Scutellarin inhibits ferroptosis by promoting cellular antioxidant capacity through regulating Nrf2 signaling

Abstract

Ferroptosis is a lytic form of regulated cell death that is driven by iron-dependent lipid peroxidation and has been implicated in various diseases, including acute kidney injury (AKI). Scutellarin is a flavonoid isolated from Erigeron breviscapus (Vant.) Hand.-Mazz. and possesses various pharmacological activities, including anti-inflammatory and antioxidative properties. However, it is unclear whether scutellarin can inhibit ferroptosis and mitigate related diseases. In this study, we show that scutellarin can inhibit ferroptosis in both human HK-2 cells and mouse bone marrow-derived macrophages stimulated with RSL3 or erastin. Mitochondrial dysfunction and reactive oxygen species generation are counteracted by scutellarin treatment, suggesting the involvement of its antioxidative activity. Furthermore, scutellarin increases the nuclear levels of Nrf2 and the expressions of its target genes, including HO-1 and GPX4. Scutellarin-mediated inhibition of ferroptosis and increases in these proteins are abrogated by co-treatment with brusatol, an Nrf2 inhibitor, indicating an essential role for Nrf2 in this process. In a mouse model of folic acid-induced AKI, scutellarin mitigates acute renal damage, as revealed by histopathological analysis and serum blood urea nitrogen and creatinine assays. Folic acid-induced acute renal injury is associated with increased ferroptosis, as revealed by elevated level of 4-hydroxynonenal (4-HNE), a surrogate marker of ferroptosis, which is diminished by scutellarin co-treatment. Specifically, the elevated 4-HNE levels in macrophages (MAC-2 positive) and other renal cells are suppressed by scutellarin. Overall, scutellarin can inhibit ferroptosis both in cultured cells and in a mouse model of AKI by regulating Nrf2 signaling.

1. Introduction

Ferroptosis is a nonapoptotic form of regulated cell death that is driven by iron-dependent lipid peroxidation [1–4]. The (phosphor)lipid peroxide-reducing enzyme glutathione peroxidase 4 (GPX4) is a central regulator of ferroptosis and prevents cell death by catalyzing the conversion of lipid peroxides into non-toxic lipid alcohols in the presence of sufficient glutathione [2,5,6]. The cystine/glutamate antiporter (Xc– also known as xCT) system is essential for maintaining the level of intracellular glutathione [6]. In addition to the system Xc–/GPX4 cellular antioxidant axis, ferroptosis suppressor protein 1 (FSP1) is the second mainstay of ferroptosis-inhibiting antioxidant enzyme. Mechanistically, FSP1 is recruited to the plasma membrane by myristoylation and acts as an oxidoreductase to reduce ubiquinone (also known as coenzyme Q10) into its reduced form ubiquinol, which functions as a lipophilic radical trapping antioxidant to block lipid peroxides [7,8]. In addition to mitochondrial GPX4, dihydroorotate dehydrogenase (DHODH) has been found to attenuate ferroptosis in mitochondria by reducing ubiquinone to ubiquinol [9]. Ferroptosis is also regulated by small antioxidative molecules such as α-tocopherol (vitamin E), which act as lipophilic radical-trapping antioxidants to terminate the propagation of lipid peroxides [3,5]. Moreover, two recent reports showed that 7-hydrocholesterol is an endogenous suppressor of ferroptosis that acts as an antioxidant to protect against lipid peroxidation, thereby regulating the sensitivity of cells to ferroptosis [10,11]. Notably, several important antioxidant proteins, such as GPX4 and heme oxygenase-1 (HO-1), are encoded by target genes of nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a critical role of Nrf2 in modulating ferroptosis [12,13]. Thus, the antioxidant systems of a cell play critical roles in protecting against ferroptosis.

Ferroptosis, an oxidative form of cell death, can be induced by the disruption of cellular antioxidant systems through the inhibition of GPX4 activity by genetic deletion of this gene or by RSL3-induced GPX4 inactivation [5]. This form of cell death can also be triggered by depletion of glutathione through inhibition of system Xc-,, thereby indirectly decreasing GPX4 activity [1,5]. Although many studies have adopted such biochemical methods to induce ferroptosis, the role of ferroptosis in disease pathogenesis and its therapeutic modulation remain areas of active investigation.