Asthma is a complex chronic respiratory disease that depends on the interaction of genetic and environmental factors [
], and it features the activation of Th2 cells. Studies in humans and in animal asthma models have shown that inflammation, obstruction, and remodeling occur not only in the proximal airways but also in the distal pulmonary parenchyma [
]. The impairment of the distal airways is recognized as an important factor contributing to airflow obstruction [
], particularly in patients with severe asthma and nocturnal asthma [
The remodeling response in asthmatics contributes to significant changes in the structures of the proximal and distal airways [
], as well as in the extent of airflow obstruction. This process involves airway smooth muscle hypertrophy and hyperplasia, mucous gland hyperplasia, and an increase in the thickness of the airway wall [
]. Currently, anti-inflammatory therapies, such as corticosteroids, are considered the gold-standard treatment for asthma, particularly during an acute asthma attack. Corticosteroids inhibit numerous pro-inflammatory responses and induce numerous anti-inflammatory pathways. However, the development of new drugs that control this disease is essential for patients with severe, corticosteroid-insensitive asthma [
] and to decrease the collateral systemic effects of steroid use.
Under physiologic conditions, smooth muscle contraction is controlled by the phosphorylation of the myosin light chains [
] via Rho-mediated Ca
], or by myosin phosphatase activity, which dephosphorylates the myosin light chains [
] independently of the cytoplasmic calcium concentration [
The inhibition of Rho/Rho-kinase pathway may be considered a potential pharmacological and therapeutic target in lung diseases [
] because it relaxes the airway smooth muscle tone and decreases airway inflammation and remodeling. These various functions are associated with changes in the actin cytoskeleton, including cell adhesion, motility, migration, and contraction [
]. The increased activity of Rho-kinase in the vascular smooth muscle under pathophysiological conditions has been reported in hypertensive animal models [
] and in humans [
]. Thus, inhibitors of Rho/Rho-kinase that relax the airway smooth muscle and reduce muscle tone are predicted to be relevant to asthma treatment [
Recently, our research group published promising results using the Rho-kinase inhibitor Y-27632 in an animal model of chronic allergic inflammation. Treatment with Y-27632 (a pyridine derivative, (+)-(R)-trans–4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide), a selective inhibitor of Rho-kinase family enzymes, in sensitized animals reduced lung mechanics, inflammation, remodeling and oxidative stress in the airway and lung tissue [
Considering the relevance of corticosteroids as the gold standard treatment for asthma and aiming to complement our previous research, the focus of the present study was to evaluate the importance of combining a new class of drugs for controlling bronchial smooth muscle contraction with a corticosteroid. Knowing that the peripheral lung tissue is involved in asthma physiopathology, especially in severely asthmatic patients [
], we decided to broaden our research focus to not only include the evaluation of the airways but also study the lung parenchyma. We believe that this approach differentiates our study from those previously conducted by comprehensively covering two different compartments that determine the pathogenesis of this disease.
In the present study, we evaluated the effects of treatment with the Rho-kinase inhibitor Y-27632, the corticosteroid dexamethasone, or both, on guinea pigs with chronic allergic inflammation. Our analysis focused on the mechanical responses, inflammation, remodeling, and production of oxidative stress in the airways and lung parenchyma. The final results showed that both of the individual treatments were effective in reducing the maximum resistance and elastance of the respiratory system and lung parenchyma when challenged with antigen, suggesting that Rho-kinase inhibition or corticosteroid treatment modulate the constriction of the airways and lung parenchyma. There was also a reduction in E
NO, eosinophilic infiltration, Th1/Th2 inflammatory cytokines, extracellular matrix remodeling, actin content, the number of NF-ĸB-positive cells and oxidative stress.
The combination of the Rho-kinase inhibitor with the corticosteroid decreased all of the functional and histological parameters to the same extent as the individual treatments, in addition to maximizing the reduction of collagen and IFN-γ in the airways, the number of cells positive for IL-2, IFN-γ, NF-ĸB and the volume fraction of 8-iso-PGF2α in the distal lung tissue.
In the lung parenchyma, the combined treatment of Y-27632 with the corticosteroid maximized the reduction in the eosinophil counts and TIMP-1-positive cells when compared to animals treated only with the corticosteroid.
Notably, no previous studies have examined repeated treatment with a Rho-kinase inhibitor combined with a corticosteroid as a therapeutic strategy for the control of hyperresponsiveness, remodeling and inflammatory alterations induced by chronic allergic inflammation in the airways and distal lung tissue.
To avoid interfering with the animals’ sensitization, we started treatment with the corticosteroid and/or Y-27632 twenty-four hours after the fourth ovalbumin inhalation exposure. Using the PCA technique, we found that the sensitized animals had an increase in specific IgG1 anaphylactic antibodies.
The inhalation time was recorded to evaluate the acute responses to antigen exposure, as previously described [
]. Treatment with the Rho-kinase inhibitor, the corticosteroid or the combination of these therapies attenuated the acute response in sensitized animals. The use of Y-27632 alone increased the contact time with the antigen when compared to the corticosteroid-treated group in sensitized animals. This most likely occurred because of the muscle-relaxing action of the Rho-kinase inhibitor (Y-27632) on the airway smooth muscle [
] and on the myocontractile elements of the lung parenchyma, resulting in diminished signs of respiratory distress and increased inhalation time. On the other hand, dexamethasone affects mainly the inflammatory component of asthma.
We believe that several mechanisms contributing to the acute responses to antigen challenge, including smooth muscle contraction, were most likely altered by treatment with Y-27632 and the protective effect of the corticosteroid [
As a confirmation that these three treatments (separate use of Y-27632 and the corticosteroid, and the combined use of the corticosteroid with Y-27632) can effectively control the inflammatory process, we observed a significant reduction in E
NO among the animals sensitized with ovalbumin.
level has been proposed as an indirect marker of lung inflammation and correlates with the severity and response to the treatments. It is also elevated in asthmatics and animal models of chronic pulmonary inflammation [
]. Nitric oxide is involved in various mechanisms of asthma physiopathology. Previous studies have demonstrated that nitric oxide derived from constitutive isoforms has protective effects on bronchoconstriction and remodeling [
], whereas nitric oxide produced by iNOS is involved in the constriction, inflammation and remodeling processes [
The maximum elastance and resistance responses after antigen challenge decreased in both the respiratory system and the distal pulmonary tissue when the sensitized animals were treated with Y-27632, the corticosteroid, or a combination of both treatments, compared to the animals that received the vehicle. To further investigate the mechanisms involved in the control of the mechanical responses, we evaluated the volume fraction of actin in the airways and in the lung parenchyma of sensitized and treated guinea pigs. There was a decrease in the volume fraction of actin in the airway walls and alveolar septa in the treated groups compared to the untreated groups.
The results from different studies using the same model of experimental asthma have shown that treatment with Y-27632 is effective in decreasing the actin content in the airways [
] and in the distal parenchyma [
], but none of the studies assessed the effects of this inhibitor combined with corticosteroids. In the current study, the Rho-kinase inhibitor, alone or in combination with a corticosteroid, was able to reduce the volume fraction of actin compared to the OVA group.
There is evidence that activated Rho-kinase regulates actin/myosin contractibility regardless of the level of free calcium [
]. Actin is part of the cytoskeleton of endothelial cells, and the presence of inflammatory agonists increases cytosolic calcium levels. Increased calcium levels diminish cAMP and activate RhoA/Rho-kinase, causing the reorganization of the cortical actin into stress fibers, which are bundles of actin/myosin necessary to induce cell contraction [
]. In the distal parenchyma, the actin is localized in the myocontractile elements, such as myofibroblasts.
Corticosteroids may directly or indirectly modulate the contraction of the airway smooth muscle by suppressing the agonistic responses induced by an increase in intracellular calcium or by down-regulation linked to uncoupling receptors (M
histaminic receptors). In addition, corticosteroids can enhance the relaxation of the airway smooth muscle by activating AMP cycle-dependent or -independent mechanisms. The effects of dexamethasone on human airway smooth muscle were studied by Goldsmith et al. [
]. These authors showed that in human bronchial cells, corticosteroids reduced the level of smooth muscle-α and that such effects were mediated, at least in part, by the attenuation of mRNA translation and increased protein degradation.
The combined treatments resulted in a reduction in the number of cells positive for IFN-γ in the airways and in the lung parenchyma. We also observed a greater reduction in eosinophils and IL-2 in the lung parenchyma when both treatments were combined. Additionally, our results showed that in the lung parenchyma, treatment with the Rho-kinase inhibitor had a greater impact on reducing the eosinophil counts than treatment with the corticosteroid, showing that use of this drug by itself is promising.
In a related study, Souza et al. [
] investigated the effects of the corticosteroid montelukast and 1400 W (a selective iNOS inhibitor) in the peripheral lung tissue of guinea pigs with chronic allergic pulmonary inflammation. The results showed that the isolated use of dexamethasone was able to reduce the eosinophilic infiltrates and the number of Th1 (IFN-γ) and Th2 (IL-4 and IL-5) cells. Hence, the combination of an iNOS inhibitor and montelukast caused a maximized reduction in IL-4, IL-5, and IFN-γ. In mice chronically exposed to ovalbumin, chronic administration of budesonide reduced airway hyperresponsiveness, as well as leukocyte infiltration, with a decrease in the production of Th2 mediators such as IL-4, IL-12, and eotaxin-1 [
The results of the present study corroborated data in the literature describing the effect of a Rho-kinase inhibitor on eosinophil recruitment. Taki et al. [
] observed that the Rho-kinase inhibitor Fasudil reduced the number of eosinophils after allergen challenge, although it did not affect the recruitment of other inflammatory cells. Hashimoto et al. [
] demonstrated that the Rho-kinase inhibitor Y-27632 had a key role in reducing the infiltration and activation of inflammatory cells.
When we evaluated the alterations of the extracellular matrix, particularly the volume fraction of collagen and elastic fibers and the numbers of TIMP-1, MMP-9 and TGF-ß positive cells, we observed a reduction in the airway and distal lung parenchyma of the sensitized animals treated with the Rho-kinase inhibitor or with the corticosteroid. The combination of the corticosteroid with Y-27632 enhanced the reduction in the volume fraction of collagen fibers present in the airways. We also observed a greater attenuation of TIMP-1-positive cells in the lung parenchyma when both drugs were combined.
These results can be explained, at least in part, by the reduction in eosinophil recruitment and Th1/Th2 inflammatory cytokine levels, as demonstrated by the correlation data.
Studies suggest the importance of Rho-kinase modulation in the remodeling process. Zhou et al. [
] showed that Rho-kinase activation is crucial to collagen synthesis, which may be related to a combination of factors including the inhibition of the c-Jun N-terminal kinase (JNK) and TGF-ß pathways. Additionally, Kondrikov et al. [
] concluded that oxygen toxicity induces ROS to separate the guanine nucleotide dissociation inhibitor (GDI, a regulator of Rho GTPase activity) from Rho-kinase, leading to the activation of the Rho-kinase pathway and contributing to an increase in type I collagen synthesis.
Other studies suggest that inhaled corticosteroids are unable to reduce the pulmonary remodeling responses completely [
]. In this regard, Goleva et al. [
] showed that in asthmatics resistant to steroid treatment, there was an MMP-9/TIMP-1 imbalance that promoted proteolysis and contributed to the chronic remodeling of the airways and the non-reversibility of the bronchial smooth muscle contraction.
Chakir et al. [
] studied bronchial biopsies of patients with moderate and severe asthma treated with oral corticosteroids for two weeks. They showed that this treatment was unable to reduce type I and II collagen or TGF-ß. In contrast, the administration of beclomethasone in daily doses of 800 mg diminished the deposition of collagen in patients with asthma [
]. Miller et al. [
] demonstrated that corticosteroids inhibit TGF beta1 expression in eosinophils and macrophages. McMillan et al. [
] demonstrated in mice chronically exposed to ovalbumin that budesonide was able to reduce collagen deposition and mucus production by regulating inflammation and TGF beta1 signaling rather than by decreasing TGF beta protein production.
Our evaluation of the importance of oxidative stress also demonstrated an attenuation of the number of iNOS-positive cells and the volume fraction of 8-iso-PGF2α in sensitized animals treated solely with the Rho-kinase inhibitor Y-27632 or with the corticosteroid. When the treatments were combined, all of these parameters diminished, but there was a greater reduction in 8-iso-PGF2α in the distal pulmonary tissue.
It has been clearly demonstrated that iNOS activation contributes to the promotion of peroxynitrite production, which leads to lipid peroxidation and isoprostane (8-iso-PGF2α) generation [
]. The isoprostanes contribute to smooth muscle contraction by acting through the tyrosine kinases and Rho/Rho-kinase, leading to the decreased activity of myosin light-chain phosphatase and increasing the level of phosphorylated myosin light-chain and contraction [
McGown et al. [
] demonstrated that Fasudil reduced LPS-induced iNOS superregulation, reducing microvascular inflammation. Jiang and George [
] demonstrated that Rho-kinase inhibition with Y-27632 prevented the reduction of NO induced by TGF-β2, thus avoiding iNOS inhibition, and suggesting that TGF-β2 inhibits iNOS expression via a Rho-kinase-dependent pathway in pulmonary epithelial cells.
We observed that the cellular expression of NF-κB was reduced in animals treated with the Rho-kinase inhibitor or with the corticosteroid in sensitized animals. The combination of these treatments allowed for a maximized reduction of NF-κB in the distal pulmonary tissue.
The reduction of NF-κB
induced transcription might result in an inhibition of RhoA upregulation induced by IL-13 and TNF-α. Meyer-Schwesinger et al. [
] observed that Rho-kinase inhibition attenuated NF-κB expression, resulting in protection against injury. These data suggest that NF-κB expression may also be Rho-kinase-dependent.
PAP designed and performed the major part of the experiments and the morphometric analysis, performed the statistical analysis and drafted the manuscript. RFR, SSP, BMSR, ASAS, DGX, and MAA assisted in performing the experiments. APDR participated in some of the experiments and contributed to the morphometric analysis. CMP, EAL, PRMR, and MAM participated in the design of the study. IFLCT supervised the study, participated in its design, and interpreted the results, as well as prepared the manuscript. All authors have read and approved the final manuscript.