(−)-Epicatechin ameliorates cigarette smoke-induced lung inflammation via inhibiting ROS/NLRP3 inflammasome pathway in rats with COPD

https://doi.org/10.1016/j.taap.2021.115674Get rights and content

Highlights

  • (−)-Epicatechin repress cigarette smoke extract induced oxidative stress by activating Nrf2.

  • (−)-Epicatechin promores TRIM25-dependent degradation of Keap1.

  • (−)-Epicatechin can improve cigarette smoking-induced lung inflammation in rats via inhibiting ROS/NLRP3 axis.

  • Nrf2 knockdown or inhibition abolished the protein effect of (−)-Epicatechin on lung tissue.

Abstract

Chronic obstructive pulmonary disease (COPD) with increased morbidity and mortality is a worldwide healthcare challenge closely associated with cigarette smoking (CS). Currently, there is no effective therapeutic strategy to control inflammation in COPD patients. The present study tested the protective effects of (−)-Epicatechin (EC), a type of flavonoid, on CS-induced COPD and the underlying mechanism. Also, EC repressed the production of reactive oxygen species (ROS) and improved human bronchial epithelial cell viability after cigarette smoke extract (CSE) treatment. Further studies demonstrated that EC promotes ubiquitin-mediated Keap1 degradation by upregulating tripartite motif-containing protein 25 (TRIM25) expression and enhances the nuclear localization of Nrf2 protein. Also, EC dramatically inhibits the activation of NLRP3 inflammasome and reduces the CSE-induced pyroptosis, as indicated by decreasing lactate dehydrogenase release and the number of caspase-1-positive cells. Importantly, Nrf2 knockdown reversed the protective effect of EC on human bronchial epithelial cells, at least partially. Consistent with the results in vitro, EC inhibits the activation of NLRP3 inflammasome and relieves the CS-induced lung inflammation, as evident from decreased interleukin (IL)-1β and IL-18 secretion in a COPD rat model. In conclusion, this study revealed the protective effect of EC on experimental COPD rats and elucidated the mechanism of EC promoting Nrf2 activity, which might provide a novel therapeutic strategy for COPD.

Graphical abstract

A schematic model depicts that (−)-Epicatechin attenuates lung inflammation via Nrf2 dependent inhibition of ROS-NLRP3 mediated pyroptosis in the rat COPD model.

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Introduction

Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder and the third cause of death globally (Lopez-Campos et al., 2016; Rabe and Watz, 2017). About 200 million people are estimated to suffer from COPD in 2020 (Negewo et al., 2015). Smoking or passive smoking significantly increases the risk of several diseases, including respiratory diseases (Borgerding and Klus, 2005). Tobacco smoke is a toxic and carcinogenic mixture of >5000 chemicals, which might be the main reason for smoking-related respiratory diseases in humans (Talhout et al., 2011). According to the World Health Organization (WHO) report on the global tobacco epidemic, 5.4 million premature deaths were attributed to tobacco smoking worldwide (Talhout et al., 2011). Although COPD also occurs among never-smokers, smoking is still the first causal and utmost risk factor for COPD (Quaderi and Hurst, 2018; Wang et al., 2018a). The increased oxidative stress caused by long-term cigarette smoking (CS) exposure contributes to inflammation in the lung, leading to the development of COPD (Domej et al., 2014; Zuo et al., 2014; Goncalves and Romeiro, 2019). Although glucocorticoids are commonly effective in suppressing inflammation of the respiratory system, they are less effective in controlling inflammation in patients with COPD (Adcock et al., 2012). Hence, it is necessary to develop new drugs with antioxidant and anti-inflammatory properties that also delay the progression of COPD.

Inflammasomes are multiprotein innate immune complexes assembled by intracellular pattern recognition receptors (PRRs), including the nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs, also known as NOD-like receptors) and absent in melanoma 2-like receptors (ALRs, also known as AIM2-like receptors; Martinon et al., 2002; Broz and Dixit, 2016; Rathinam and Fitzgerald, 2016). In 2002, Martinon et al. reported that NLR family pyrin domain-containing 1 (NLRP1) forms an inflammasome complex (Martinon et al., 2002). Since then, the other members of the NLR families have also been discovered, including NLR family pyrin domain-containing 3 (NLRP3; Rathinam and Fitzgerald, 2016). The inflammasomes are activated by microbial infection or endogenous danger signals, which regulate the maturation of the proteolytic enzyme caspase-1. Then, caspase-1 triggers cell pyroptosis by regulating proteolytic maturation and release of interleukin-1β and IL-18 (Guo et al., 2015). Accmulating evidence showed that pyroptosis mediated by inflammasome activation was involved in the initiation or progression of diseases, severely affecting human health (Szabo and Petrasek, 2015; Heneka, 2017; Liu et al., 2018a; Moossavi et al., 2018). Emerging data have also suggested that NLRP3 deficiency or inhibited inflammasome-dependent activation of caspase-1 or IL-1β alleviates inflammation in some inflammatory diseases (Satoh et al., 2015; Wu et al., 2018a; Dapaah-Siakwan et al., 2019). Recently, increasing clinical and experimental studies have shown that inflammasome activation induced by exogenous factors, such as smoke exposure, promotes inflammation and plays a key role in many chronic respiratory diseases, including COPD (Brusselle et al., 2014; Abderrazak et al., 2015; Hosseinian et al., 2015; Sayan and Mossman, 2016; Peng et al., 2018; Wang et al., 2018b). Since smoke exposure and the subsequent production of reactive oxygen species (ROS) is one of the upstream factors that trigger inflammation activation, suppressing intracellular oxidative stress levels might be a suitable alternative for COPD treatment.

Natural plants are used as an alternative medicinal resource for the treatment of several diseases (Rodrigues et al., 2016). (−)-Epicatechin (EC) is a natural flavonoid derived from various plants with blue, red, or purple pigments. Also, it has various benefits, including antioxidant, anti-inflammation, or anti-apoptotic properties (Rodrigues et al., 2016; Bernatova, 2018; Borges et al., 2018). Notably, the bioavailability of EC in rats is up to 34.2% after oral administration (Roura et al., 2005). Previous studies reported that EC protects the traumatic brain injury or intestine injury from radiation by repressing oxidative stress (Cheng et al., 2016; Li et al., 2019). Moreover, EC alleviates inflammation in lipopolysaccharide (LPS)-induced acute lung injury or renal inflammation (Prince et al., 2017; Xing et al., 2019). However, whether EC treatment can efficiently delay the progression of COPD is yet to be elucidated. In addition, the mechanism of antioxidant or anti-inflammation effects of EC is yet unclear.

In the present study, we aimed to investigate the antioxidant and anti-inflammatory effects of EC on CS-induced lung inflammation and dissect the therapeutic mechanisms underlying EC in COPD rats. We found that EC represses oxidative stress and NLRP3 activation induced by CSE or CS in vitro and in vivo. Also, for the first time, this study demonstrated that EC promotes ubiquitin-mediated degradation of keap1 by upregulating TRIM25 expression, which enhances Nrf2 stability.

Section snippets

Animal model administration and treatment

Wistar male rats (250–300 g, 12–13 weeks) were fed at the Laboratory Animal Centre of Shanghai General Hospital and maintained at 25 ± 2 °C and relative humidity of 55 ± 10% in a controlled room with a 12 h light/dark cycle daily. All animal experiments were conducted in accordance with the guidelines of the Chinese Council on Animal Care and approved by the Ethics Committee of Shanghai General Hospital (Animal ethical clearance number: 2019AW037). The rats were placed in a self-produced

EC represses oxidative stress induced by CSE and improves BEAS-2B cell viability

First, we detected the effect of different concentrations of CSE (from 3% to 20%) on the viability of BEAS-2B cells and identified the optimal concentration using CCK-8 assay based on previous studies (Peng et al., 2018; Wu et al., 2014b; Zhou et al., 2019). As shown in Fig. 1B, the viability of BEAS-2B cells was decreased after treatment with CSE for 24 h in a dose-dependent manner. The viability of BEAS-2B cells in 5% CSE group (53 ± 2.1%) was significantly decreased compared to the control

Discussion

CS has been considered as a primary risk factor for COPD (Lopez-Campos et al., 2016). Oxidative stress is increased in patients with COPD due to chronic exposure to CS (Kirkham and Barnes, 2013; Choudhury and MacNee, 2017). Oxidant-induced alveolar epithelial cell injury is a critical event that may account for lung damage caused by CS exposure (Negewo et al., 2015; Domej et al., 2014). A previous study showed that exposure to CSE induced human type II alveolar epithelial A549 cell damage (

Conclusion

Together, EC inhibits NLRP3-mediated inflammation via repressed oxidant stress, which depends on enhancing TRIM25-mediated Keap1 degradation. The study also indicated that EC might be a potential compound for COPD treatment.

Declaration of Competing Interest

The authors have no conflict of interest to declare.

Acknowledgements

This work was financially sponsored by grant from the National Natural Science Foundation of China (Grant No. 81803891; Grant No. 81873402).

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