Rosiglitazone Prevents Autophagy by Regulating Nrf2-Antioxidant Response Element in a Rat Model of Lithium-pilocarpine-induced Status Epilepticus

Highlights

• Autophagy and oxidative stress are activated in SE rats.

• Rosiglitazone exerts neuroprotection in SE-induced brain injury.

• Rosiglitazone regulates autophagy and oxidative stress in SE rats.

• Rosiglitazone prevents autophagy via increasing antioxidant factor Nrf2, which is demonstrated by siNrf2 transfection in SE rats.

Abstract

Status epilepticus (SE) leads to irreversible neuronal damage and consists of a complex pathogenesis that involves oxidative stress and subsequent autophagy. Rosiglitazone has recently been considered as a potential neuroprotective factor in epilepsy because of its antioxidative function. The aim of this study was to assess the effects of rosiglitazone in SE rat models and investigate whether its mechanisms of action involve autophagy via the antioxidant factor, nuclear factor erythroid 2-related factor 2 (Nrf2). The male Sprague-Dawley rats (200–220 g) were used to establish lithium-pilocarpine-induced SE model. We found that rosiglitazone markedly improved neuronal survival at 24-h post-SE as indicated via Hematoxylin-Eosin and Nissl staining. Furthermore, along with a reduction in reactive oxygen species, rosiglitazone pretreatment enhanced the antioxidative activity of superoxide dismutase and the expression level of Nrf2, as detected via chemical assay kits and Western blotting, respectively. In addition, the microtubule-associated protein light chain 3II (LC3II)/LC3I ratio was increased and peaked at 24 h after SE, whereas p62 mRNA levels were sharply elevated at 72 h after SE, both SE-induced increases of which were reversed via rosiglitazone pretreatment. To further test our hypothesis of the key role of Nrf2 in this process, small-interfering RNA for Nrf2 (siNrf2) was then transfected into SE rats to knockdown Nrf2 expression. We found that siNrf2 partially blocked the above effects of rosiglitazone on autophagy-related proteins in SE rats. Taken together, our findings suggest that rosiglitazone attenuates oxidative-stress-induced autophagy via increasing Nrf2 in SE rats and may be used as a promising therapeutic strategy for SE treatment.

Introduction

Epilepsy is one of the most common central-nervous-system diseases and adversely impacts affected patients due to long-term recurrent seizures. The complex pathogenesis of epilepsy converges upon oxidative stress injury (Pauletti et al., 2019), glutamate excitotoxicity (Wasterlain and Chen, 2008), calcium overload (Xu and Tang, 2018), and genetic as well as immune abnormalities (Granata et al., 2011; Nabbout, 2012). Status epilepticus (SE) is defined as an abnormally prolonged seizure that lasts longer than five minutes or two or more seizures in which the person does not regain full consciousness in the interim between seizures, according to the International League Against Epilepsy (ILAE) in 2015 (Trinka et al., 2015). Since SE can result in irreversible neuronal damage that can induce significant disabilities and even mortality, there is an urgent need to further elucidate the complex pathogenesis of SE and develop more effective treatments.

Many previous studies have investigated the relationship between SE and oxidative stress (Pearson-Smith et al., 2017, Abdel-Salam et al., 2020) and demonstrated that they interact and further lead to neuronal damage in the brains of SE patients. Epileptic seizures can aberrantly increase oxidative stress (Puttachary et al., 2015), which can lead to excitotoxicity and neuronal death via various mechanisms involving impairment of membrane-bound proteins. Oxidative stress is characterized as the overproduction of free radicals. Increased reactive oxygen species (ROS) impair cytoarchitecture (Kovac et al., 2012) and are a contributing factor in seizure-induced neuronal damage (Williams et al., 2015). Therefore, inhibition of oxidative stress may be a critical strategy for the treatment of SE. As a consequence, various endogenous antioxidant defense enzymes, such as superoxide dismutase (SOD), function to partially counteract SE-induced increases in ROS (Si et al., 2016). Neurons also express a series of transcription factors that comprise an antioxidant defense to provide protection from oxidative injury. Nuclear factor erythroid 2-related factor 2 (Nrf2), one of the CNC (cap-'n-'cohar) leucine zipper transcription factor family members, critically activates cell-defense responses to provide self-protection during oxidative stress damage in vivo. As an antioxidant factor, Nrf2 expression is increased in epilepsy models and ameliorates cognitive impairment and neuronal injury following epileptic seizure (Wang et al., 2014, Shao et al., 2017).

In recent years, many studies have determined a strong association between oxidative stress and autophagy. Specifically, oxidative stress induces autophagy (Chen et al., 2008) and ROS affect autophagic activity (Azad et al., 2009). Furthermore, several studies have shown that SE induces autophagic activation (Wang et al., 2017a, Wang et al., 2017b, Rami and Benz, 2018). Microtubule-associated protein light chain 3 (LC3), expressed on autophagic membranes, is coupled with p62. Moreover, both LC3 and p62 represent indicators for evaluating autophagic activity. Although the underlying mechanisms are not fully understood, regulation of autophagic activity may mitigate SE-induced neuronal loss and further augment any anti-convulsive effects. Therefore, antioxidant compounds may serve as promising treatments for regulating autophagy in SE.

Rosiglitazone, a member of thiazolidinediones (TZDs), is a potent exogenous agonist of peroxisome proliferator-activated receptor gamma (PPARγ). In the past several years, PPARγ agonists have been shown to exert neuroprotective antiepileptic properties that include anti-inflammatory and anti-oxidative-stress components (Sun et al., 2012, San et al., 2015). However, whether rosiglitazone has an effect on autophagy via anti-oxidative-stress pathways in SE has remained unclear. Hence, the objective of the present study was to assess the effects of rosiglitazone in SE rat models and investigate whether its mechanisms of action involve regulation of autophagy via Nrf2. Collectively, our findings may reveal novel targets for future treatments and may provide a theoretical basis for the development of PPARγ agonists in the treatment of SE.