We explored the potential benefits of stigmasterol in the treatment of asthma, an airway disorder characterized by immune pathophysiology and with an ever-increasing worldwide prevalence. We assessed the modulatory effect of the intraperitoneal administration of stigmasterol on experimentally induced airway inflammation in guinea pigs. The effect of stigmasterol on inflammatory cell proliferation, oxidative stress, lung histopathology, and remodeling was investigated. The results showed significant suppressive effects on ovalbumin-induced airway inflammatory damage. Stigmasterol at 10–100 mg/kg reduced proliferation of eosinophils, lymphocytes, and monocytes while reducing peribronchiolar, perivascular, and alveolar infiltration of inflammatory cells. Histopathology revealed stigmasterol maintained lung architecture and reversed collagen deposition, an index of lung remodeling. Overexpression of serum vascular cell adhesion molecule-1 (VCAM-1) and ovalbumin-specific immunoglobulin E (OVA sIgE) elicited by ovalbumin sensitization and challenge was significantly controlled with stigmasterol. Taken together, stigmasterol possessed significant antiasthmatic properties and had suppressive effects on key features of allergen-induced asthma.
Stigmasterol, a naturally occurring steroid alcohol, belongs to a larger class of plant compounds called phytosterols [
Asthma is a chronic pulmonary disorder associated with airway hyperresponsiveness (AHR), inflammation, and airway obstruction. The pathophysiology of asthma is characterized by severe inflammatory cell activation and accumulation, airway muscle hypertrophy, submucosal fibrosis, and excessive mucus production resulting in permanent airway remodeling [
The search for novel medications for asthma spans across synthetic molecules, molecular interventions, and alternatives from natural sources. Particular interest in the latter has taken center stage, with some interesting findings already reported from both experimental and clinical investigations [
In this study, we investigate the potential benefits of stigmasterol in the treatment of asthma. We assess its possible anti-inflammatory or immunomodulatory effects in ovalbumin-induced asthma in guinea pig model of inflammation.
Stigmasterol (98%), ovalbumin (OVA), and dexamethasone were obtained from Sigma Aldrich (St. Louis, USA). Guinea pig VCAM-1 and OVA sIgE ELISA quantification kits were purchased from MLBio Biotechnology Company Limited (Shanghai, China).
Guinea pigs (300–350 g) of either sex were obtained from Noguchi Memorial Institute for Medical Research, Legon, Ghana. Animals were kept under standard temperature and humidity conditions (temperature 23 ± 2°C with a 12 h light-dark cycle) at the Animal House facility of the Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST, and allowed access to commercial chow and distilled water ad libitum. All protocols used in this study were approved by the Faculty of Pharmacy Ethics Committee, and animal handling was done in compliance with the National Institute of Health Guidelines for Care and Use of Animals.
Five groups (
SOD level was expressed in units per mg protein, where 1 unit of enzyme activity is the quantity of enzyme required to prevent the auto-oxidation of adrenaline at 25°C, and calculated with the equation
24 h after the last OVA exposure, the lung tissues were carefully removed and fixed in 10% formalin. Tissues were serially dehydrated in increasing concentrations of ethanol, cleared in xylene in a TP 1020 Tissue processor (Leica Biosystems, Wetzlar, Germany), and embedded in paraffin using a Leica EG 1160 Embedding machine (Leica Biosystems, Wetzlar, Germany). Transverse sections of 3
All results are presented as mean ± SEM. Data analysis was done using one-way analysis of variance (ANOVA). Multiple comparisons between the treatment groups were performed using Dunnet’s post hoc test. All statistical analyses were done using GraphPad for Windows version 6 (GraphPad Prism Software, San Diego, USA).
Ovalbumin challenge of previously OVA-sensitized guinea pigs was characterized by significant increase of blood eosinophils, lymphocytes, and monocytes relative to the naïve control animals (Figures
Effect of stigmasterol on inflammatory cell count in blood. Guinea pigs were sensitized and challenged with ovalbumin as described in the methods. Animals received either saline, dexamethasone, or stigmasterol 1 h prior to each challenge. Naïve controls received normal saline only. Animals were sacrificed 24 h after the last challenge, and blood was collected for counts of eosinophil (a), lymphocyte (b), and monocyte (c). Data is expressed as cell count (103/
Serum analysis showed a significantly increased mean expression of soluble vascular cell adhesion molecule-1 (VCAM-1) in the saline-treated OVA-sensitized and challenged group to 205.20 ± 25.82 × 10−12 g/ml from a mean value of 10.58 ± 3.18 × 10−12 g/ml for the naïve control group (Figure
Effect of stigmasterol on serum vascular cell adhesion molecule-1 (VCAM-1) and serum OVA-specific immunoglobulin E (OVA sIgE). Guinea pigs were sensitized and challenged with ovalbumin as described in the methods. Animals received either saline, dexamethasone, or stigmasterol 1 h prior to each challenge. Naïve controls received normal saline only. 24 h after the last challenge, animals were bled by dissection of the jugular vein. Blood collected was allowed to clot and centrifuged at 1000 rpm for 15 min. Serum vascular cell adhesion molecule-1, VCAM-1 (a), and serum OVA-specific immunoglobulin E, OVA sIgE (b), levels were quantified with sandwich ELISA. Data is expressed as VCAM-1 or OVA sIgE concentration (pg/ml) ± SEM (
The mean OVA sIgE was significantly increased to 81.75 ± 7.5 × 10−9 g/ml in the saline-treated OVA-sensitized and challenged group compared to the naïve control animals with values below detectable levels (Figure
Analysis of supernatant showed an antioxidant profile consistent with severe inflammation. Levels of malondialdehyde (MDA), a direct product of lipid peroxidation, were significantly elevated to 56.39 ± 5.15 nmol/mg protein in the saline-treated OVA-sensitized and challenged animals relative to 9.30 ± 1.01 nmol/mg protein in the naïve animals (Figure
Effect of stigmasterol on BALF oxidative stress markers. Guinea pigs were sensitized and challenged with ovalbumin as described in the methods. Animals received either saline, dexamethasone, or stigmasterol 1 h prior to each challenge. Naïve controls received normal saline only. Bronchoalveolar fluid was collected by aspiration 24 h after the last ovalbumin challenge and centrifuged for 1000 rpm for 10 min. The supernatant was analyzed quantitatively for level of malondialdehyde (a), reduced glutathione (b), superoxide dismutase (c), and catalase (d). Data is expressed as mean concentration/mg protein ± SEM (
Lung architecture in naïve animals was consistent with normal guinea pig lung structure. Alveolar spaces were clear with little or no accumulation of cells around the bronchioles (Figure
Effect of stigmasterol on inflammatory cell infiltration. Guinea pigs were sensitized and challenged with ovalbumin as described in the methods. Animals received either saline, dexamethasone, or stigmasterol 1 h prior to each challenge. Naïve controls received normal saline only. Animals were sacrificed 24 h after the last ovalbumin challenge. The lungs were excised, fixed, and embedded in paraffin. 3
Subepithelial collagen deposition (blue stain) was significantly pronounced in the saline-treated OVA-sensitized and challenged group mostly in the perivascular and peribronchiolar regions (Figure
Effect of stigmasterol on inflammatory collagen deposition. Guinea pigs were sensitized and challenged with ovalbumin as described in the methods. Animals received either saline, dexamethasone, or stigmasterol 1 h prior to each challenge. Naïve controls received normal saline only. Animals were sacrificed 24 h after the last ovalbumin challenge. The lungs were excised, fixed, and embedded in paraffin. 3
We explored the effects of stigmasterol on chronic airway inflammation induced by aerosolized ovalbumin and investigated its potential inhibitory effect on inflammatory features triggered by repeated challenge with ovalbumin in previously sensitized guinea pigs noting the possible mechanisms involved in the inhibition.
Cell infiltration into the lung tissues and alveolar fluids, elevation of inflammatory cells in blood, and changes to lung histology are features largely consistent with asthma [
In this study, we could show that stigmasterol inhibited early phase immune responses to allergen exposure. Analysis of serum collected from guinea pigs 24 h after the last ovalbumin exposure showed elevated levels of ovalbumin-specific immunoglobulin E, OVA sIgE in saline-treated OVA-sensitized and challenged controls and significantly reduced in stigmasterol-treated animals. Elevated blood inflammatory cell count induced by OVA sensitization and challenge in the saline-treated animals was also suppressed by stigmasterol. Similar to dexamethasone, stigmasterol could significantly control eosinophil, lymphocyte, and monocyte proliferation. In asthma, the proliferation of blood borne inflammatory cells and their subsequent migration into airway tissue drives epithelial tissue damage caused by chemical and inflammatory mediator release, leading to severe inflammation. Indeed previous studies have established a direct link between Th2 cell control and a good asthma prognosis [
Prior to tissue invasion, inflammatory cell movement and subsequent adherence to endothelial cells are mediated by several adhesion molecules. VCAM-1, identified as a major adhesion molecule in this process, is shed from cytokine-activated endothelial cells to promote subsequent leucocyte attachment [
Significant correlation between asthma severity and either systemic or airway oxidative stress has been established [
Hematoxylin and eosin (H&E) staining revealed excessive infiltration of inflammatory cells, mostly eosinophils and lymphocytes, in the ovalbumin challenged groups compared to that in untreated naïve animals. Lung sections of saline-treated OVA-sensitized and challenged animals showed high cellularity especially in peribronchiolar and perivascular regions. Dexamethasone and stigmasterol lowered cell accumulation in the peribronchiolar, perivascular, and alveolar regions, obtaining lower cell infiltration scores compared to the saline-treated OVA-sensitized and challenged asthmatic control guinea pigs. Stigmasterol treatment was associated with less congestion, sparsely distributed inflammatory cells in the alveolar region, and reduced thickening of alveolar septa. Persistent uncontrolled airway inflammation and cell infiltration lead to a cycle of tissue damage and repair eventually causing permanent damage to the lung tissues referred to as lung remodeling. It is associated with airway smooth muscle thickening, epithelial and goblet cell hyperplasia, basement membrane thickening, and collagen deposition [
Taken together, our data demonstrates for the first time that stigmasterol suppresses airway inflammation and remodeling by inhibiting allergen-induced immunoglobulin E-mediated responses and also abolishes VCAM-1-aided cellular migration into the lung tissues. Again, we show in part here that stigmasterol controls oxidative stress and preserves lung tissue antioxidant capacity, and this mechanism is a key factor responsible for its anti-inflammatory action.
Stigmasterol inhibits OVA-induced asthma in guinea pigs and has potential as a molecule of interest for the treatment of asthma.
The authors declare that they have no conflicts of interest.