Psoriasis is a widespread chronic skin disorder with a 2–3% prevalence rate worldwide. The condition occurs as a result of an inflammatory reaction mediated by the immune system. It usually starts by chemokines and cytokines drawing cells of the immune system to the psoriatic area of the skin. This is quickly followed by invading and local cell proliferation [1
]. However, psoriasis can also be caused by systemic low-grade inflammation, which may be associated with co-morbidities such as metabolic syndrome and cardiovascular disease [4
]. Although studies are yet to reveal the precise cause of the condition, genotypic dependency, and environmental stimuli such as infection, stress, drugs and trauma have been implicated as possible causes of the diseases [6
]. Psoriasis affects the skin’s appearance, therefore compromising the quality of patients’ life considerably [7
]. Although several short-term and long-term treatment options for the disease exist, a complete cure is yet to be achieved. The disease was considered initially as an autoimmune disease mediated by Th1 [8
]. However, accumulating evidence suggests that the cytokines interleukin (IL)-17, Foxp3 induced by Th17 or neutrophils and Treg cells, respectively, are liable for the increased prevalence of psoriatic dermatitis skin and could play an essential role in the disease development [9
Metabonomics is a current high-throughput technique [12
], which offers quantitative measures of biological systems involving global alterations in individuals’ metabolic profiles in response to genetic modification or pathophysiological stimuli [13
]. Metabolic product profiling i.e., metabotyping or metabolic phenotyping has offered newfangled insights into the metabolic syndrome [14
]. The metabonomics approach has gained application in diagnosis of diseases, evaluation of the efficacy, and toxicity of drugs and investigations on pathogenesis of diseases [15
]. Using metabolomics, Ueharaguchi Y et al. elucidated the possible pro-psoriatic dermatitis mechanism of Thromboxane A2 [17
]. Arnald Alonso et al. studied the mechanism of immune-mediated inflammatory diseases [18
]. To date, however, metabolomics analysis in mouse psoriasis has been rarely performed.
The use of β2
adrenergic receptor agonists is a popular treatment option for obstructive diseases of the lungs and owns a number of anti-inflammatory effects [19
]. Topical use of (R
)-salbutamol cream (0.5%) exhibits effectiveness in treatment of sub-acute lupus erythematosus (SCLE) and discoid lupus erythematosus (DLE) [21
]. However, the effects of (R
)-salbutamol in treatment psoriasis has not been reported, and its effects on the metabolomics and biochemistry analysis in psoriasis has yet to be done.
Imiquimod (IMQ), which is a toll-like receptor 7/8 agonist, is a robust immune activator [22
]. The topical application of IMQ in mice causes psoriasis-like skin, which is similar to human psoriasis dermatitis phenotypically and histologically [23
]. The present study aimed to evaluate (R
)-salbutamol effects on IMQ-induced skin dermatitis (psoriasis-like) in BALB/c mice. The effect of (R
)-salbutamol on the regulation of Th17/Tregs cells and one profile of metabolomics were also investigated. This study provides a new use of (R
)-salbutamol in treatment of psoriasis and the underlying mechanism.
2. Materials and Methods
Animal experiments were conducted using 8–11 weeks-old BALB/c female mice weighing 20–25 g (Certification No. 44007200057513, SPF grade) purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China). This study was approved the Institutional Animal Care and Research Advisory Committee of the Institute of Biomedical and Pharmaceutical Sciences at Sun Yat-Sen University (permit number IACUC-2013-1204; approved 1 December 2013). Imiquimod cream (19040540) was obtained from Med-Shine Pharmaceutical Co., Ltd. (Sichuan, China) and (R)-salbutamol (>99% purity, 99.85% ee) was from Dongguan Key-Pharma Biomedical Co., Ltd. (Dongguan, China). ELISA kits of IL-17 (M181112-008a) was from Neobioscience, (Shenzhen, China). All of antibodies were acquired from Biolegend (San Diego, CA, USA).
2.2. Mice Treatments
The mice were divided into six groups (8 mice/group), i.e., imiquimod-treated group (IMQ), IMQ plus high dose of (R)-salbutamol group (H, 2 mg/kg), IMQ plus medium dose of (R)-salbutamol group (M, 1 mg/kg), IMQ plus low dose of (R)-salbutamol group (L, 0.5 mg/kg), IMQ plus dexamethasone group (Dex, 1 mg/kg) and control group (C). The mice were given water and normal forage for three days. An area of about 5 cm × 4 cm on the back skin of each mouse was shaved. In IMQ treatment, the mice received a daily topical application of 62.5 mg of 5% imiquimod cream for seven days. The control group received Vaseline. (R)-salbutamol and Dex were administered to mice orally (p.o.) twice a day for seven days. Mice in the IMQ group were administered with distilled water instead of the drugs. On the 8th day, mice were sacrificed to collect plasma, spleen and skins samples.
2.3. Evaluating the Severity of Skin Inflammation
The degree of inflammation on the back skin was determined using the clinical Psoriasis Area and Severity Index (PASI) (Supplementary Table S1
). Of note, we did not include the affected skin area in the calculation of overall scores. Scores ranging from 0 to 4 were used to rank the scaling, thickening, and erythema separately. In detail, scores 4, 3, 2, 1, and 0 were interpreted as very striking, moderate, slight, and none, respectively. The assessment of the morphological changes of dermatitis in mice was based on the PASI scoring system, with scores ranging from 0 to 12. Finally, skin dermatitis was evaluated using average scores for each group.
2.4. Histopathological Examination
After harvesting, the back skin specimen was fixed in 4% paraformaldehyde for 24 h prior to embedding in paraffin. Thereafter, they were cut into appropriate sections that were subjected to hematoxylin and eosin (HE) staining. The histopathological features of the sections were examined under a microscope (Nikon, Tokyo, Japan). The Baker scoring criteria were used to analyze the histopathological changes (Supplementary Table S2
2.5. Hematological Analysis
The IDEXX ProCyte DX hematology analyzer (IDEXX, Westbrook, ME, USA) was employed for hematological analysis. This test was performed using whole blood samples preserved in EDTA tubes. The measurement of the white blood cells (WBC), neutrophil (NEUT) and mononuclear cells (MONO) were made in the hematology analyzer.
2.6. IL-17 ELISA
An enzyme-linked immunosorbent assay (ELISA) kit (Neobioscience, Shenzhen, China) was employed to measure the serum levels of Plasma IL-17 based on a standard curve created from serial dilutions of anti-IL-17 antibody.
2.7. Intracellular Staining and Flow Cytometry
Mice spleens were excised and bathed in PBS solution in a culture dish. The specimens were homogenized and filtered via a 70 µm nylon mesh. The tissue homogenate was mechanically dissociated to release single cells which were lysed in erythrocyte lysis buffer. Finally, they were suspended in PBS (without magnesium and calcium ions).
To perform intracellular staining, Splenocytes were harvested and incubated with magnetic-activated cell sorting buffer (MACS, 0.5% BSA and 2 mM EDTA in PBS) blocking with Fc receptors. This was followed by staining with the following antibodies: Alexa Fluor 488–anti-CD3 (B284975), BV605–anti-CD4 (B279163), BV510–anti-IL-17 (B263584) and PE-anti-Foxp3 (B275698). The cell activation cocktail (with Brefeldin A) containing phorbol myristate acetate (PMA, protein kinase C activator, 40.5 µM), ionomycin (Ca2+ ionophore, 669.3 µM) and brefeldinA (Golgi inhibitor, 2.5 mg/mL) (B265097) was used to stimulate the splenocytes for 6 h. After incubation, we blocked the harvested splenocytes by treating them with MACS, followed by surface-staining, fixing, and permeabilization with the Fixation/Permeabilization Kit (B280105).
2.8. Metabolomic Analysis
Metabolite extraction of plasma: A three solvent biphasic system: water: methanol: methyl-T-butyl-ether at the volumetric ratio of 1:3:6 (MTBE solution) was used to extract metabolites in this study [24
]. Briefly, 160 µL of MTBE solution was mixed with 40 µL of plasma and vortexed for 30 min at 4 °C. The mixture was centrifugated (3000× g
, 30 min, 4 °C) which formed two fractions, (1) a hydrophilic fraction comprising water and methanol (2) and an organic hydrophobic fraction comprising methanol and MTBE. These fractions were vacuum-dried and then suspended in 45 µL 0.1% formic acid in water in preparation for analysis. The quality control (QC) test was performed using 600 µL from each sample to accurately represent the breadth of metabolites [26
]. The blank was injected during the initial run to condition the column. The QC sample was injected as six replicates to increase the precision of injection. To test the stability of the system, a QC sample from every five experimental samples was analyzed.
UHPLC/ESI-TIMS TOF-MS/MS data acquisition and analysis: The UHPLC/ESI-TIMS TOF-MS/MS system in conjunction with the Trapped Ion Mobility spectrometry and TOF mass spectrometry (Bruker Daltonics Inc., Billerica, MA, USA) in positive and negative ion mode and the Dionex UltiMate 3000 RSLC system (Thermo Scientific/Dionex, Amsterdam, The Netherlands) equipped with an Acquity UPLC BEH-C18 column (2.1 mm × 50 mm, 1.7 µm) were used to analyze the samples. We acquired MS/MS data by combining the AutoMS/MS scanning experiment with data-dependent acquisition (DDA) model, which allowed the precursor ion to be selected as the most intense peak during LC–MS analyses. The samples used in this experiment were stored at 4 °C and analyzed using 5 µL.
Sample injection description: for each sample, we prepared two fractions from biphasic extractions [28
], an aqueous layer and an organic layer. We first injected 5 µL of the organic layer, followed by an injection of 5 µL of the aqueous phase to the same column to prepare the aforementioned gradient. This step lasted for 1 min and the gradient was not initiated. Meanwhile, the concentration of acetonitrile (B solvent) in the mobile phase was not increased, this was done to ensure that the hydrophobic lipids were ahead in the column. Immediately following (via the next line in the sequence table), we injected 5 µL of the aqueous phase to the same column, after which a full gradient was initiated.
2.9. Statistical Analysis
The data of UHPLC/ESI-TIMS TOF-MSMS were processed for peak alignment, picking, and normalization using Progenesis QI software (Waters, Manchester, United Kingdom) to generate peak intensity for m
data pairs and retention time (TR). The resultant data matrix was analyzed using EZinfo 3.0 software for PCA, PLS-DA, and OPLS-DA. We screened potential biomarkers that discriminate the IMQ/(R)-salbutamol and control group by metabolomics analysis. The max fold change >2 values p
< 0.05 and variable importance in projection (VIP) values >1.0 were used as the cutoff values. The metabolite peaks were identified by MSMS analysis or by screening the biochemical databases, such as KEGG, Lipidmaps and HMDB. The OmicShare tools (www.omicshare.com/tools
) were used to establish the Venn diagrams. The MetaboAnalyst 4.0 software (McGill University, Montreal, QC, Canada) and KEGG pathway database were employed for pathway analysis.
Other statistical analyses were performed as means ± standard deviation (SD). Data statistics were performed and presented by GraphPad prism-5 software (GraphPad Software Inc., La Jolla, CA, USA). The one-way ANOVA and t Student’s-test were used to compare differences between the IMQ and (R)-salbutamol plus imiquimod-induced groups. p < 0.05 was considered statistically significant.
The current study evaluated the effects of commonly used 1 mg/kg (R
)-salbutamol in treatment of psoriasis and in regulating Th17/Tregs cells response and metabolism in IMQ-induced psoriasis in mice. β2
receptor agonists are bronchodilators used in the treatment of obstructive pulmonary diseases [19
]. It is reported that the (R
)-salbutamol of β2
receptor agonists can also reduce inflammation of the respiratory tract, suppress proinflammatory mediators, thus preventing exudate production and tissue edema [30
]. Racemic salbutamol relieves carrageenan-induced paw edema in rats via a β2
receptor-dependent mechanism [33
]. An explorative clinic investigation indicated that (R
)-salbutamol can treat discoid and subacute lupus erythematosus [21
]. This study established a significant alleviation effects of (R
)-salbutamol on IMQ-induced psoriasis-like skin dermatitis in mice. The anti-psoriatic effects of (R
)-salbutamol involves in regulating the Th17/Tregs blance and glycerophospholipid metabolism.
Previous research has suggested that psoriasis could be caused through complex interactions between the immune system, autoantigens, several environmental factors, and susceptible loci associated with psoriasis [34
]. InIMQ-induced mouse psoriasis. IMQ activates WBCs, neutrophils and monocyte cells through TLRs, initiating cytokine signaling cascade, and induce IL-17 expression and other cytokines [37
]. Cellular infiltration represents an essential feature of skin inflammation, while WBCs, neutrophils, and monocytes are the main cell types that infiltrate the area [38
]. These cells playan essential roles in the development of skin dermatitis. Herein, the application of imiquimod cream resulted in the formation of clear psoriasis-like pathological alterations. These observations agreed with a study conducted previously [39
]. Our results showed that (R
)-salbutamol reduced erythema, skin thickness, scaling, and confer protective effects against psoriasis dermatitis induced by IMQ. In addition, histopathological results confirmed that (R
)-salbutamol prevented the proliferation and abnormal differentiation of IMQ-induced keratinocytes. Hematological evaluation revealed significant reduction in IMQ-stimulated increases in the number of WBCs, neutrophils and monocytes, which lead to a significant suppression in inflammatory responses Moreover, (R
)-salbutamol reduced the number of neutrophils and the levels of IL-17 in mice plasma, the latter produced from either Th17 or neutrophils T cells [11
Psoriasis is an autoimmune disease that is mediated by T cell dysfunction [40
]. Th17 cells, Treg cells, and associated cytokines were thought to be vital for psoriasis development [41
]. The Th17/Treg axis is crucial for the development of chronic disease and has been the focus of drug development [42
]. The Treg-foxp3-driven disorder is regarded as a potentially more critical pathway in the initial stage of psoriasis [43
]. In this study, IMQ application results in significantly increased levels of CD4+ Th17+ T cells (Th17) and decreased levels of CD3+CD4+ T cells and CD25+ Foxp3+Tregs in comparisons with control groups. These results are consistent with the findings of an earlier study [45
]. In the IMQ induced psoriasis-like mice, (R
)-Salbutamol administration reduced the expression of IL-17 in plasma along with a downregulation of the CD4+ Th17+ T cells (Th17) and upregulation of the CD3+CD4+ T cells and CD25+ Foxp3+ Tregs in the spleens. Collectively, these findings indicated that (R
)-salbutamol attenuates psoriasis-like skin dermatitis induced by IMQ by regulating Th17 and Treg differentiation and inhibiting cytokines secretion. These results are not in agreement with reports in which treatment of racemic salbutamol regulates the balance between Th1 and Th2 cytokines instead of Th17 and Treg [46
]. Additionally, Dex markedly reduces the levels of IL-17 in mice plasma (Figure 4
D) and neutrophils (Figure 4
C) in mice blood, suggesting dexamethasone alleviates psoriasis by reducing the production of IL-17 from neutrophils rather than from Th17 cells [46
]. However, the inhibition effects of Dexamethasone on total WBC, neutrophil, and monocyte in blood were much more less significant in comparing (R
)-salbutamol in IMQ induced Psoriasis mice.
The spleen is a major organ of the immune system that reflects the immune system of the human body and producing a variety of immune-active cytokines, hence it plays an important role in immune activities [47
]. In this study, splenomegaly was induced by this systemic effects of topical IMQ application and with an increase of to body weight ratio. This is due to an increase in cellular proliferation in the spleen, activated by inflammatory immune responses and it was verified in the current study. This psoriasis-induced splenomegaly was greatly ameliorated by (R
)-salbutamol, indicating systemic anti-psoriatic effects of (R
)-salbutamol by regulating inflammatory immune cells production of spleen in inflammatory immunoreactions.
Previous studies showed that altered small molecules metabolism play key roles in the immune dysfunction in autoimmune diseases, including psoriasis, systemic lupus erythematosus, and rheumatoid arthritis [48
]. In the untargeted metabolomics, employing multivariate statistical analysis, alterations in metabolites that are involved in the IMQ-induced psoriasis were identified. PCA and PLS-DA showed significant differences in the metabolic profiles among the (R
)-salbutamol-treated (L, M, H), IMQ-induced, and control groups. The metabolic profiles of IMQ treated was significantly different in comparing to the control group, while the (R
)-salbutamol treatment were very similar to that of the control group. These observations provided further evidences for the therapeutic effects of (R
)-salbutamol against IMQ-induced psoriasis in mice. The untargeted metabolomics data of the present study identified 40 new potential biomarkers related to metabolic dynamics in IMQ-induced e psoriasis before and after treated with (R
)-salbutamol. Glycerophospholipids, sphingolipid and arachidonic acid were the primary metabolites that affect IMQ induced psoriasis. These may be the major metabolic pathway that regulated the pathological process and the underlying molecular mechanism of this psoriasis. Glycerophospholipids are vital for maintain normal physiological cellular functions and as necessary as proteins and genes [50
]. Its downregulation may contribute to cell membrane damage. Phosphatidylcholines (PC) and phosphatidylserine (PS) are the main glycerophospholipids found in the phospholipid membranes [50
]. Recent studies revealed that the higher proliferation rates of skin cells could also explain the lower concentrations of PC in psoriasis plasma, which is an essential component of cell membranes [49
]. Furthermore, reduced levels of PS were found in active psoriasis [51
]. In our study, PC and PS had lower concentrations in the IMQ-induced groups, but which were up-regulated by (R
)-salbutamoltreatment, indicating that (R
)-salbutamol conferred anti-psoriatic effects by modulating the metabolism of glycerophospholipid. Several studies [51
] have reported the importance of sphingolipids in innate immunity regulation, especially in T cells differentiation and programming. Our results discovered that (R
)-salbutamol against IMQ induced psoriasis by regulates the Th17/Treg axis. The metabolomics results suggest that the regulation of Th17/Tregs by (R
)-salbutamol may involve in sphingolipids. The biomarkers indentified in this study either in the IMQ-induced mouse psoriasis or after, (R
)-salbutamol treatment could be useful in identify pathways and mechanism mediating the pathogenesis of psoriasis.
In summary, this study demonstrates a significant anti-psoriasis effects by (R
)-salbutamol. This may involve regulateing the Th17/Tregs axis balances and glycerophospholipid metabolism in response to IMQ induced psoriasis (Figure 9
)-salbutamol also revealed a better therapeutic effect than corticosteroid in against IMQ-induced psoriasis in this study. (R
)-salbutamol has been used years in clinic for anti-asthma and COPD with well accepted safety-profile. Therefore, the findings of the this study provide a better alternative for the unmet medical need in treatment of psoriasis.