonstrates that WCR exhibits promising potential for attenuating inflammatory and psoriatic symptoms underlying psoriasis-like dermatitis in mice. This preclinical evidence suggests that WCR may have the pharmacological actions of regulating Th17 related cytokines, making it potentially useful as an alternative therapeutic strategy for psoriasis. Further investigation is needed to evaluate acute and chronic toxicities in animal models prior to performing a clinical study.
All experiments were conducted according to the international and Thai national guidelines for ethical conduct in the care and use of animals and were approved by the Animal Ethics Committee of the Faculty of Medicine, Chiang Mai University (Protocol no. 14/2559).
The authors declare that they have no conflicts of interest.
Mingkwan Na Takuathung conducted the experiments, data analyses and interpretation, and figures and manuscript preparation. Ariyaphong Wongnoppavich and Seewaboon Sireeratawong contributed to critical revisions. Ampai Panthong, Parirat Khonsung, and Natthakarn Chiranthanut revised the manuscript. Noppamas Soonthornchareonnon provided the Wannachawee Recipe. All authors have read and approved the final manuscript.
The authors wish to thank Kanjana Jaijoy, Ph.D., for her help with serum collection in their laboratory. The authors would like to thank the Graduate School, Chiang Mai University, for providing academic and documentation support. The authors would also like to express their sincere gratitude to Chatchai Swaddichai, M.D., PhD., and Surasak Im-iam, B.ATM, for providing the herbal plants of WCR and starting the use of WCR treatment in patients as well as initiating preclinical and clinical research of WCR. This work is supported by the Faculty of Medicine, Chiang Mai University (Grant no. 023-2560), and Agricultural Research Development Agency (Public Organization) (Grant no. CRP5705020550). This research was also supported by the Faculty of Medicine Graduate Student Scholarship and Faculty of Medicine Research Fund, Chiang Mai University, Chiang Mai, Thailand.