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.