original article effects of artemether on the ... · abstract: artemether exhibits diverse...

Int J Clin Exp Med 2015;8(5):7069-7078www.ijcem.com /ISSN:1940-5901/IJCEM0007408

Original ArticleEffects of artemether on the proliferation, apoptosis, and differentiation of keratinocytes: potential application for psoriasis treatment

Jie Wu1, Hong Li2*, Ming Li1*

1Department of Dermatology, Zhongshan Hospital, Fudan University, Shanghai 200032, China; 2Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China. *Equal contributors.

Received February 27, 2015; Accepted May 2, 2015; Epub May 15, 2015; Published May 30, 2015

Abstract: Artemether exhibits diverse pharmacological effects and has multiple applications. This study aimed to investigate its antiproliferative and apoptogenic effects on HaCaT cells and keratinocyte differentiation-inducing activity in vivo. WST-8 analysis demonstrated that Artemether can inhibit the proliferation of cultured HaCaT cells in a time- and dose-dependent manner. Annexin V/PI dual staining and JC-1 staining further revealed that Artemether can dose-dependently augment HaCaT apoptosis. To investigate the keratinocyte differentiation-inducing activity of Artemether, it was prepared as topical creams at concentrations of 1%, 3%, and 5%. During the 4 weeks of topi-cal treatment, no evidence of irritation was observed in the mouse tail test. Artemether cream dose-dependently increased the degree of orthokeratosis and the relative epidermal thickness of mouse tail skin, indicative of the keratinocyte differentiation-inducing activity. Taking the in vitro and in vivo findings together, the present study sug-gests that Artemether may be a promising antipsoriatic agent worthy of further investigation.

Keywords: Artemether, psoriasis, antipsoriatic, keratinocyte, HaCaT cells, proliferation, apoptosis, differentiation, mouse tail test

Introduction

Psoriasis is a chronic, remitting, and immune-mediated inflammatory skin disease with a worldwide incidence of approximately 2% to 3% [1]. Aside from the infiltration of immune cells, expression of inflammatory cytokines, hyperp-roliferation and altered differentiation of epi-dermal keratinocytes, and subepidermal angio-genesis are also observed in psoriatic lesions [2]. Patients seldom die from psoriasis, but the itchy skin, slightly raised erythema, silvery white scales, and comorbidities, such as car-diovascular disease and metabolic syndrome, seriously impair their quality of life [3, 4]. Currently, drug treatment remains the main option for most psoriasis patients. However, the long-term therapeutic efficacy of existing drugs is not ideal because of side effects and the development of pharmacoresistance [5]. Therefore, identification of new effective anti-psoriatic agents with less adverse effects remains a hotspot in dermatology to date.

Artemisia annua L., also known as Qinghao in Chinese, has been used in China for more than two thousand years. Artemether (ART) is a semi-synthetic derivative of artemisinin isolat-ed from A. annua. Artemisinin and its deriva-tives are known for their antimalarial effects, but they can also exert antiproliferative, apop-totic, anti-angiogenic, anti-inflammatory, and immunomodulatory activities [6]. In particular, they can inhibit the activation and proliferation of T cells [7], and they can reduce the produc-tion of tumor necrosis factor-alpha [8], a key cytokine that is substantially increased in pso-riasis lesions [9].

Considering the pharmacological effects of artemisinin and its derivatives, we speculated that these substances can block the pathologi-cal changes brought about by psoriasis in vari-ous aspects. In this study, we determined whether or not artemisinin and its derivatives exhibit antipsoriatic activity in an experimental model. This study is the first to evaluate the in

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Effects of artemether on keratinocytes

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vitro and in vivo pharmacodynamic effects of ART on the proliferation, apoptosis, and differ-entiation of keratinocytes.

Materials and methods

Materials for in vitro experiment

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA (1×), phosphate-buffered saline (PBS, pH 7.4), and a Dead Cell Apoptosis Kit with Annexin V Alexa Fluor® 488 and propidium iodide (PI) for flow cytometry were purchased from Invitrogen (Carlsbad, USA). A WST-8 assay Kit was pur-chased from Dojindo Molecular Technologies, Inc. (Tokyo, Japan). ART (purity ≥ 99.3%) was purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China) and was dissolved in dimethylsulfoxide (final concentration < 0.25% v/v).

General cell culture

HaCaT cells, an immortalized line of human epi-dermal keratinocytes extensively used in psori-asis research [10], were obtained from the Experimental Research Center of Changhai Hospital (Shanghai, China). Cells were routinely maintained in DMEM culture medium with 10% FBS, 100 U/mL penicillin, and 100 U/mL of streptomycin, and then maintained at 37°C in a humidified atmosphere containing 5% CO2.

Antiproliferative effects of ART on HaCaT cells

The antiproliferative effects of ART on HaCaT cells were examined with a WST-8 assay Kit because it is more reproducible and sensitive than the MTT assay [11]. In brief, cells were cul-tured in a 96-well plate at a density of 3 × 103 cells per well. After 24 h of culture, serial dou-bling dilutions of ART (10, 20, 40, 80, 160, and 320 μg/mL) were introduced into the treatment wells, whereas the negative controls were rou-tinely maintained in DMEM without ART. At 24, 48, and 72 h, 10 μL of WST-8 solution was added to each well, and then the cells were incubated for another 3 h. A microplate reader (FlexStation 3, Molecular Devices) was used to determine the absorbance of each group at 450 nm with a reference wavelength of 630 nm.

Apoptogenic effects of ART on HaCaT cells

HaCaT cells were cultured in six-well plates at a density of 5 × 105 cells/well in 2 mL of DMEM culture medium. After 24 h of culture, the unat-tached cells were removed by washing twice with PBS. Subsequently, they were incubated with or without ART (0, 14.5, 29, 58, and 116 μg/mL) for an additional 48 h. At the end of the treatment, both floating and adherent cells were harvested, pooled together, and then washed twice with PBS. Subsequently, cell apoptosis was assayed by Annexin V/PI dual staining and JC-1 (5,5”,6,6”-tetrachloro-1,1”, 3,3”-tetraethylbenzimidazolylcarbocyanineio-dide). The results of these two assays were suf-ficient to establish the occurrence of ART-induced apoptosis.

Annexin V/PI dual staining. Approximately 1 × 106 cells were collected, suspended in 100 μL of binding buffer, and then stained with 5 μL of Annexin V staining solution and 1 μL of PI work-ing solution (100 μg/mL). After incubation at room temperature for 15 min, an additional 400 μL of binding buffer was introduced into the cell suspension and gently mixed. The cells were then analyzed on a flow cytometer (FACSCalibur, BD), measuring the fluorescence emission at 530 nm and 575 nm using 488 nm excitation.

JC-1 staining. Approximately 1 × 106 cells were incubated with 200 μL of diluted JC-1 (2 μM final concentration) for 20 min at 37°C in the dark and then washed with warm PBS. The cells were pelleted by centrifugation, resus-pended in 500 μL of PBS, and then measured with a flow cytometer (FACSCalibur, BD). Green fluorescence (480 nm to 530 nm) was mea-sured in the FL-1 channel, and red fluorescence (580 nm to 630 nm) was measured in the FL-2 channel. The mitochondrial membrane poten-tial (MMP) in HaCaT cells was represented by their red/green fluorescence intensity ratio. Meanwhile, their fluorescence images were observed and obtained under ultraviolet illumi-nation with a BX51 fluorescence microscope (Olympus, Japan).

Preparation of ART cream

All experimental reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. Hyaluronic acid was added to an adequate vol-


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ume of water and heated at 85°C until com-plete dissolution. Ultrez 10 was soaked in the above solution to achieve complete swelling, and the mixture changed into a transparent non-mass gel after adding triethanolamine. Glycerol, propylene glycol, and tween 80 were gradually added and mixed well. The gel was weighed to calculate water loss. Sodium sulfite was dissolved by water with the volume equiva-lent to the water loss and slowly added to the gel. After complete mixing, the blank hydrogel was obtained, and its aqueous phase was pre-pared by heating to 85°C. Monoglyceride, hexa-decanol, geroil GTCC, silicone oil, vitamin E, solid paraffin, albolene, nipagin, and laurocap-ram were heated in 85°C water and melted into the oil phase. The two phases were completely mixed, and then the vehicle cream matrix was prepared. An adequate amount of ART was added to dimethylsulfoxide and heated in an 85°C water bath to dissolve. The mixture was added to the vehicle cream matrix and weighed to replenish the lost water. After stirring, the ART cream was prepared. The prepared vehicle cream and ART cream (1%, 3%, and 5%) were stored separately at 4°C.

All these formulated topical preparations, together with the negative control of normal saline solution and positive control of 0.1% taz-arotene cream (Huapont Pharm, Chongqing, China. Lot 2011005), were later evaluated for their keratinocyte differentiation-modulating activities in mouse tail test.

Animals

Male ICR mice weighing 22 g to 25 g were pur-chased from the Shanghai Lab Animal Research Center. All mice were kept in a specific patho-gen-free animal room and had free access to typical mouse food and water. An artificial light cycle (12 h light and 12 h dark) and controlled temperature (23 ± 2°C) were maintained. The mice were allowed to acclimatize for 1 week prior to the experiment. Animal care and treat-ment were conducted in accordance with the guidelines of the Animal Care and Use Ethical Committee of Zhongshan Hospital, Fudan University.

Experimental protocol of mouse tail test

Sixty mice were divided into six groups com-posed of 10 animals each. The animals received

either normal saline solution (negative control) or 0.1% tazarotene cream (positive control). The remaining four groups were treated with the test samples, namely, vehicle cream, 1% ART cream, 3% ART cream, and 5% ART cream.

In a typical procedure, approximately 0.1 g of topical preparation was thinly coated on the dorsal surface of the mouse tail. To prevent the topical agents from rapidly rubbing off, the mice were individually housed for 2 h and then returned to their animal cages. The treatment was administered twice a day (8:00 and 16:00) for four consecutive weeks. Sixteen hours after the last treatment, the mice were sacrificed by cervical dislocation. Subsequently, the dorsal surface of the mice tails, measuring approxi-mately 2 cm in length starting 1 cm from the anus, were removed from the underlying carti-lage and stained with hematoxylin-eosin.

Histological measurement of mouse tail skin

The histological sections were inspected under a BX60 light microscope (Olympus, Japan) and analyzed as described by Bosman et al [12]:

(A) The horizontal length of the fully developed granular layer within an individual scale lying the turning point of the beginning and ending of two adjacent hair follicles (n = 10 scales per animal, n = 10 animals per group; a total of 100 measurements per group).

(B) The whole horizontal length of an individual scale lying the turning point of the beginning and ending of two adjacent hair follicles (n = 10 scales per animal, n = 10 animals per group; a total of 100 measurements per group).

(C) The vertical epidermal thickness between the dermoepidermal borderline and the begin-ning of the horny layer (n = 5 measurements per scale, n = 10 scales per animal, n = 10 ani-mals per group; a total of 500 measurements per group).

Evaluation of antipsoriatic activity

With the raw data from (A)-(C) mentioned above, the antipsoriatic activities of topical prepara-tions were further evaluated as described by Bosman et al [12]:

(1) The degree of orthokeratosis (OK) per scale was calculated as OK (%) = (A/B) × 100.


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(2) The drug activity was calculated as Drug activity = (OK of the treated group - mean OK of the negative control group)/(100 - mean OK of the control group) × 100.

(3) Out of the raw data of (C), the mean epider-mal thickness of the negative control was set to 100% and served as the baseline data. The relative epidermal thickness of the other treat-ed animals was defined as the percentage ratio of their mean epidermal thickness to that of the baseline data.

Statistical analysis

Data are presented as mean ± standard devia-tion (SD). Statistical significance was deter-mined with ANOVA, and subsequent significant difference was employed using LSD for multiple comparisons. All analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Values of P < 0.05 were considered significant.

Results

Effects of ART on HaCaT cell proliferation

Treatment with ART at different concentrations (10, 20, 40, 80, 160, and 320 μg/mL) for 24, 48, and 72 h decreased the proliferation rate of HaCaT cells compared with that of untreated cells (P < 0.05). WST-8 analysis showed that

the antiproliferative effects of ART on HaCaT cells were dose- and time- dependent (Figure 1).

Quantification of apoptosis by Annexin-V/PI dual staining

ART-induced apoptosis in HaCaT cells was ana-lyzed by Annexin V/PI dual staining. As shown in Figure 2A, early apoptosis in the HaCaT cells treated with 14.5 μg/mL ART was similar to that in the vehicle control. When the concentra-tion of ART was increased to 29, 58, and 116 μg/mL, the percentage of early apoptotic cells continued to substantially increase. Late apop-totic cells also revealed the same trend. These results revealed that ART can increase the apoptosis ratio in a dose-dependent manner as compared with the vehicle control. The typical images of flow cytometric analysis are present-ed in Figure 2B.

Assessment of apoptosis by JC-1 staining

The loss of MMP is a hallmark of apoptosis. JC-1 is primarily used to detect mitochondrial depolarization in the early stages of apoptosis [13]. It accumulates as aggregates in healthy cells and exists as monomers in apoptotic cells. With the mitochondrial membrane becomes more polarized, the fluorescence of the JC-1 dye changes from red to green. Hence, cells that shifted in fluorescence were classified as apoptotic, and their MMP was represented by the red/green fluorescence intensity ratio.

As shown in Figure 3A, the red/green fluores-cence intensity ratio in the untreated HaCaT cells was 1.70, whereas that in the cells treated with 14.5 μg/mL ART was 1.61 (P > 0.05). As the concentration of ART was increased from 29 μg/mL to 116 μg/mL, the fluorescence intensity ratios in the treated cells significantly decreased from 1.24 to 0.31 (P < 0.05). These results not only revealed the ART-induced depo-larization in HaCaT cells but also confirmed its dose-dependent apoptogenic effect.

Similar reductions in red fluorescence were also observed by fluorescence microscopy. The typical images are presented in Figure 3B. ART reduced the number of red JC-1 aggregates and increased the number of green JC-1 mono-mers, suggesting loss of MMP in HaCaT cells. As the concentration of ART was increased, this

Figure 1. Antiproliferative effects of Artemether (ART) on HaCaT cells. HaCaT cells were treated with or without ART for 24, 48, and 72 h. Cell prolifera-tion was analyzed by the WST-8 assay and defined as 100% when cultured in DMEM alone. Meanwhile, data pooled from three independent experiments were expressed as the percentage of the vehicle con-trol. Compared with the vehicle control, ART inhibited the proliferation of HaCaT cells in a dose- and time-dependent manner (P < 0.05).


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Figure 2. ART-induced apoptosis in HaCaT cells analyzed by An-nexin-V/PI dual staining. HaCaT cells were treated with or with-out ART for 48 h. Cell apoptosis was analyzed using Annexin V/PI dual staining and expressed as the percentage of total cell popu-lations. A. ART increased the apoptosis ratio of HaCaT cells in a dose-dependent manner as compared with the vehicle control (P < 0.05). B. Cells in the upper right portion are late apoptotic cells, whereas those in the lower right portion are early apoptotic cells. Results are representative of one of three independent ex-periments.


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Figure 3. ART decreased mitochondrial membrane potential (MMP) in HaCaT cells as measured by JC-1 staining. HaCaT cells were treated with or without ART for 48 h. After staining with JC-1 dye, the MMP in HaCaT cells was analyzed by flow cytometry and observed by fluorescence microscopy. A. As the concentration of ART was increased, decreased ratios of red/green fluorescence intensities were observed, suggesting loss of MMP in HaCaT cells. Data shown are the means ± SD of three independent experiments. * P < 0.05 compared with the vehicle control. B. A similar reduction in red fluorescence was also observed in HaCaT cells by fluorescence microscopy. Cells treated with high concentrations of ART showed a marked re-duction in red fluorescence (seen in healthy cells) and a recip-rocal increase in green fluorescence (sign for MMP collapse) compared with that of the vehicle control. Images are repre-sentative of one of three independent experiments.


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phenomenon became prominent. These results revealed the occurrence of mitochondrial per-turbation in the ART-treated HaCaT cells.

Keratinocyte differentiation-modulating activity in mouse tail test

No visible skin irritations, such as erythema or edema, were found in any mouse during the 4 weeks of tropical treatment. The keratinocyte differentiation-modulating action in mouse tail tests are shown in Figure 4. Among the topical preparations, 0.1% tazarotene cream produced the most evident and striking increase in the degree of OK and the relative epidermal thick-ness. Compared with the negative control, vehi-cle cream increased the degree of OK but did not change the relative epidermal thickness. ART cream dose-dependently increased both the degree of OK and the relative epidermal thickness in the mouse tail skin.

Consistent with the measurement results men-tioned above, histomorphological changes in the mouse tail also revealed the same trend. The typical pathology images of mouse tail skin are shown in Figure 5. Parakeratotic skin with-

out an apparent granular layer was observed in the mice treated with normal saline solution. By contrast, a well-developed granular layer was clearly observed in the tazarotene-treated mouse tail skin. The OK of the epidermal granu-lar layer slightly increased in the vehicle cream group compared with that in the negative con-trol group. When treated with ART cream at dif-ferent concentrations, a mild to moderate dif-ferentiation of the epidermal granular layer in the mouse tail skin were observed. Their degree of OK increased in an ART dose-dependent manner.

Discussion

ART is widely known as an anti-malarial agent [14]. It also has multiple applications because of diverse pharmacological effects [15]. This study is the first to explore the antipsoriatic activity of ART. Results revealed that ART can restore homeostasis to the epidermis and may be a promising agent for psoriasis treatment.

Whereas psoriasis is primarily a T-cell-mediated disease, intrinsic alterations in epidermal kera-tinocytes also play an important role to induce

Figure 4. Keratinocyte differentiation-modulating ac-tion of ART cream in the mouse tail test. Topical treat-ment was administered twice daily for four consecutive weeks. Compared with the vehicle control-treated ani-mals, mice exposed to ART cream displayed a dose-de-pendently increased orthokeratosis (OK) of the granular layer, drug activity, and relative epidermal thickness, in-dicating the keratinocyte differentiation-inducing activ-ity of ART. Data are expressed as mean ± SD (n = 10). *P < 0.05 compared with the vehicle cream.


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psoriasis [16, 17]. Exposure of altered autoanti-gens by keratinocytes may be directly respon-sible for the activation and expansion of certain T-cell subpopulations in psoriatic skin [18]. Furthermore, intrinsic multigenic altered kerati-nocytes can express inflammatory cytokines and growth factors that modify the microenvi-ronment in psoriatic lesions [19]. Therefore, keratinocytes are closely associated with the development of psoriasis by amplifying the inflammatory response and contributing to angiogenesis.

As mentioned above, regulation of the patho-logical changes initiated or perpetuated by keratinocytes may be effective in psoriasis management [20]. Given their capability to attenuate inflammatory responses and bring homeostasis to the epidermis, drugs such as dithranol and vitamin D analogs are currently used in clinical settings to treat psoriasis with satisfactory results [21, 22]. Laboratory stud-ies have demonstrated the immunosuppres-sive effects of ART on T cells both in vitro and in vivo [7]. The present study further revealed that this drug can inhibit keratinocyte proliferation and induce keratinocyte apoptosis. Considering the effects of ART against inflammation and keratinocytes, we speculate that ART may be a potential antipsoriatic agent.

Altered keratinocyte differentiation, including parakeratosis and absence of the granular layer, play important roles in the clinical appear-ance of psoriatic lesions [23]. Such pathologi-cal changes can be partially mimicked by the mouse tail model because parakeratosis occurs in the normal adult mouse tail [24]. This model also has the advantages of easy access to animals and experimental procedure. Similar to most pharmacodynamic studies on antipso-riatic drugs [25, 26], our study also evaluated the inducing effect of ART on epidermal differ-entiation. As expected, 0.1% tazarotene cream significantly enhanced the degree of OK in mouse tail skin. These results not only indicat-ed the rationality of using 0.1% tazarotene cream as a positive control but also further confirmed the reliability of the mouse tail model.

Repeated scratching as a result of itchiness can impair the skin barrier function and increase the transepidermal water loss in pso-riatic lesions [27]. A moisturizer can reduce the transepidermal water loss, attenuate their epi-dermal hyperproliferation, and induce differen-tiation in psoriasis [28, 29]. Therefore, we added moisturizing ingredients, including sili-cone oil, solid paraffin, and albolene, into the vehicle cream matrix. Similar to our findings in

Figure 5. Histological sections of mouse tail skin treated topically for four consecutive weeks (HE, ×200). Note: A. The granular layer is missing in most parts of the tail specimen. B. Tazarotene-induced OK is clearly observed over the whole horizontal length of the scale as a black layer. C. The granular layer is less developed in most parts of the mouse tail skin treated with vehicle cream. D-F. The degrees of OK increased in an ART dose-dependent manner. A well developed granular layer is detected in the mouse treated with 5% ART cream.


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another study [30], the vehicle cream also increased the degree of OK as compared with the negative control. Considering the absence of any anti-psoriatic drug in the vehicle cream matrix, we hypothesized that OK is related to these moisturizing ingredients. The detailed mechanism of such activity will be investigated in another project in the future.

After 4 weeks of treatment with ART cream, the degree of OK and relative epidermal thickness-es were significantly increased compared with that of the vehicle cream. The keratinocyte dif-ferentiation-modulating activity of ART cream increased in a dose-dependent manner (i.e., from 1% to 5% ART cream). In the histological sections, a continuous granular cell layer was clearly observed in the mice treated with 5% ART cream. These results indicated that the ART cream promoted the differentiation of mouse tail epidermis and exerted antipsoriatic activity in a dose-dependent manner.

Psoriasis is an autoimmune disease with mul-tiple disorders, and keratinocyte abnormality is only one of its various pathological changes [31]. Hence, despite the weaker keratinocyte differentiation-modulating activity of ART cream than 0.1% tazarotene cream, ART cream is still a promising antipsoriatic drug because of its immunomodulatory, anti-inflammatory, and anti-angiogenic effects, among others [32, 33]. These effects might be synergistic in pso-riasis treatment, and further studies on these areas are warranted.

Conclusions

This study revealed that ART possessed antip-roliferative action and apoptogenic effects on HaCaT cells and exhibited keratinocyte differ-entiation-modulating activity in the mouse tail test. Therefore, ART may be a promising agent for psoriasis treatment and is worthy of further development. To better understand the poten-tial antipsoriatic effects of ART, future studies must develop new experimental methods and animal models.

Acknowledgements

We gratefully acknowledge Dr. Xin Hong for the experiment design, as well as Dr. Weng Weiyu and Jiang Dongbo for formulating the topical preparations. We also thank Xu Yuansheng,

Sheng Dexin, Sun Shuhui, Yang Lili, and Wei Yi for their technical assistance.

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Ming Li, Department of Dermatology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China. Tel: +86 21 64041990-2587; Fax: +86 21 64037247; E-mail: [email protected]

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