Carboxyamidotriazole ameliorates inflammation in experimental allergic asthma by inhibiting the expression of phosphodiesterase 4

doi:10.21203/rs.3.rs-3130449/v1

Download PDF

Research Article

Carboxyamidotriazole ameliorates inflammation in experimental allergic asthma by inhibiting the expression of phosphodiesterase 4

https://doi.org/10.21203/rs.3.rs-3130449/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Objective

Carboxyamidotriazole (CAI) is a small molecular drug with anti-inflammatory and anti-tumor effects, which can inhibit a variety of phosphodiesterases (PDEs) isolated from multiple tissue sources. The present study aims to explore the effect of CAI on allergic asthma of experimental mice model, and further verify the relation between CAI and specific PDE.

Methods

An in vitro model of lipopolysaccharide (LPS) stimulated-rat alveolar macrophages NR8383 and an in vivo model of ovalbumin (OVA) sensitized mice were used to examine the anti-inflammation effect of CAI. Airway hyperresponsiveness (AHR) was measured by Forced Maneuvers Pulmonary Function Testing (BUXCO). Fixative lung tissue was stained to observe the airway remodelling and the mucous secretion. mRNA expression of PDE-4A/B/D were detected by RT-qPCR.

Key findings:

In rat alveolar macrophage NR8383, CAI significantly reduced the inflammation induced by LPS and restrained the expression of PDE-4B. In mice with allergic airway inflammation, CAI can effectively reduce airway hyperresponsiveness, inhibit inflammatory factor secretion, alleviate the accumulation of inflammatory cells and reduce the levels of IgE in plasma. Airway remodeling manifested by smooth muscle thickening, cell proliferation, pro-inflammatory cytokines secretion and inflammatory cells gathering around the airway in mice was significantly prevented by CAI. CAI effectively restrained the expression of PDE-4A, PDE-4B and PDE-4D in lung tissue.

Conclusion

CAI can inhibit inflammatory activities both in activated macrophages and allergic asthma animal models, which might be related to reduction the expression of PDE4.

Allergic asthma

Carboxyamidotriazole

Phosphodiesterase

NR8383

Asthma is a common respiratory disease with complex causes, such as genetics, respiratory infections and airborne environment. As air pollution has increased in recent years, the incidence of asthma shows a trend of rising^[1]. Globally, the morbidity rate of asthma is 5%-10% which equates to about 2 million people dying every year. Allergic asthma is associated with IgE, activated type 2 T helper (Th2) cells and eosinophilic inflammation in the airways^[2]. The proinflammatory mediators derived from eosinophils are major contributors to inflammation in asthma. IgE accumulation can augment humoral and cellular immune responses to allergens with a further worsen medical condition. The severity of asthma can be measured by inflammatory factors, inflammatory cells and IgE accumulation which also is the measure of a treatment. Airway remodeling can be observed in asthma models, which is characterized by thickening of the subepithelial reticular basement membrane (RBM), angiogenesis and goblet cell hyperplasia^[3].

At present, glucocorticoids are the most effective and common therapy available for patients with mild or moderate asthma, such as prednisolone. However, 5%~10% of asthmatic patients are relatively insensitive to the treatment of glucocorticoids. And drug resistance or dependence might develop in patients receiving long-term high-dose glucocorticoids therapy. In addition, long-term glucocorticoids therapy may lead to osteoporosis susceptibility to infection and metabolic syndrome^[4–5]. Therefore, we need to find an effective medicine which can target airway inflammation, aim at its potential targets or against glucocorticoid insensitivity.

Phosphodiesterase (PDEs) is a big family composed of at least 11 isozymes (PDE1-PDE11) existing in kinds of inflammatory cells. It has been reported that PDE inhibitors play an important role in inflammatory diseases through regulating intracellular level of some inflammatory mediators ^[6]. Among PDEs family, PDE4 is over expressed in leukocytes and other inflammatory cells involved in the pathogenesis of inflammatory pulmonary disease, PDE4 inhibition might be a promising approach for the treatment of pulmonary inflammatory conditions such as asthma, chronic organized pulmonary disease (COPD), etc^[7–9]. Roflumilast, a PDE4 inhibitor to treat COPD, is authorized for clinical use in the European Union and the USA in 2010^[10]. From the above, we’d like to explore the effect of selective or non- selective PDE4 inhibitor in pulmonary inflammatory disease, such as asthma, COPD and so on.

Carboxyamidotriazole (CAI) named L651582, is a carboxyamide-amino-imidazole, as a non-cytotoxic drug inhibiting tumor cell proliferation^[11]. The dosage range of CAI tested in early experiments showed appropriate range was 10–50µM in vitro and 10-50mg/kg in vivo^[12]. Recently we found that CAI shows great promising in treating acute or chronic inflammatory and autoimmune diseases through its anti-inflammatory effects^[13–14]. However, the treatment effect of CAI on pulmonary inflammatory disease had never been reported. Meanwhile, we confirmed that CAI is a non-selective PDE inhibitor in vitro against different subtypes of PDEs^[15]. Previous studies have not confirmed whether CAI has a beneficial effect on allergic airway inflammation like other PDE4 inhibitors. Combining the anti-inflammatory with inhibition effect of PDE, we suppose that CAI may also have a similar effect on treating asthma comparing with glucocorticoids, through different mechanisms with fewer side effects. Here we reported some valuable evidence indicating that CAI may ameliorate inflammation in experimental allergic asthma as well as its inhibitory effect on PDE4 expression both in vitro and in vivo. An alternative or optimizing role of CAI to the glucocorticoids therapy might be expected.

2.1 Cells culture and inflammatory model

Rat alveolar macrophages cell line (NR8383) was obtained from China Center for Type Culture Collection, cultured in F12-k supplemented with 10% fetal bovine serum, 0.5%(200×) antibiotics(penicillin and streptomycin) and 1mmol/mL L-glutamine. The cells were maintained at 37℃ in a 5%CO₂-95%O₂ atmosphere. There were 5groups design: CON(Group 1) means normal group with no operation; LPS(Group 2) means 1.0µl lipopolysaccharide (LPS) induced NR8383 to secrete inflammatory medium. CAI20/40(Group 3/4) and DEX(Group 5) respectively means treating with 20µM CAI, 40µM CAI, 100nM dexamethasone after LPS inducing^[16]. The cell supernatant were collected to measure the level of IL-1、IL-6、TNF-α by ELISA, 24h and 48h after treatment. Carboxyamidotriazole(CAI) was synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences. Dexamethasone acetate injection(1ml:5mg) was purchased from Guangzhou Baiyunshan Tianxin Pharmaceutical Co.,LTD.

2.2 Animals and group

Female SPF C57BL/6 mice (aged 6-8weeks) were purchased from Beijing HFK Bioscience Co., Ltd and maintained in animal facility under specific, pathogen-free conditions with a 12h light-dark cycle. The mice had free access to food and water and acclimated for 7 days before doing the experiments. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College.

Mice were randomly divided into 5 groups: CON (normal mice group); OVA (model group); CAI20 (20mg/kg CAI-treated group); CAI40 (40mg/kg CAI-treated group) and DEX (0.5mg/kg dexamethasone-treated group). Each group has 8 mice.

2.3 Induction of allergic asthma and treatment

The allergic airway inflammation in mice was induced with ovalbumin(OVA). All mice were sensitized by intraperitoneal injection of 20µg ovalbumin (grade V, Sigma-Aldrich, USA) and 200µL aluminum adjuvant (2%, InvivoGen USA) on days 0 and 14. On day 21–26, the OVA-sensitized mice(except CON group mice) were challenged by nebulization of 5% ovalbumin in saline solution for 1hour. Mice in CON group were sensitized by intraperitoneal injection of saline solution on day 0 and 14 and challenged by saline solution for 1 hour on day 21–26. Half an hour before challenging on day 21–26, mice in treatment groups were administrated with the CAI and those in model group and control group received PEG400 (the vehicle) intragastrically. All mice were killed 24 hours after the last nebulization^[17–20].

2.4 Airway hyperresponsiveness test

Airway hyperresponsiveness test had been done 24 hours after the last nebulization. Airway hyperresponsiveness (AHR) was measured by Forced Maneuvers Pulmonary Function Testing (BUXCO). After anesthesia, inserting a cannulas into the airway and inhaling aerosolized methacholine at increasing concentrations (0,2.5,5,10,20mg/mL) for 10min, the airway resistance and the dynamic airway compliance were measured .

2.5 Bronchoalveolar lavage and cell counts

The bronchoalveolar lavage fluid (BALF) was obtained by infusion and extraction three times of 1.5mL cold PBS(4℃). The BALF was centrifuged at 4℃ for 10min at 2500rpm. The sediment was resuspended with 1.0mL PBS. The total cell counts in BALF were determined by a hemocytometer using trypan blue staining. A part of the resuspended BALF (100µL) was smeared onto glass slide and stained with Wright-Giemsa for eosinophil counts. Eosinophils were selected from counting 200 total cells to obtain the ratio of eosinophil to total cell count.

2.6 Measurement of cytokine levels in BALF and IgE levels in plasma

Concentrations of IL-4, IL-13 and IFN-γ in supernatant of BALF were assessed by ELISA method using commercially available kits (Excell Biotech (Taicang)Co.,Ltd). Exposing the heart and lung of mice to obtain the blood samples. The sample was centrifuged at 4℃ for 25min at 3000rpm to collect the plasma. The levels of total and OVA-specific IgE in plasma were measured by ELISA (Cayman Chemical Company,USA).

2.7 Lung tissue histopathology

The left lung was soaked in 4% paraformaldehyde for 24h Fixation. Fixative lung issue was stained with means of routine H&E staining and PAS staining to observe the Airway remodelling and the mucous secretion by light microscope.

2.8 Measure the level of PDE4s mRNA expression both in cells and mice by RT-qPCR

The primers for PDE4-A/B/D and beta-actin were designed as GenBank standard. They were synthesised by Shanghai GeneCore BioTechnologies Co., Ltd. Cell-related RNA was obtained from NR8383 24h after inducing and treatment. Mice-related RNA was obtained from right lung tissue. After extracting RNA and reverse transcription by TransScript First-Strand cDNA Synthesis SuperMix kit(Transgen Co.,Ltd) and cDNA amplified by SYBR GREEN I Real Time PCR Kit(Transgen Co.,Ltd), Reverse Transcription Quantitative PCRs detection and analysis were performed on BIO-RAD.

2.9 Statistical analysis

The data were expressed as mean ± SEM;n represents the number of animals. Statistical analyses were performed by using ANOVA followed by Dunnett’s test, and p < 0.05 was considered significant.

3.1 CAI suppressed proinflammatory cytokines production in NR8383

To investigate the effect of CAI on airway inflammatory response, we observed its effect on the release of inflammatory mediators from LPS-stimulated NR8383 alveolar macrophages. Compared with the normal group(CON), the level of IL-1β in LPS-induced group increased significantly (P < 0.01). After 24h treatment of 20µM CAI, IL-1β level dropped significantly (P < 0.05), and 40µM CAI and DEX were fairly more effective in inhibiting IL-1β production (P < 0.01). While longer treatment with CAI or dexamethasone (48h) seemingly didn’t show stronger inhibitory effect on IL-1β production (Fig. 1A). For LPS-induced IL-6 and TNF-α productions in NR8383 cells CAI showed more dramatic inhibitory effect in a dose-dependent manner after 24h or 48h treatment (P < 0.001) (Fig. 1B/C). And it is noteworthy that the anti-inflammatory effect of 40µM CAI in term of IL-6 and TNF-α-blocking is comparable with that of dexamethasone.

3.2 CAI-treated mice were protected from methacholine (MCh) induced airway hyper-responsiveness

Airway hyper-responsiveness is one of airway inflammation characteristic that when sensitized by stimuli airway narrows excessively. Airway hyper-responsiveness changes were tested with the lung function instrument. Without treatment, OVA group reacted dramatically after inhaling aerosolized methacholine including airway resistance increasing(Fig. 2A) and the dynamic airway compliance decreasing(Fig. 2B) obviously. After CAI(40mg/kg) treatment, the airway resistance and the dynamic airway compliance became gentle(P < 0.05). According to the figure, the effect of treating with 40mg/kg CAI was better than 0.5mg/kg dexamethasone. However, treating with 20mg/kg CAI was inferior to dexamethasone. The results suggested CAI might protect mice from airway hyper-responsiveness.

3.3 CAI relieved Th1/ Th2 unbalance in BALF

Th1/Th2 unbalance will lead to allergic inflammation. OVA-stimulation increased the levels of IL-4, IL-13 in BALF of mice significantly (P < 0.001) with IFN-γ production decreasing dramatically (P < 0.001) (Fig. 3). Whereas CAI treatment, at 20mg/kg or 40mg/kg, reduced the level of IL-4 markedly (P < 0.01) (Fig. 3A). For another Th2 cell-related cytokine, IL-13 production was only inhibited by 40mg/kg (P < 0.01) (Fig. 3B). Besides, CAI40 also can obviously increase the content of IFN-γ (P < 0.001) compared with DEX (Fig. 3C). The results suggest that CAI may hinder the abnormal activation of Th2 cells, activate Th1 cells and relieve Th1/Th2 unbalance.

3.4 CAI reduced inflammatory cells infiltration and eosinophils ratio in BALF

Th2 cytokines may activate eosinophils and induce inflammatory cells infiltration which was confirmed in the present study. Inflammatory cell numbers and eosinophil ratio in BALF of mice in model group were obviously increased following sensitization of ovalbumin(OVA) (P < 0.001). Treatment with 40mg/kg CAI, inflammatory cells count decreased significantly (P < 0.01) (Fig. 4A) and eosinophils ratio also declined significantly dramatically (P < 0.001) (Fig. 4B). Besides just treat with 20mg/kg CAI can also significantly decreased the eosinophil ratio (P < 0.01).

3.5 The treatment of CAI decreased the level of total and OVA-specific IgE in plasma

Relief of increasing the level of IgE by B cells is an important indicator of allergic disease treatment. To investigate the inhibition of CAI in TH2-allergic inflammation, we determined the levels of IgE. OVA-challenged induced the levels of total IgE and OVA-special IgE in the serum both significantly increased (P < 0.001). While treating with CAI can ease that. The data showed 40mg/kg CAI can reduce the content of total IgE (P < 0.01) (Fig. 5A) and obviously reduce the content OVA-specific IgE(P < 0.001) (Fig. 5B). Allergic asthma is a type I hypersensitivity induced by IgE. Therefore reducing the level of IgE is an important index to evaluate the treatment effect on the allergic asthma. CAI can obviously decrease two types of IgE revealing that the treatment effect of CAI on the allergic asthma is tremendous.

3.6 CAI can ease airway remodeling

Airway remodeling can be observed in asthma models, which is characterized by thicken of the subepithelial reticular basement membrane (RBM), angiogenesis and goblet cell hyperplasia^[3]. Whether or not improve airway remodeling is also a measure standard to judge the effect of a treatment on asthma.

Evaluation of lung pathological tissue staining with H&E reveal infiltration of inflammatory cells. The model group showed bronchial wall and smooth muscle thickening, epithelium falling off and the goblet cells hyperplasia. We also found the aggregation and infiltration of a large number of eosinophils and macrophages around bronchus. However, treating with CAI made the bronchial wall and smooth muscle thinning with the goblet cells back to normal. And the number of inflammatory cells around bronchus showed an obvious decrease (Fig. 6).

3.7 CAI can reduce mucus secretion

The mucus in the airway included mucopolysaccharide which can react with the PAS dye to show a kind of purple material. By staining with PAS, there were a wide range of purple materials in the airway of model group mice, suggesting mucus secretion increased obviously. While there were little purple materials in the airway of CAI40 and DEX group mice, it meaned the mucus secretion induced by allergic inflammation of CAI40 and DEX group significantly decreased (Fig. 7).

3.8 CAI reduces PDE-4B mRNA expression in NR8383 cells and PDE4A/B/D mRNA expressions in mice

LPS-induced NR8383 showed a high expression of PDE-4B mRNA compared with normal group (P < 0.001). CAI can reduce this high expression significantly (P < 0.001) (Fig. 8A). So we speculated that CAI might be a PDE-4B inhibitor. But there was no obvious difference on the expression of PDE4A and PDE4D in the NR8383.

In lung tissue, OVA-challenged mice with no treating also revealed a high expression of PDE4A/B/D (P < 0.05). We found PDE-4A (Fig. 8B) and PDE-4D (Fig. 8D) expression in CAI40 group significantly lower (P < 0.05). While the PDE-4B expression in CAI20 group and CAI40 group were both significantly lower (P < 0.05)(Fig. 8C). PDE-4C has no expression in asthma and COPD^[8]. Thus we suggested that the Carboxyamidotriazole is a PDE4 inhibitor.

Asthma is a chronic airway inflammatory disease characterized by the infiltration of T cells, mast cells, basophils, macrophages and eosinophils. Multiple inflammatory mediators and modulators have been implicated in airway inflammation and obstruction. Thus, one of the key strategies for fighting asthma is balancing the cytokines induced by Th cells. In allergic asthma, CD4⁺ T helper (Th) lymphocytes play a central role in orchestrating inflammatory responses by secreting cytokines such as interleukins and interferons and can be categorized into phenotypes (e.g., Th0, Th1, Th2) on the basis of their products. The immediate allergic response to antigens is primarily driven by Th2-type cells that produce a unique cytokine profile including IL-3, IL-4, IL-5 and IL-13. These cytokines induce switching of B cells towards IgE-producing cells, stimulate eosinophilopoiesis and eosinophils activation, and promote airway inflammation and remodeling^[23–24]. Th1 cells producing IFN-γ are primarily involved in classic delayed hypersensitivity and have been proposed to inhibit Th2-driven processes. Th1/Th2 unbalance has been a classic pathogenesis of immune-mediated inflammatory disease. Abnormal Th1 activation results in an increased Th1/Th2 ratio that might increase the risk of autoimmune diseases. In contrast, the shift in Th1/Th2 balance toward a polarized Th2 response might lead to allergic diseases. Recently, a new ‘pathogenic Th population disease induction model’ has been widely accepted based on ‘classical Th1/Th2 balance disease induction mode’. In the present study, we observed a series of airway inflammation and airway hyperresponsiveness characteristics in an OVA-induced mouse model of allergic asthma, including increased number of eosinophils, increased mucus secretion, OVA-specific IgE production, excess production of Th2 cytokines and decreased dynamic airway compliance. We investigated the effect of CAI on airway inflammation and airway hyperresponsiveness. Our data suggest that CAI-treated groups showed suppressed allergic airway inflammation and relief of airway hyperresponsiveness. CAI decreased the levels of Th2 cell-related cytokines IL-4 and IL-13 and increase the content of the major effector cytokine IFN-γ of Th1 cells indicating that the therapeutic effect of CAI might be mediated by balancing Th1/Th2 cytokines.

In OVA-challenged mouse model of allergic asthma, the level of total and OVA-specific IgE in plasma increased dramatically. It has been well-established that IgE has causal relationship with asthma and elevated IgE level in plasma is the major characteristic of allergy. And anti-IgE therapy has been verified an effective way to prevent exacerbations of asthma^[25]. Though IgE-related pathophysiology of allergic airway inflammation might be far more complex, several recent developments have revealed that anti-IgE treatment might be a disease modifying pharmacological intervention by preventing or reversing airway remodelling changes, as represented by omalizumab ^[23], decreasing airway eosinophilic infiltration and reducing OVA-specific IgE level. CAI reduces the total IgE level in the plasma of allergic asthmatic mice in a dose-dependent manner, and its ability to reduce OVA-specific IgE is even comparable to dexamethasone predicting a therapeutic potential for allergic asthma. The LPS-stimulated rat NR8383 was a commonly used model for in vitro evaluating the anti-inflammatory properties of a certain drug in airway^[21–22]. In the present study, CAI displayed high anti-inflammatory potential by decreasing cytokines secretion in NR8383 cells. It is worth noting that levels of TNF and IL-6 after treatment with CAI and DEX are below the CON. It may be due to NR8383 itself which is a alveolar macrophage cell line secreting a little inflammatory medium without stimulated. Treatment with CAI and DEX may inhibit its own secretory function. Through measure of RNA, the expression of PDE-4B in NR8383 cells also be inhibited. However, the expression of PDE-4A and PDE-4D were almost unchanged after treatment with CAI. The reason needs to be explored further.

Phosphodiesterase (PDEs) is a superfamily formed of at least 11 intracellular isoenzymes^[8] which involves in lots of physiological processes via catalyzing the hydrolysis of cAMP and cGMP. Increasing intracellular concentrations of cAMP/cGMP may inhibit production of proinflammatory cytokines and alleviate the process of inflammatory diseases, which is more important for the activity of a variety of inflammatory cells, such as eosinophils, neutrophils, basophils, lymphocytes and so on. PDE4 is a member of PDEs which is the primary cAMP-hydrolyzing enzyme in inflammatory and immune cells especially macrophages, eosinophils, and neutrophils. PDE4 includes four subtypes (PDE-4A, PDE-4B, PDE-4C, PDE-4D), which are widely expressed in many tissues. And PDE 4B is highly expressed in lung tissue. Upregulation of the PDE 4B enzyme in response to pro-inflammatory agents suggest that it has a role in inflammatory processes^[26]. PDE 4D is highly expressed in T-cells and also plays an important role in airway smooth muscle contraction. A great deal of studies have shown the pathogenesis of asthma is associated with upregulated expression of PDE4 enzyme in lung tissues which promotes pro-inflammatory cell infiltration, increases cytokine secretion, stimulates IgE release and degranulation^[8]. Some studies confirmed that PDE-4A expression in the alveolar macrophages of the COPD patients is increased. And the expression of PDE-4B is relative to TNF-α secretion in alveolar macrophages induced by LPS^[27], which also regulates Th2 cells function in the experimental mice allergic asthma model. Moreover, the mice with PDE-4D gene deletion won't develop airway hyperresponsiveness on methacholine stimulation^[28]. Above all, inhibition of PDE4 leads to down-regulating the inflammatory response and may be a new pharmacological strategy for airway inflammation control. It should be noted that PDE 4C is expressed lowly in lung and not in circulating inflammatory cells.

Previously we have reported the inhibitory effect of CAI on cAMP-PDEs in various tissues of mice and different types of cells^[15]. In the present study, we further demonstrated that 40mg/kg of CAI significantly inhibited the expression of PDE-4A, PDE-4B, and PDE-4D in lung tissue of the mice allergic asthma model. At the same time, CAI also significantly inhibited the expression of the PDE-4B in LPS-stimulated alveolar macrophages of rats. It can be speculated that the beneficial effect of CAI against airway inflammation in experimental allergic asthma might attributable to inhibiting the expression of PDE4. It has been proved there is a crosstalk between NF-κB pathway and cyclic AMP pathways^[29]. We also have demonstrated CAI can inhibit NLRP3 inflammasome and NF-κB activation according to our group's study^[30–31]. Based on these theoretical bases, we can speculate that anti-inflammation mechanism of CAI in asthma may relate to breaking the crosstalk between PDE4 gene expression and NF-κB pathway activation. That is, decreased PDE4 gene expression and activity lead to the activation of cyclic AMP signaling cascade that largely inhibits NF-κB pathway, which we will study next step.

In all of asthma treatments, glucocorticoids are the most effective anti-inflammatory treatments available for asthma which mild or moderate asthma may be controlled well. However, as the disease worsens and dosage increases, some patients show glucocorticoid resistance with disease uncontrolled. In order to avoid using glucocorticoid and achieve the same therapeutic effect, we selected a high concerntration of glucocorticoid as the positive control group to verify the effection of CAI. The results suggest the high concerntration of CAI works just as glucocorticoid. Cause CAI aims at different targets with glucocorticoids, it might be a new type of anti-inflammatory agent for asthma therapy. And we even consider that combining CAI with glucocorticoids might improve glucocorticoids resistance and alleviate the adverse reactions when used by elderly and children, which certainly need further investigation.

In vitro, carboxyamidotriazole showed a clear anti-inflammatory effect in rat NR8383 alveolar macrophages as evidenced by significantly reduced inflammatory cytokines secretion. In vivo, CAI has profound inhibitory effects on allergic airway inflammation in an OVA-induced mouse model of asthma. It decreases inflammatory cells infiltration, balances Th1/Th2 cytokines, reduces IgE concentration in plasma, relieve airway remodeling and ease airway hyperresponsiveness. Moreover, CAI inhibited PDE4-B mRNA expression in NR8383 cells, and the inhibitory effect of CAI on PDE4-A/B/D mRNA expressions was further confirmed in mice lung tissues, indicating it to be a potential nonselective PDE4 inhibitor. We conjecture the inhibition of inflammatory activities might be related to reduction the expression of PDE4 which needs subsequent verification.

Conflict of interest

The authors did not have any conflict of interest.

Acknowledgments

This work was supported by the CAMS Innovation Fund for Medical Sciences（CIFMS）2021-I2M-C&T-B-049；Beijing Hope Run Special Fund of Cancer Foundation of China LC2020A34; CAMS Major Collaborative Innovation Project 2022-I2M-2-002.

Authors' contributions

Conceptualization, Peng Li, Fangrui Sun, Lei Guo, Hongliang Wu; methodology, Peng Li, Fangrui Sun, Jie Yu; software, Peng Li, Rui Ju; validation, Fangrui Sun, Jie Yu; formal analysis, Peng Li, Meikui Wu; resources, Hongjiang Guo，Diya Zhang; data curation, Jie Yu, Rui Ju; writing—original draft preparation, Peng Li, Fangrui Sun; writing—review and editing, Lei Guo, Hongliang Wu; supervision, Meikui Wu; project administration, Lei Guo; funding acquisition, Hongliang Wu. All authors have read and agreed to the published version of the manuscript.

D’Amato G, Vitale C, Martino A D, et al. Effects on asthma and respiratory allergy of Climate change and air pollution[J]. Multidisciplinary Respiratory Medicine, 2015, 10(1):39.
Patel T R, Sur S. IgE and eosinophils as therapeutic targets in asthma[J]. Current Opinion in Allergy & Clinical Immunology, 2016, 17(1):42.
Saglani S, Lloyd C M. Novel concepts in airway inflammation and remodelling in asthma[J]. European Respiratory Journal, 2015, 46(6): 1796–1804.
Rodriguez J M, Monsalves-Alvarez M, Henriquez S, et al. Glucocorticoid resistance in chronic diseases[J]. Steroids, 2016, 115:182–192.
Yutaka H, Hiromichi M, Lawrence L, et al. Side effects of corticosteroids in patients with advanced cancer: a systematic review[J]. Supportive Care in Cancer, 2018.
Zebda R, Paller A S. Phosphodiesterase 4 (PDE4) Inhibitors[J]. Journal of the American Academy of Dermatology, 2017, 78(3S1): S0190962217328232.
Chakraborti A K, Parikh N. Phosphodiesterase 4 (PDE4) Inhibitors in the Treatment of COPD: Promising Drug Candidates and Future Directions[J]. Current Medicinal Chemistry, 2016, 23(2):-.
Moretto N, Caruso P, Bosco R, et al. CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration[J]. Journal of Pharmacology & Experimental Therapeutics, 2015, 352(3):559–567.
Giembycz M A, Newton R. Harnessing the Clinical Efficacy of Phosphodiesterase 4 Inhibitors in Inflammatory Lung Diseases: Dual-Selective Phosphodiesterase Inhibitors and Novel Combination Therapies[M]// Phosphodiesterases as Drug Targets. Springer Berlin Heidelberg, 2011:415–446.
Murad H A, Habib H S, Rafeeq M M, et al. Co-inhalation of roflumilast, rather than formoterol, with fluticasone more effectively improves asthma in asthmatic mice[J]. Experimental Biology & Medicine, 2017, 242(5):1535370216685006.
Felder C C, Ma A L, Liotta L A, et al. The antiproliferative and antimetastatic compound L651582 inhibits muscarinic acetylcholine receptor-stimulated calcium influx and arachidonic acid release.[J]. Journal of Pharmacology & Experimental Therapeutics, 1991, 257(3):967–971.
Teicher B A, Holden S A, Chen Y N, et al. CAI: effects on cytotoxic therapies in vitro and in vivo[J]. Cancer chemotherapy and pharmacology, 1994, 34(6): 515–521.
Guo L, Li J, Ye H, et al. Anti-inflammatory and analgesic potency of carboxyamidotriazole, a tumoristatic agent[J]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao Acta Academiae Medicinae Sinicae, 2009, 31(3):315.
Lei ZHU, Juan LI, Lei GUO, et al. Therapeutic Effect of Carboxyamidotriazole on Adjuvant Arthritis in Rats[J]. Chinese Academy of Medical Sciences,2016,38(1):49–54.
Lei G, Luo L, Rui J, et al. Carboxyamidotriazole: A novel inhibitor of both cAMP-phosphodiesterases and cGMP-phosphodiesterases[J]. European Journal of Pharmacology, 2015, 746(746C):14–21.
Ju R, Wu D, Guo L, et al. Inhibition of pro-inflammatory cytokines in tumour associated macrophages is a potential anti-cancer mechanism of carboxyamidotriazole[J]. European Journal of Cancer, 2012, 48(7): 1085–1095.
Liang P, Peng S, Zhang M, et al. Huai Qi Huang corrects the balance of Th1 /Th2 and Treg /Th17 in an ovalbumin -induced asthma mouse model[J]. Bioscience Reports, 2017: BSR20171071.
Youm J, Lee H, Chang H B, et al. Justicia procumbens Extract (DW2008) Selectively Suppresses Th2 Cytokines in Splenocytes and Ameliorates Ovalbumin-Induced Airway Inflammation in a Mouse Model of Asthma[J]. Biological & Pharmaceutical Bulletin, 2017, 40(9):1416–1422.
Yun-Ho H, Seong-Gyeol H, Seul-Ki M, et al. The Protective Effects of Astaxanthin on the OVA-Induced Asthma Mice Model[J]. Molecules, 2017, 22(11):2019-.
Jeon W Y, Shin H K, Shin I S, et al. Soshiho-tang water extract inhibits ovalbumin-induced airway inflammation via the regulation of heme oxygenase-1[J]. Bmc Complementary & Alternative Medicine, 2015, 15(1):1–10.
Huang D, Chen Y, Chen W, et al. Anti-inflammatory effects of the extract of Gnaphalium affine D. Don in vivo and in vitro[J]. Journal of Ethnopharmacology, 2015, 176(28):356–364.
Ren W, Wang Z, Hua F, et al. Plasminogen activator inhibitor-1 regulates LPS-induced TLR4/MD-2 pathway activation and inflammation in alveolar macrophages[J]. Inflammation, 2015, 38(1): 384–393.
Samitas K, Delimpoura V, Zervas E, et al. Anti-IgE treatment, airway inflammation and remodelling in severe allergic asthma: current knowledge and future perspectives[J]. European Respiratory Review An Official Journal of the European Respiratory Society, 2015, 24(138):594.
Hirahara K, Nakayama T.CD4 + T-cell subsets in inflammatory diseases: beyond the Th1/Th2 paradigm. Int Immunol. 2016 Apr;28(4):163–71
Tam J S, Grayson M H. IgE and antiviral immune response in asthma[J]. Journal of Allergy and Clinical Immunology, 2017: S0091674917302105.
Baskaran P, Parthasarathy A, Myong S J. A Novel Catecholopyrimidine Based Small Molecule PDE4B Inhibitor Suppresses Inflammatory Cytokines in Atopic Mice[J]. Frontiers in Pharmacology, 2018, 9:485-.
Avila DV, Myers SA, Zhang JW, et al. Phosphodiesterase 4b expression plays a major role in alcohol-induced neuro-inflammation[J]. Neuropharmacology, 2017, 125:376–385.
Lin AH, Shang Y, Mitzner W, et al. Aberrant DNA Methylation of Phosphodiesterase 4D: Effect on Airway Smooth Muscle Cell Phenotypes[J]. American Journal of Respiratory Cell & Molecular Biology, 2016, 54(2):241.
Ghosh M, Aguirre V, Wai K, et al. The Interplay between Cyclic AMP, MAPK, and NF-κB Pathways in Response to Proinflammatory Signals in Microglia[J]. Biomed Res Int, 2015, 2015:308461.
Du X, Chen W, Wang Y, et al. Therapeutic efficacy of carboxyamidotriazole on 2,4,6-trinitrobenzene sulfonic acid-induced colitis model is associated with the inhibition of NLRP3 inflammasome and NF-κB activation[J]. International Immunopharmacology, 2017, 45:16–25.
Guo L, Ye C, Hao X, et al. Carboxyamidotriazole ameliorates experimental colitis by inhibition of cytokine production, nuclear factor-κB activation, and colonic fibrosis[J]. Journal of Pharmacology & Experimental Therapeutics, 2012, 342(2):356.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Carboxyamidotriazole ameliorates inflammation in experimental allergic asthma by inhibiting the expression of phosphodiesterase 4

Status:

Version 1

Abstract

Objective

Methods

Key findings:

Conclusion

Figures

1. Introduction

2. Methods

2.1 Cells culture and inflammatory model

2.2 Animals and group

2.3 Induction of allergic asthma and treatment

2.4 Airway hyperresponsiveness test

2.5 Bronchoalveolar lavage and cell counts

2.6 Measurement of cytokine levels in BALF and IgE levels in plasma

2.7 Lung tissue histopathology

2.8 Measure the level of PDE4s mRNA expression both in cells and mice by RT-qPCR

2.9 Statistical analysis

3. Results

3.1 CAI suppressed proinflammatory cytokines production in NR8383

3.2 CAI-treated mice were protected from methacholine (MCh) induced airway hyper-responsiveness

3.3 CAI relieved Th1/ Th2 unbalance in BALF

3.4 CAI reduced inflammatory cells infiltration and eosinophils ratio in BALF

3.5 The treatment of CAI decreased the level of total and OVA-specific IgE in plasma

3.6 CAI can ease airway remodeling

3.7 CAI can reduce mucus secretion

3.8 CAI reduces PDE-4B mRNA expression in NR8383 cells and PDE4A/B/D mRNA expressions in mice

4 Discussion

5 Conclusions

Declarations

References

Additional Declarations

Status:

Version 1