3.10 Nrf2 deficiency prohibits the regulatory effect of leonurine on ferroptosis
We further investigated whether the ability of leonurine to inhibit ferroptosis, as well as its cytoprotective potential, was dependent on Nrf2. In Nrf2 KO mice, cisplatin significantly increased the Fe and MDA levels and decreased the GSH levels compared to those in the wild-type mice (Fig. 10A-C). Moreover, treatment with leonurine markedly reversed the above changes in wild-type mice, while this effect was almost abolished in the Nrf2 KO mice. Moreover, the alleviating effects of leonurine on FTH-1 and TFR were eliminated (Fig. 10D-E). The protective effects of leonurine on the cisplatin-induced expression of the ferroptosis-related proteins GPX4 and xCT were also abolished in Nrf2 KO mice (Fig. 10D-F). DAB staining indicated that the reduced iron deposition in the leonurine treatment group was restored by the KO of Nrf2 (Fig. 10G). Furthermore, electron microscopy analysis revealed that the mitochondrial damage mitigated by leonurine was reversed in Nrf2 KO mice (Fig. 10H).
Discussion and conclusions
Ferroptosis and its related components are now better understood, and Nrf2 has been shown to play a key role in mediating this process. In particular, the antioxidant, iron, and intermediate metabolic statuses of cells are mediated by Nrf2 target genes. While the mechanisms of Nrf2 and ferroptosis in cisplatin-induced AKI have been elucidated, the relationship between them remains unclear(Hu et al., 2020; Ikeda et al., 2021; Shelton et al., 2013). In this study, we aimed to determine whether the nuclear transcription factor Nrf2, a critical regulator of the cellular antioxidant response, could inhibit cisplatin-induced ferroptosis in subjects with kidney injury. Moreover, we evaluated whether leonurine, an Nrf2 activator, could alleviate cisplatin-induced kidney injury by inhibiting Nrf2-mediated lipid peroxidation and ferroptosis.
Iron has been reported to play a key role in cisplatin-induced nephrotoxicity both in vitro and in vivo (Baliga et al., 1998). The abnormal accumulation of iron produces large amounts of free radicals that damage DNA, proteins, and other biomolecules (Stoyanovsky et al., 2019). Nrf2 is a key factor regulating the cellular antioxidant response, as it controls the expression of HO-1, NQO1 and other genes related to antioxidant stress. In addition to its important role in maintaining cellular redox balance, Nrf2 helps to mediate lipid peroxidation and ferroptosis (Abdalkader et al., 2018). The injection of cisplatin significantly increased lipid peroxidation and ROS production, which were reversed by the activation of Nrf2 or its downstream target genes. Notably, increased lipid oxidation and downstream Nrf2 target inactivation significantly enhance the overall protein lipid oxidation and ferroptosis in disease environments with low Nrf2, further promoting disease progression (La Rosa et al., 2021). However, whether Nrf2 levels are directly related to ferroptosis sensitivity in cisplatin-induced AKI has not been clarified. To elucidate the effect of Nrf2 on ferroptosis sensitivity in CI-AKI, we analyzed the iron accumulation, lipid peroxidation and expression of ferroptosis-related proteins in cisplatin-treated wild-type and Nrf2 KO mice. Both the wild-type and Nrf2 KO mice exhibited altered iron accumulation, upregulated MDA levels, and downregulated protein levels of GPX4 and xCT, which are biomarkers of ferroptosis. However, the abovementioned biomarkers were altered more significantly in Nrf2 KO mice than in the wild-type mice (Figs. 1-2). Therefore, we propose for the first time that the inhibition of Nrf2 aggravates ferroptosis and further enhances the progression of CI-AKI.
Considering the mitigated effect of Nrf2 on ferroptosis, targeting the upstream regulators of the ferroptotic cascade, including dysregulated iron levels and ROS production, by pharmacologically modulating the Nrf2 signaling pathway remains one of the best strategies for treating ferroptosis-related pathologies. We found that leonurine, an Nrf2 activator, significantly increased the cell viability and decreased the iron accumulation, ROS and cellular lipid ROS induced by erastin and RSL3 (Fig. 3). Iron is an essential element that is regulated by a variety of proteins. In general, iron is loaded onto transferrin, which binds to transferrin receptor 1 (TFR) on the cytoplasmic membrane and delivers iron to numerous tissues via endocytosis (Torti and Torti, 2013). Excess iron is stored in the protein ferritin, which is composed of 24 subunits of FTH-1 and FTL. Here, leonurine significantly reduced the protein expression levels of TFR, FTH-1 and FTL induced by RSL3 (Fig. 3). Next, we measured the levels of several other well-established biomarkers of ferroptosis, including GSH and lipid peroxidase-derived MDA, as well as those of GPX4 and xCT. As GSH depletion can trigger ferroptosis and MDA is the end-product of ferroptosis, we herein showed that leonurine inhibited the GSH depletion induced by RSL3 and the upregulation of MDA. Importantly, xCT and its key component Slc7a11 are responsible for the generation of intracellular GSH in response to oxidative stress. Inhibition of xCT reduces the level of GSH and the activity of glutathione peroxidase 4 (GPX4), thereby increasing lipid peroxidation. GPX4 deficiency is considered to be a biomarker of ferroptosis, and the depletion of GPX4 induces the ferroptosis of numerous renal tubular epithelial cells (Friedmann Angeli et al., 2014) (Seibt et al., 2019). In the present study, the downregulation of xCT and GPX4 induced by RSL3 was reversed by leonurine treatment in a dose-dependent manner (Fig. 4). Based on the ability of Nrf2 to prevent lipid peroxidation and ferroptosis, we hypothesized that the inhibitory effect of leonurine on ferroptosis is mediated by Nrf2. We further showed that the siRNA-mediated knockdown of Nrf2 ameliorated the effects of leonurine on the ROS production, lipid peroxidation, cellular iron level and expression of ferroptosis-related proteins induced by RSL3 (Fig. 5). In summary, our data showed that leonurine significantly activated Nrf2 and inhibited the ferroptosis of HK-2 cells induced by RSL3. The above effects were not observed after Nrf2 knockdown, which indicates that the protective effect of leonurine on ferroptosis in vitro may be mediated via the Nrf2 pathway.
As reported, leonurine is a therapeutic candidate for LPS-induced AKI and renal fibrosis (Cheng et al., 2015; Xu et al., 2014). However, the potential effects of leonurine on cisplatin-induced AKI have not yet been elucidated. Our results showed that leonurine significantly decreased the serum levels of Bun and SCr induced by cisplatin as well as the levels of KIM1 and NGAL and the histological extent of kidney injury (Fig. 6). Considering the abovementioned inhibitory effect of leonurine on ferroptosis induced by RSL3 in vitro, we further examined its effects on related ferroptosis biomarkers in the kidney. Consistent with the in vitro results, leonurine significantly inhibited the cisplatin-induced increases in iron accumulation and the TFR, FTL and FTH-1 levels in the kidney (Fig. 7). Furthermore, leonurine inhibited morphological and biochemical changes in factors related to ferroptosis, such as MDA, SOD and GSH depletion and the downregulation of GPX4 and xCT in cisplatin-induced AKI. In addition, leonurine markedly upregulated the expression of Nrf2, HO-1 and NQO1 (Fig. 8). Given that Nrf2 is a principal regulator of antioxidant responses that suppresses ferroptosis, we further investigated the potential for Nrf2 deficiency to ameliorate the protective effects of leonurine against cisplatin-induced ferroptosis. We treated Nrf2 KO mice with leonurine and then observed the histological and molecular parameters of cisplatin-induced AKI. The treatment of Nrf2 KO mice with leonurine did not rescue cisplatin-induced renal damage, as determined by the serum levels of Bun and Cre, the levels of KIM1 and NGAL, and the histological extent of kidney injury (Fig. 9). Furthermore, the treatment of Nrf2 KO mice with leonurine did not inhibit ferroptosis-related morphological or biochemical changes in cisplatin-induced AKI, as reflected by the MDA levels, SOD and GSH depletion, and downregulation of GPX4 and xCT (Fig. 10). Taken together, our results support that leonurine provides renal protection predominantly by activating the Nrf2-mediated inhibition of ferroptosis.
In conclusion, our study revealed the substantial significance of upregulating Nrf2 to prevent ferroptosis, thereby alleviating CI-AKI. This conclusion is derived from three key findings. First, Nrf2 KO mice were more susceptible to cisplatin-induced renal injury and ferroptosis. Second, the protective effect of leonurine against cisplatin-induced AKI was achieved by activating the antioxidant signaling molecule Nrf2 and inhibiting ferroptosis-related morphological and biochemical changes. Finally, the treatment of Nrf2 KO mice and Nrf2 siRNA HK-2 cells with leonurine nearly failed to rescue cisplatin- and RSL3-induced ferroptosis and renal injury in vivo and in vitro. The present study furthers our understanding of the mechanism by which Nrf2 inhibits ferroptosis and provides a strategy for investigating the antiferroptotic activity of Nrf2 activators in the context of cisplatin-induced AKI.