Elsevier

Phytomedicine

Volume 91, October 2021, 153701
Phytomedicine

Original Article
Isoforskolin, an adenylyl cyclase activator, attenuates cigarette smoke-induced COPD in rats

https://doi.org/10.1016/j.phymed.2021.153701Get rights and content

Abstract

Background

Chronic obstructive pulmonary disease (COPD) is characterized by limited airflow due to pulmonary and alveolar abnormalities from exposure to cigarette smoke (CS). Current therapeutic drugs are limited and the development of novel treatments to prevent disease progression is challenging. Isoforskolin (ISOF) from the plant Coleus forskohlii is an effective activator of adenylyl cyclase (AC) isoforms. Previously we found ISOF could attenuate acute lung injury in animal models, while the effect of ISOF on COPD has not been elucidated.

Purpose

In this study, we aimed to evaluate the efficacy of ISOF on COPD and reveal its potential mechanisms.

Methods

A rat model of COPD was established by long-term exposure to CS, then the rats were orally administered with ISOF (0.5, 1 and 2 mg/kg). The pulmonary function, lung morphology, inflammatory cells and cytokines in serum or bronchoalveolar lavage fluid (BALF) were evaluated. Transcriptomics, proteomics and network pharmacology analysis were utilized to identify potential mechanisms of ISOF. Droplet digital PCR was used to detect the mRNA expression of AC1-10 in donor lung tissues. AC activation was determined in recombinant human embryonic kidney 293 (HEK293) cells stably expressing human AC isoforms. In addition, ISOF caused trachea relaxation ex vivo were assessed in isolated trachea rings from guinea pigs.

Results

ISOF significantly ameliorated pathological damage of lung tissue and improved pulmonary function in COPD rats. ISOF treatment decreased the number of inflammatory cells in peripheral blood, and also the levels of pro-inflammatory cytokines in serum and BALF. Consistent with omics-based analyses, ISOF markedly downregulated the mTOR level in lung tissue. Flow cytometry analysis revealed that ISOF treatment reduced the ratio of Th17/Treg cells in peripheral blood. Furthermore, the expression levels of AC1 and AC2 are relatively higher than other AC isoforms in normal lung tissues, and ISOF could potently activate AC1 and AC2 in vitro and significantly relax isolated guinea pig trachea.

Conclusion

Collectively, our studies suggest that ISOF exerts its anti-COPD effect by improving lung function, anti-inflammation and trachea relaxation, which may be related to AC activation, mTOR signaling and Th17/Treg balance.

Introduction

Chronic obstructive pulmonary disease (COPD) is a common preventable and treatable disease that is characterized by airflow obstruction, and it is now the third largest cause of human mortality worldwide (GOLD, 2021; Rabe and Watz, 2017; Whittaker Brown and Braman, 2020). Cigarette smoke (CS) is the main risk factor for the development of this disease (Rabe and Watz, 2017). The pathological hallmarks of COPD include pulmonary emphysema, chronic bronchitis, small airway remodeling and hypersecretion of mucus (Barnes, 2008). Currently, the pathogenesis of this complex disease is still incompletely elucidated. Oxidative stress, protease-antiprotease imbalance, inflammatory cells and mediators all contribute to the pathological changes observed in COPD (GOLD, 2021). It is now recognized that neutrophils, macrophages and dendritic cells of the innate immune system and also T cells and B cells of the adaptive immune system are involved in COPD pathogenesis (Ni and Dong, 2018).

At present, the drugs for the prevention and treatment of COPD include inhaled corticosteroids (ICSs), long-acting β2-adrenoceptor agonists (LABAs), and long-acting muscarinic antagonists (LAMAs) (GOLD, 2021). Combination therapy was recommended to treat COPD patients with severe symptoms and/or high-risk exacerbation history (GOLD, 2021). Roflumilast is a selective phosphodiesterase 4 (PDE4) inhibitor licensed for COPD treatment which could reduce airway inflammation and improve lung function in patients (Rabe, 2011). PDE4 is the principal cyclic adenosine monophosphate (cAMP) degrading enzyme and inhibition of PDE4 could lead to intracellular cAMP accumulation, then activating downstream signaling pathway to regulate the inflammatory responses and maintain immune homeostasis (Maurice et al., 2014; Raker et al., 2016). The side effects of roflumilast, including weight loss, diarrhea, nausea and headache, have limited its clinical application (Rabe and Watz, 2017). Although progress has been made in the treatment of symptoms and acute exacerbations prevention of COPD, the development of novel treatments to reduce the relentless progression of COPD remains challenging (Rabe and Watz, 2017; Whittaker Brown and Braman, 2020).

Adenylyl cyclase (AC) plays an important role in cAMP signaling and compartmentation (Johnstone et al., 2018). Mammalian cells express nine transmembrane AC isoforms (AC1∼AC9) which play key roles in cellular responses to extracellular signals and one soluble AC (AC10) acts as an intracellular metabolic sensor (Pavan et al., 2009). With distinct regulatory properties and tissue distribution, AC isoforms have been considered as potential drug targets in different diseases (Pavan et al., 2009; Pierre et al., 2009). Intracellular cAMP homeostasis is regulated by AC which catalyzes cAMP formation from ATP and PDE that degrade cyclic nucleotides (Raker et al., 2016). A growing number of evidence suggests that enhance intracellular cAMP generation has beneficial effects in lung diseases such as COPD (Maurice et al., 2014; Schmidt et al., 2020; Zuo et al., 2019). Indeed, the bronchorelaxation effects of β2-adrenoceptor agonists are partially attribute to the increased intracellular cAMP (Billington and Hall, 2012). Moreover, both selective PDE4 and dual PDE3/4 inhibitors are considered as potential therapeutic agents for the treatment of COPD (Abbott-Banner and Page, 2014; Franciosi et al., 2013; Schmidt et al., 2020).

Isoforskolin (ISOF) is the principal active component from the plant Coleus forskohlii native to Yunnan in China, and it is an effective AC activator which induces increasing intracellular cAMP (Yang et al., 2011; Yang et al., 2001). We previously found that ISOF pretreatment could attenuate lipopolysaccharide (LPS) induced acute lung injury by downregulation of inflammatory responses and pro-inflammatory cytokines (Yang et al., 2011). We also reported that ISOF could downregulate inflammatory cytokine levels in LPS induced human mononuclear leukocytes through TLR4/MyD88/NF-κB signaling pathway (Du et al., 2019). However, the therapeutic effect and mechanisms of ISOF on COPD remain to be elucidated.

In this study, we evaluated the efficacy of ISOF on a COPD rat model established by long-term exposure to cigarette smoke using roflumilast as a positive control. The pulmonary function, lung morphology, inflammatory cells or cytokines in serum and bronchoalveolar lavage fluid (BALF) were detected. Then we analyzed and validated the potential mechanisms of ISOF by network pharmacology, transcriptome and proteomics. We further detected the expression of AC isoforms in normal lung tissues and investigated the AC activation and spasmolytic activity of ISOF. The results indicate that ISOF potently activates AC1 and AC2 in vitro, improves lung ventilation function and attenuates inflammation in COPD model rats, supporting that ISOF might be a potential drug to prevent and treat COPD.

Section snippets

Reagents and antibodies

ISOF (purity > 99.9%) was synthesized by Basilea Pharmaceutica (Jiangsu, China) according to the patent method of ISOF synthesis (ZL201610044817.8). Roflumilast (ROFL) was purchased from BioChemPartner (Shanghai, China). Vecuronium Bromide was from Zhejiang Xianju Pharmaceutical (Zhejiang, China). Cigarettes (Hongqi Canal® Filter tip cigarette, smoke of each cigarette contain 11 mg tar, 0.7 mg nicotine and 13 mg carbon monoxide) was obtained from Henan Tobacco Industry (Zhengzhou, China).

ISOF improved the pulmonary function in COPD model rats

CS exposure is the most appropriate approach to develop the characteristic features of COPD in animal models. In this study, we established a rat model of COPD by long-term nose-only exposure to cigarette smoke (1 cigarette each rat, once a day for 14 weeks). The lung function in conscious rats was detected once a week (Fig. 1). Compared to control group, the Te and Ti increased while PEF, PIF, EF50, and MV decreased in model rats from 11 to 14 weeks, indicating that the pulmonary function

Discussion

The development of novel treatments for COPD, other than bronchodilators, remains challenging (Jarnicki et al., 2016; Rabe and Watz, 2017). Roflumilast is the only selective PDE4 inhibitor that has been approved for the treatment of COPD in clinic, while the narrow therapeutic window and side effects have hampered its clinical application (Rabe and Watz, 2017; Wedzicha et al., 2016). Elevating intracellular cAMP level is considered a promising strategy to improve multiple lung diseases (

Conclusion

In summary, we report here for the first time that ISOF potently activates AC1 and AC2 in vitro, significantly downregulates the mTOR level and restores the Th17/Treg balance in COPD model rats, decreases the inflammatory cells and cytokines, and consequently ameliorates pathological damage of lung tissue and improves pulmonary function in COPD model rats. Taken together, our findings support ISOF as a promising candidate for the treatment of COPD.

Authors' contribution

Chuang Xiao: Conceptualization, Methodology, Validation, Formal analysis, Writing-original draft, Funding acquisition; Sha Cheng, Haochang Lin: Investigation, Methodology, Validation, Formal analysis, Writing-original draft; Zhiying Weng: Resources, Formal analysis, Funding acquisition; Peihua Peng, Deyou Zeng: Investigation, Methodology; Xiaohua Du: Investigation, Methodology, Resources; Xiujuan Zhang, Yaqing Yang, Yaping Liang, Rong Huang, Lueli Wang: Investigation, Formal analysis; Chen Chen:

Declaration of Competing Interest

The authors declare no conflicts of interest.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (No. 81870037, 81860012, 81560589, 81760869, 81402991, 82060656), National Key Research and Development Program of China (2017YFC1309302), Yunnan Provincial Science and Technology Department in China (No. 2017FA043, 2017IC041, 2018HC007, 202005AF150043, 2014BC012, 2014IA033, 202005AE160004, 2019FD020, 2019HB025), China Postdoctoral Science Foundation (No. 2019M663579), and partially supported by the Open Project of

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