| Backgroud Psoriasis is a common chronic inflammatory disease in the skin with typical clinical features of silvery-white scales on the thick and red plaques. Pathologically, psoriasis is characterized by the epidermal hyperplasia, parakeratosis, acanthosis, formation of new blood vessels (angiogenesis), and accumulation of inflammatory cells such as dendritic cells, neutrophils, T cells, and macrophages in the epidermis and dermis. However, mechanisms in the pathogenesis of psoriasis are largely unclear and treatment remains ineffective.It has been shown that T cells are a key player in the pathogenesis of psoriasis. Recent studies also suggested that the Th17-dependent inflammatory pathway plays a crucial role in psoriasis.It is now clear that TGF-β1, together with the proinflammatory cytokine IL-6, regulates the differentiation and functions of Th17responses. In patients with psoriasis, increased levels of TGF-β1, IL-6, IL-23, and IL-17A in the skin lesion and/or in the plasma are closely correlated with the disease activities, suggesting a close link between TGF-(31/IL-6and Th17immunity in the pathogenesis of psoriasis.TGF-β1is generally regarded as a multipotent cytokine involved in many biological processes. TGF-β1is widely expressed by many cell types and plays a critical role in the T cell development including Th17cells. This is demonstrated by the finding that T cells lacking TGF-β1fail to differentiate into the Th17lineage. In contrast, mice overexpressing of human TGF-β1in epidermal keratinocytes driven by the keratin-5promoter develop a skin phenotype with strong similarities to human psoriasis, suggesting a pathogenic role for TGF-β1in the pathogenesis of psoriasis. However, the signaling mechanism of TGF-β1in the pathogenesis of psoriasis remains unclear. It is well documented that after binding of TGF-β1to its receptors, it activates the downstream mediators, Smad2and Smad3, to regulate the biological activities. Among them, Smad3is generally considered to be a key regulator in immune and fibrosis responses, while Smad2has an opposite role in term of tissue fibrosis.Based on the known role of Smad3in diseases associated with inflammation and fibrosis, we therefore hypothesized that targeting Smad3may offer a new therapeutic strategy for psoriasis. This hypothesis was tested in a mouse model of psoriasis in K5.TGF-β1WT transgenic (Tg) mice by administering a Smad3inhibitor that has been shown to specifically inhibit Smad3phosphorylation in vitro.ObjectiveThe aim of this study was to observe the role and molecular mechanisms of Smad3inhibitor on psoriasis in mice through analyzing the protein level and mRNA level of cytokines involved in TGF-β/Smad-mediated skin fibrosis and Th17-mediated skin inflammation. This study intended to illustrate the therapeutic potential of Smad3inhibitor and suggested the pathogenic role of Smad3in psoriasis. Therefore, blockade of TGF-β/Smad3signaling may represent a novel treatment strategy for psoriasis. Chinese medicine with Smad3inhibitor-like effect will develop in psoriasis and this will promote the combination of Chinese medicine and modern science in the therapy of psoriasis.Methods 1.K5-TGFβ1transgenic mice and normal mice began to be used for this study at the age of12weeks. TGFβ1wild-type (TGFβ1WT) mice were the normal control mice without psoriatic lesion. Littermates TGFβ1transgenic mice were randomly grouping to disease control group, the control-treated group and the Smad3inhibitor treatment group, while the wild-type littermates TGFβ1control mice were the normal mice group. Each group developed6mice.2.Smad3inhibitor was dissolved with DMSO and became the ointment to treat the mice. Smad3inhibitor ointment (5ug/ml) was applied on the psoriatic lesion twice daily for2months, while the control-treated group treated with DMSO ointment without Smad3inhibitor.3. A standard mouse psoriasis skin-scoring system was used to score the severity of skin disease and the double skin-fold thickness (DSFT) of the dorsal skin was measured by a micrometer before and once for every2weeks after treatment over the8-week period.4. By the end of experiment, mice were killed and blood and the dorsal skin tissues were collected for analysis.5. Changes in histology were examined in paraffin-sections by hematoxylin and eosin (H&E).6. Collagen-like matrix deposition was stained with Masson’s trichrome staining.7. The protein level of cytokines involved in TGF-β/Smad signaling and inflammation was performed with immunohistochemistry and western blot.8. Two-color immunofluorescence was used to detect Th17cells.9. mRNA levels of TGF-1, IL-6, IL-23p19, IL-17A, and β-actin were analyzed by real-time PCR.10. Plasma levels of IL-17, IL-6and IL-23and TGF-1were measured by specific ELISA kits.11. Data obtained from this study are expressed as mean±SE. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Newman-Keuls Post Test using Prism5.0.ResultsWe first determined an effective and safe dose of a Smad3inhibitor (SIS3) for treatment of psoriasis in K5.TGF-β1Tg mice. Groups of3K5.TGF-β1Tg and littermate wild-type (WT) mice were topically treated with the ointment containing three dosages of SIS3at2.5,5,10μg/ml (weight/volume) or the DMSO control twice daily for3months. Results revealed that treatment with SIS3at a dose of5μg/ml produced a better inhibitory effect on the progression of psoriatic lesions, including a significant reduction in the disease severity score and DSFT score, and less extent of CD3+T cells, F4/80+macrophages, and collagen-like matrix deposition in the dermis. These inhibitory effects on the skin plaque lesions were clearly evident as early as1month after the SIS3treatment and maintained over the entire period of3months. We then tested the potential toxicity of SIS3in groups of3WT mice following the same protocol as described in Tg mice. Results from WT mice clearly demonstrated that there were no detectable toxicities in all three dosages of SIS3ointments, although a trend increase in serum levels of lactate dehydrogenase (LDH) was seen in those treated with the highest dose of SIS3(10μg/ml).Based on the results obtained from these pilot studies, SIS3at5μg/ml was used as an optimal treatment dose and groups of6Tg mice were treated with the SIS3for2months (the mean effective time). Compared to the age-control WT mice where no skin lesions were observed, Tg mice without SI S3treatment or treated with control DMSO ointment developed psoriasis-like plaque lesions, including a typical clinical feature of silvery-white scales on the thick and red plaques, a higher level of the mean disease severity score, epidermal hyperproliferation and parakeratosis, and a marked thickening of the epidermis as determined by a significant increase in the epidermal thickness score and DSFT score. After treatment with Smad3inhibitor ointment, the mean disease severity score(F=4.413, P<0.05), epidermal thickness (F=24.57, P<0.01) and the DSFT (F=8.423, P <0.05) were attnuated significantly. Immunohistochemistry also detected a massive infiltration of CD3+T lymphocytes and F4/80+macrophages to the epidermis and dermis, which were suppressed by Smad3inhibitor(F=19.18, P<0.05; F=22.35, P<0.01). In addition, examinations by Masson’s trichrome and immunostaining with the anti-α-SMA antibody also revealed a large amount of collagen-like matrix deposition in the dermis, which was associated with a massive α-SMA+myofibroblast accumulation. In contrast, Tg mice treated with a Smad3inhibitor exhibited a significant inhibitory effect on collagen deposition (F=16.01, P<0.01) and α-SMA accumulation (F=32.23, P<0.01) compared with untreated or DMSO control treated Tg mice.We next examined the potential mechanisms by which Smad3inhibitor suppressed psoriasis in K5.TGF-(β1Tg mice. We first determined whether the development of psoriasis-like lesions in TGF-β1Tg is associated with the activation of TGF-β/Smad3and the inhibitory effect of SIS3on psoriasis is attributed to the blockade of TGF-β/Smad3signaling. As shown from the results of immunohistochemistry, real-time PCR, and ELISA detected that TGF-pi at both mRNA and protein levels was highly upregulated locally in psoriatic lesions and systemically in plasma in Tg mice received DMSO ointment or non-treatment. Immunohistochemistry and Western blot analysis also revealed that increased TGF-β1expression locally in the skin plaque lesions was associated with a marked activation of Smad3as determined by high levels of phospho-Smad3and its nuclear translocation. In contrast, Tg mice treated with SIS3exhibited a marked inhibition of TGF-β1mRNA (F=8.891, P<0.05) and protein expression by both immunohistochemistry (F=36.74, P<0.01) and Western blot (F=34.07, P<0.05). In addition, the activation of Smad3was also suppressed by both Immunohistochemistry (F=21.88, P<0.05) and Western blot analysis (F=15.05, P<0.05) after the treatment of Smad3inhibitor, compared with untreated or DMSO control treated Tg mice.Increasing evidence shows that the IL-23/Th17axis plays crucial roles in psoriasis. It is also well accepted that differentiation of Th17cells depends on the stimulation of TGF-β1and IL-6, we, therefore, studied whether the inhibitory effect of Smad3inhibitor on psoriasis is associated with blockade of the Th17inflammation pathway beside the inhibition of TGF-β/Smad3signaling. Real-time PCR showed that compared to the WT mice, mRNA levels of IL-6, IL-23and IL-17A were highly upregulated in the psoriatic lesions in Tg mice received DMSO control or non-treatment. These changes in the psoriatic lesions were also associated with an increase in plasma levels of IL-6, IL-23, and IL-17A as determined by ELISA. In contrast, Tg mice topically treated with the SIS3ointment showed a substantial inhibition of IL-6, IL-23and IL-17A both locally in mRNA level as IL-6(F=6.397, P<0.05), IL-23(F=4.921, P<0.05),IL-17A (F=7.583, P<0.05) and systemically in plasma as IL-6(F=5.119, P<0.05), IL-23(F=5.402, P<0.05), IL-17A (F=7.046, P<0.05). Further studies by Western blot analysis also showed a marked upregulation of a Thl7transcriptional factor RORyt in the psoriatic lesions from untreated or DMSO-treated Tg mice, which was blocked in Tg mice treated with Smad3inhibitor (F=5.154, P<0.05). Further study by two-color immunofluorescence also clearly showed that there were many CD4+IL-17A+Th17 cells in the skin lesions from the untreated or DMSO control-treated Tg mice, which was significantly inhibited in Tg mice treated with the Smad3inhibitor (F=5.644, P <0.05).DiscussionThe present study provided the first evidence for Smad3inhibitor as a novel therapeutic agent for psoriasis. Smad3inhibitor could effectively attenuate the collagen deposition and the overexpression of TGFβ1in psoriatic skin lesions, inhibit the up-regulation of Smad3phosphorylation, and effectively regulate the IL-6, IL-17A, IL-23content, reduce Th17cells differentiation, suppress inflammation. Thus, Smad3inhibitor might play a therapeutic role in psoriasis. Results from this study suggested that targeting the TGF-β/Smad3pathway might represent an effective therapy for psoriasis. Furthermore, we also identified that inhibition of the TGF-β/Smad3signaling pathway and the IL-23/Th17inflammatory pathway were mechanisms by which Smad3inhibitor suppressed psoriatic skin disease in K5.TGF-β1Tg mice.Research sourceThis work was cooperated by Southern medical university and the Chiniese University of Hong Kong. It was done in Li Ka Shing Institute of Health Sciences and Department of Medicine and Therapeutics, the Chinese University of Hong Kong, and supported by grants from Research Grant Council of Hong Kong (RGC GRF469110, and CUHK3/CRF/12R). |
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