Fer-1

Inhibition of ferroptosis by up-regulating Nrf2 delayed the progression of diabetic nephropathy

Shuangwen Li a, Lisi Zheng a, Jun Zhang a, Xuejun Liu b,**, Zhongming Wu a,*

a NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital & Tianjin Institute of
Endocrinology, Tianjin Medical University, Tianjin, 300134, China
b Department of Neurology, Chu Hsien-I Memorial Hospital, Tianjin Medical University, Tianjin, 300134, China

Abstract

Diabetic nephropathy (DN) is now considered the leading cause of end-stage renal disease. In diabetes, the accumulation of reactive oxygen species (ROS) and iron overload are important determinants that promote the occurrence of DN. However, the underlying mechanism of how they cause diabetic kidney damage remains unclear. Ferroptosis, characterized by iron-dependent lipid peroxidation, provided us with a new idea to explore the progression of DN. Iron overload, reduced antioxidant capability, massive ROS and lipid peroxidation were detected in the kidneys of streptozotocin-induced DBA/2J diabetic mice and high-glucose cultured human renal proximal tubular (HK-2) cells, which were the symbolic changes of ferroptosis. Furthermore, the characteristic mitochondrial morphological changes of ferroptosis were observed in high glucose cultured cells. Additional treatment of Ferrostatin-1 (Fer-1) in DN models significantly rescued these changes and alleviated the renal pathological injuries in diabetic mice. Besides, the decreased NFE2-related factor 2 (Nrf2) was observed in DN models. The specific knockdown of Nrf2 increased the sensitivity of cells to ferroptosis in the high glucose condition. In Nrf2 knockdown cells, up-regulating Nrf2 by treating with fenofibrate improved the situation of ferroptosis, which was verified in RSL-3 induced cells. Moreover, the ferroptosis-related changes were inhibited by increasing Nrf2 in fenofibrate treated diabetic mice, which delayed the progression of DN. Collectively, we demonstrated that ferroptosis was involved in the development of DN, and up-regulating Nrf2 by treating with fenofibrate inhibited diabetes-related ferroptosis, delaying the progression of DN. Our research revealed the development mechanism of DN from a new perspective, and provide a new approach delaying the progression of DN.

1. Introduction

DN is a major complication of diabetes and has become the leading cause of most end-stage renal diseases, delaying its progression still a worldwide problem [1]. Iron homeostasis is essential for the normal function of the renal cells [2]. Iron functions as a cofactor for vital iron-containing enzymes, involving ATP production, DNA and heme synthesis and many other physiological activities [3]. However, the deposition of iron in the cells causes tissue dysfunction, which detected in the proximal tubular lysosomes of DN patients and diabetic mice [4–7]. The research showed that a low-iron diet or iron chelators could delay the progress of DN in diabetic rats [8]. It is well-known that excessive iron is destructive because iron pool of cells can produce ROS via Fenton reaction, which may be more pronounced in diabetes-induced highly redox-active mitochondrion [9]. However, the precise mechanism of excessive iron on accelerating the development of DN remains to be elucidated. It is noted that iron overload can cause a specific cell death termed as ferroptosis [10], which provides us a new angle to explore the progression of DN.
Ferroptosis is a newly discovered regulated cell death caused by iron- dependent lipid peroxidation, and it is genetically, biochemically and morphologically different from other regulatory cell deaths, including apoptosis, necroptosis and pyroptosis [11]. The characteristic changes of ferroptosis, such as the accumulation of redox-active iron, the loss of antioxidant capacity and the peroxidation of phospholipid-containing polyunsaturated fatty acids, can be used to evaluate the degree of fer- roptosis [12]. In the case of ferroptosis, the low expression of FTH-1 and overexpression of TFR-1 lead to excessive accumulation of ferrous iron and then promote the production of massive ROS through the Fenton reaction, which is considered to be one of the crucial mechanisms of iron biotoxicity [13]. Simultaneously, during ferroptosis, the disrupted function of System Xc-depletes the production of sufficient GSH, which prevents GPX4 from exerting its normal antioxidant capacity [14,15]. Therefore, the cell lipid membranes with phospholipids are extremely vulnerable to the attack of ROS. The end products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), easily form adducts with proteins and DNA, leading to significant cytotoxic effects and inducing cell ferroptosis [16].

We have made it clear that ROS occupies a central position in the entire ferroptosis process [17]. Moreover, in diabetes, a variety of pathways that generate ROS have been identified as potentially major factors in the pathogenesis of diabetic kidney disease [18]. Although researchers have tried to use antioxidants (vitamin C or E) for diabetic patients, expecting to delay the progression of DN from the aspect of anti-oxidative stress, the results demonstrated minimal renoprotection in humans [19]. It is likely that the current method of simply resisting oxidative stress may not be an ideal strategy for treating human DN. Herein, considering the closely relationship between ferroptosis and ROS, we speculated that controlling ferroptosis might be an effective way to improve kidney injurers in diabetes.

Nrf2 regulates many vital genes related to iron storage and transport at the transcriptional level [20]. The knockdown of Nrf2 could lead to an increase in iron content in the spleen and liver of mice [21]. At the same time, Nrf2 maintains the balance of cellular redox homeostasis and oxidative medium by regulating the expression of a series of signaling proteins and enzymes, playing an important regulatory role in anti-oxidative stress [22]. Therefore, we proposed that it was possible to suppress diabetes-related ferroptosis by up-regulating Nrf2.

Fenofibrate is widely used in the treatment of hypertriglyceridemia for its effect on improving blood lipids [23]. Many studies have shown that fenofibrate could improve diabetes complications, but this effect does not depend on its ability to reduce lipids [24]. The researches have proposed that fenofibrate inhibited the progression of diabetic compli- cations by protecting cells from oxidative stress, but there were still controversies about its exact mechanism [25]. Given the central role of Nrf2 in regulating oxidative stress, we investigated whether fenofibrate could delay the development of DN by regulating Nrf2.

In this study, we firstly explored the association between the devel- opment of DN and ferroptosis, revealing a new perspective on the developmental mechanism of DN. We also investigated whether the diabetes-related ferroptosis could be inhibited via up-regulating the expression of Nrf2 by treating with fenofibrate, thereby delaying the progression of DN. This research is expected to provide a new way for the control and treatment of DN.

2. Material and methods

2.1. Animal model and experimental groups

We induced diabetes models according to the protocols of Animal Models of Diabetic Complications Consortium. Eight-week-old male DBA/2J mice (n = 36, HFK Bioscience, Beijing, China), weighing 22–26 g were induced to diabetes by intraperitoneal injection of freshly prepared streptozotocin (dissolved in 0.1 M citrate buffer, pH 4.5; Sigma- Aldrich, St Louis, MO, USA) at 40 mg/kg for 5 consecutive days, while normal control animals received the same volume of citrate buffer. We selected DBA/2J mice in this study because this strain has been shown to be more susceptible to hyperglycemia-induced renal injury compared with other strains in the streptozotocin-induced diabetic mice. Blood glucose levels were assayed from tail vein blood. After the last injection of streptozotocin for 1 week, only mice with random blood glucose concentration >16.7 mmol/L (300 mg/dL) for 3 days were further used in the study. All mice acclimatization for one week and maintained in polycarbonate cages at 22–24 ◦C with a 12/12 h light-dark cycle and
10% humidity with ad libitum access to diet. The mice were randomly divided into six groups (n = 6): normal mice group (NC); diabetic mice group (DM); Fer-1 (Selleck, Houston, TX, USA) intraperitoneal injected diabetic mice group (Fer-1); 1% dimethyl sulfoxide (DMSO) intraperi- toneal injected diabetic mice group (vehicle-P); fenofibrate (Sigma- Aldrich, St Louis, MO, USA) intragastric administrated diabetic mice group (Fn); 0.5% sodium carboxymethyl cellulose (Na-CMC; Solarbio, Beijing, China) intragastric administrated diabetic mice group (vehicle- G). During the duration of 12-week treatment, the diabetic mice in the Fer-1 and vehicle-P groups received intraperitoneal injections of Fer-1 (2.5 μmol/kg, dissolved in 1% DMSO) and 1% DMSO every day, respectively. And the diabetic mice in the Fn and vehicle-G groups received intragastric treatment of fenofibrate (100 mg/kg, dissolved in 0.5% Na-CMC) and 0.5% Na-CMC every day, respectively. Mice blood glucose levels and body weight were monitored at week 0, 2, 4, 8 and 12. Urine specimens were collected at week 0 and 12. Kidney tissues were collected before the time of euthanization and preserved for examination.All procedures were conducted following the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, as well as the Animal Welfare Act guidelines. Experimental protocols were approved by the ethical committee of Tianjin Medical University.

2.2. Urine albumin and creatinine assay

Mice were individually housed in metabolic cages for 24 h, and urine samples were collected and urine output was recorded at week 0 and 12. Urine samples were centrifuged for 20 min to collect the supernatant for detecting the concentrations of urinary albumin and urinary creatinine by using the Mouse uALb ELISA Kit (Fankewei, Shanghai, China) and Mouse Cr ELISA Kit (Fankewei, Shanghai, China), respectively. Subse- quently, the 24-h urinary albumin and urinary albumin/creatinine ratio (ACR) were calculated.

2.3. Histologic evaluation

Anesthetized mice were perfused with 0.01 mol/L phosphate- buffered saline for 10 min, then the kidneys were collected, fixed in 4% paraformaldehyde, paraffin-embedded and cut into 4 μm thick slices. The sections were deparaffinized with xylene and then rehydrated with ethanol. After staining, sections were sealed with neutral balsam and observed under an optical microscope. The extent of renal injury was analyzed by Image-Pro Plus 6.0 software of the glomerular hyper- trophy, tubular damage, mesangial matrix expansion and interstitial fibrosis. Slices were stained with hematoxylin and eosin (H&E) for the scoring of glomerular hypertrophy, tubular epithelial disruption and tubular dilation. Glomerular size was detected to illustrate the degree of glomerular hypertrophy. Twenty stained glomeruli from per section (3 mice per group) were chosen randomly for analysis. The damaged de- gree of tubules was evaluated for their widened lumen, atrophy and thickened basement membranes. Periodic acid-Schiff’s (PAS) staining illustrated the mesangial matrix expansion. Mesangial matrix expansion was semi-quantitatively evaluated by measuring the relative number of pixels (pink or red area) divided by the total area of glomerulus. Twenty stained glomeruli from per section (3 mice/group) were chosen randomly for analysis. Masson staining evaluated the degree of renal fibrosis by measuring the relative number of pixels (blue).

2.4. Immunohistochemical and immunofluorescent staining

The sections were washed 3 times with phosphate buffer saline and blocked in 10% goat serum for 30 min at room temperature after deparaffinization, rehydration and heat-induced epitope retrieval. Immunohistochemical staining was performed by incubating primary antibodies for GPX4 (ab125066; Abcam, Cambridge, MA, USA), FTH-1 (DF6278; Affinity, Cincinnati, OH, USA), TFR-1 (ab214039; Abcam), and SLC7A11 (ab175186; Abcam) overnight at 4 ◦C. Subsequently, an HRP-DAB system (Proteintech, Wuhan, China) was used to detect the immunoactivity, followed by counterstaining with hematoxylin. Goat anti-rabbit immunoglobulin G (IgG; Proteintech, Wuhan, China) was used as the negative control. The sections were imaged under a light microscope. Twenty images under brightfield were randomly taken for per section in a blinded fashion (3 mice per group). Images quantified by Image-Pro Plus 6.0 analysis software.Immunofluorescence analysis of Nrf2 in the renal tissue was per- formed using the primary antibodies Nrf2 (YT3189; Immunoway, Plano, TX, USA) and the secondary antibodies goat anti-rabbit IgG were applied for 1 h and counterstained with 4,6-diamidino-2-phenylindole (DAPI; Solarbio, Beijing, China) at the room temperature. After being washed 3 times with PBS, sections were mounted with an anti-fading mounting medium and covered with coverslips and analyzed under a fluorescence microscope (Olympus, Tokyo, Japan). Twenty images were randomly taken for each section in a blind fashion (3 mice per group).

2.5. Cell culture

HK-2 human kidney proximal tubular cells (American Type Culture Collection, Rockville, MD) in Ctrl group were cultured in Dulbecco’s Modified Eagle Media (DMEM; Gibco, Carlsbad, CA, USA) containing 5.5 mmol/L glucose, 10% fetal bovine serum (FBS; Biological Industries, Cromwell, CT, USA), 100 U/mL penicillin and streptomycin (Solarbio, Beijing, China) at 37 ◦C, 95% humidity, and 5% CO2. Cells in HG group were cultured in DMEM containing 30 mmol/L glucose, 10% FBS and 100 U/mL penicillin and streptomycin. Cells in Mannitol group were cultured in DMEM with 24.5 mmol/L mannitol, 5.5 mmol/L glucose, 10% FBS and 100 U/mL penicillin and streptomycin. Mannitol group was used for controlling cell osmolality. Cells for Fer-1 group, Fn group and DMSO group were cultured in 30 mmol/L glucose medium con- taining 1 μM ferrostatin-1, 25 μM fenofibrate and 0.1% DMSO for 48 h, respectively. Cells for Si-Nrf2 + Fer-1 group and Si-Nrf2 + DMSO group were treated with Nrf2 transfections and cultured in 30 mmol/L glucose medium containing 1 μM ferrostatin-1 and 0.1% DMSO for 48 h, respectively. And cells in Si-Nrf2 + Fn group and Si-Nrf2 + DMSO group were treated with Nrf2 transfections and cultured in 5.5 mmol/L glucose medium containing 25 μM fenofibrate and 0.1% DMSO for 48 h. Cells for RSL-3 group, RSL-3 + Fn group and DMSO group were cultured in 5.5 mmol/L glucose medium containing 0.1 μM RSL-3, 0.1 μM RSL-3 plus 25 μM fenofibrate and 0.1% DMSO for 24 h respectively.

2.6. Transmission electron microscopy of cells

HK-2 cells pellets were fixed with 2.5% glutaraldehyde (Alfa Aesar, Ward Hill, MA, USA) in 0.1 M phosphate buffer saline (pH 7.4) for 3 h at 4 ◦C. Postfixed in 1% aqueous osmium tetraoxide 0.1 M phosphate buffer saline (pH 7.4) for 2 h at room temperature, dehydrated in gradual ethanol (50%–100%), embedded in epoxy resin monomer and cured for 48 h at 60 ◦C. Ultrathin sections of 50 nm were cut, stained with uranyl acetate and lead citrate. We prepared two samples for each group, five fields were randomly selected for each sample and examined by transmission electron microscopy (HT7700-SS; HITACHI, Tokyo, Japan).

2.7. Cell viability assay

Cell viability was measured using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Cells were seeded in 96-well plates at a density of 3000 cells per well and exposed to various concentrations of the compounds for indicating times. 10 μL of work reagent was added to each well and incubated for 2 h at 37 ◦C. The absorbance was measured on a microplate reader (Synergy HT, Bio-Tek, United States) at 450 nm. The optical density is proportional to the number of living cells in the plate.

2.8. Iron and MDA assay

10 mg kidney tissues for each group were cut and washed with cold saline. Tissues were homogenized by vibrating homogenizer with cold saline immediately. In addition, the collected cells were homogenized by the ultrasonic cell disrupter. The supernatant was collected after centrifugation for 10 min to measure the iron and MDA concentrations of tissues and cells. The iron and MDA concentrations were determined by the Iron Assay Kit (BioAssay, Hayward, CA, USA) and Micro Malondialdehyde Assay Kit (Solarbio, Beijing, China), respectively. Enough working reagents was prepared according to the manufacturer’s instructions, then transferred the working reagents and collected supernatants into 96-well plate. The optical density of iron assay kit was detected at the wavelength of 590 nm, and the optical density was measured at 600, 532 and 450 nm, respectively, for further calculation of the MDA concentration.

2.9. Determination of ROS generation

Intracellular ROS level was evaluated by 2′,7′-dichlorofluorescin diacetate (Solarbio, Beijing, China). Cells grown on coverslips were incubated with 20 μmol/L 2′,7′-dichlorofluorescin diacetate at 37 ◦C for 30 min in the dark and then were washed with phosphate buffer saline.Cells were fixed in 4% paraformaldehyde, counterstained with DAPI, then washed 3 times with phosphate buffer saline. Cells were mounted with an anti-fading mounting medium and fluorescence intensity was detected by a fluorescence microscope with an excitation wavelength at 488 nm and an emission wavelength at 525 nm.

2.10. 4- HNE and GSH assay

Kidney tissues were homogenized by the above described way, and the supernatant was collected after centrifugation. The working agents of the both of kits were prepared according to the manufacturer’s instructions. 4-HNE was detected by double antibody sandwich method
using 4-HNE ELISA Kit (Fankewei, Shanghai, China). The optical density was measured by microplate reader at the wavelength of 450 nm. GSH was measured by Micro Reduced Glutathione GSH Assay Kit (Solarbio,Beijing, China). The optical density was determined at the wavelength of
412 nm. And the concentrations of 4-HNE and GSH in the tissues were then acquired by comparing the optical density of the samples to the standard curve, respectively.

2.11. Quantitative real-time PCR (qRT-PCR)

Total RNA of renal tissues and HK-2 cells was extracted and purified by using the HP Total RNA Kit (Omega Bio-Tek, Norcross, GA, USA). After quantifying the concentrations and purities of RNA, 1 μg RNA was reverse-transcribed into cDNA by M-MuLV First Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China) in a volume of 20 μL. Subse- quently, cDNA from the samples was amplified with SYBR Green fluorescence PCR kit (Sangon Biotech, Shanghai, China) on the C1000 Touch Thermocycler CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). The relative amount of the target genes was standardized against GAPDH and determined using the 2 ΔΔCT method. PCR primers are listed in Table S1.

2.12. Transfections

To generate Nrf2 knockdown studies, cells were transfected with 80 nM of siRNA (Genepharma, Shanghai, China) respectively against Nrf2
(Si-Nrf2) and control siRNA (Si-Ctrl), performing with lipofectamine RNAimax (Invitrogen, Carlsbad, CA, USA). Target sequences for pre- paring siRNAs of human Nrf2 are listed in Table S1. 8 μL of 20 μM siRNAs was combined with 250 μL of Opti-MEM serum-free media (Gibco, Carlsbad, CA, USA) in one tube. 8 μL of lipofectamine 2000 was combined with 250 μL of Opti-Mem media in another tube. They were equilibrated at regular temperature for 5 min. Two tubes were gently mixed and incubated at 37 ◦C for 20 min, transferred into 6-well plate combined with 1.5 mL of Opti-MEM serum-free media. The siRNA induced gene silencing was confirmed using quantitative real-time PCR.

2.13. Western blotting analysis

HK-2 cells were lysed by RIPA lysis buffer (Solarbio, Beijing, China), total proteins were extracted from cells in the presence of protease in- hibitors. Proteins were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking in 0.1% tris buffered saline tween that contained 5% skimmed milk for 1 h at room temperature, the polyvinylidene difluoride membranes were incubated overnight at 4 ◦C with primary antibodies against Nrf2 (YT3189; Immunoway), GPX4 (ab125066; Abcam), FTH-1 (DF6278; Affinity), TFR-1 (ab214039; Abcam), SLC7A11 (ab175186; Abcam) and GAPDH (60004-1-Ig; Proteintech, Wuhan, China), following incubation with the normal isotype-matched horseradish peroxide-labeled IgG (1:1000) for 1 h. Finally, immunoblots were visualized by chemiluminescence reagent (Advansta, Menlo Park, CA, USA) and quantified by Image J software v1.49.

Fig. 1. The ferroptosis-related changes in the kidney of DN mice. The mRNA expression of GPX4 (a), SLC7A11 (b), FTH-1 (c) and TFR-1 (d) in kidney tissue of mice. Immunohistochemical staining results of GPX4, SLC7A11, FTH-1 and TFR-1 in kidney tissues (e). Semi-quantification analyses of immunohistochemical staining of GPX4, SLC7A11, FTH-1 and TFR-1 (f). The MDA (g), 4-HNE (h), GSH (i) and iron (j) concentrations in the kidney tissue lysates. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05, ##p < 0.01 vs. DM group. 3. Statistical analysis Statistical analysis was performed using Prism 8.0 software (GraphPad, La Jolla, CA, USA). Values are shown as the means ± S.D. For two group comparison, Student’s t-test was performed. For multiple-group comparison, one-way ANOVA analysis was performed. Statistically significance was defined as P < 0.05. 4. Results 4.1. Characteristic changes of ferroptosis in the kidneys of DN mice Both of GPX4 and SLC7A11 play important roles in antioxidant stress. In this study, the mRNA expression levels of GPX4 (Fig. 1a) and SLC7A11 (Fig. 1b) in the DM group were significantly decreased compared with NC group. The results of immunohistochemical staining and semi-quantification analyses (Fig. 1e and f) also showed that the protein expression levels of GPX4 and SLC7A11 in the proximal tubules of kidneys were lower in the DM group. And the results indicated that the GSH was also significantly dropped in DM group mice (Fig. 1i). The above results denoted that the capacity of peroxidation repair in the kidneys of diabetic mice was dramatically reduced. Meanwhile, the data displayed that the end products of lipid peroxidation, MDA (Fig. 1g) and4-HNE (Fig. 1h), were s ignificantly increased in DM group, which was one of the characteristic changes of ferroptosis. We found that the expression of iron metabolism-related genes, FTH-1 and TFR-1, was changed in streptozotocin-treated mice. The expression of FTH-1 (Fig. 1c) was obviously decreased and TFR-1 (Fig. 1d) was dramati- cally increased in the DM group. And the weaker staining of FTH-1 and stronger staining of TFR-1 in renal sections were detected in DM group by immunohistochemical staining (Fig. 1e and f), which were consistent with the changes of corresponding mRNA expression. And we found the concentration of iron (Fig. 1j) in the kidneys of DM group mice was almost three times that of NC group mice, indicating that the situation of iron was overloaded in the kidneys of diabetic mice. Interestingly, the above ferroptosis-related changes in diabetic mice were obviously rescued after being treated with the ferroptosis inhibitor Fer-1. The antioxidant capacity was repaired, simultaneously, and the iron depo- sition and lipid peroxidation products were also reduced in the Fer-1 group (Fig. 1a–j). 4.2. Ferroptosis was observed in high-glucose cultured HK-2 cells The cell viability was measured in the media with various glucose concentrations (5.5, 25, 20, 40 and 50 mmol/L) at pre-determined time, respectively. Compared with HK-2 cells being treated with 5.5 mmol/L of glucose, the viability of cells being cultured in the medium with 25 mmol/L was similar or slightly higher. However, the cell viability was decreased as the glucose concentration further increased (Fig. 2a). After being treated with the different concentrations of Fer-1, the cell viability was increased in all Fer-1 treated groups, and reached a peak when the Fer-1 concentration was 1 μM (Fig. 2b). The ultrastructural analysis demonstrated that the lone distinctive morphological feature (Fig. 2c) of the HG group cells, involving shrunken mitochondria with increased membrane density and mitochondrial ridge reduction or even disap- pearance, possibly marked the point of no-return in ferroptosis. The mRNA expression of GPX4 (Fig. 2d), SLC7A11 (Fig. 2e) and FTH-1 (Fig. 2f) were decreased and the expression of TFR-1 (Fig. 2g) was increased in HG group cells compared with the Ctrl group. In addition, western blotting results showed a parallel change of protein expression level of GPX4, SLC7A11, FTH-1 and TFR-1 with the changes of corre- sponding mRNA (Fig. 2h–k). Furthermore, HK-2 cells induced by high glucose were stained by 2′,7′-dichlorofluorescein diacetate to reflect the generation of ROS. There was a minimal background fluorescence in the Ctrl group cells and cells being cultured with high glucose alone yielded the maximal degree of fluorescence (Fig. 2l). In the HG group, the production of MDA was increased by 40% (Fig. 2m) and the concen- tration of iron (Fig. 2n) was increased by 80% compared with the NC group. These results were consistent with previous research results in vivo. The ferroptosis-related changes described above were reversed in the Fer-1 group (Fig. 2c–n). There was no significant difference in the mannitol treated group which excluded the effect of high osmotic pressure on HK-2 cells (Fig. 2d–h, 2j, 2 m and 2n). 4.3. Inhibition of ferroptosis alleviated the renal pathological changes in DN mice After 5 days of continuous injection of streptozotocin, the random blood glucose level of Fer-1, DM and vehicle-P groups was gradually rising and reached 25 mmol/L at week 8, then the rate of rise was slowing down and nearly at a plateau, while the random blood glucose level of NC group was maintained at about 7 mmol/L (Fig. 3a). The bodyweight of the NC group was slowly increased, but that of Fer-1, DM and vehicle-P groups was gradually decreased (Fig. 3b). According to the Animal Models of Diabetic Complications Consortium criterion, urine volume, urinary albuminuria, urinary creatinine and ACRs are the important biomarkers to judge the success of the DN mouse model. Compared with the NC group, the 24-h urine volume (Figs. 3c), 24-h urinary albuminuria (Fig. 3d), urinary creatinine (Fig. 3e) and ACRs (Fig. 3f) of diabetic mice in Fer-1, DM and Vehicle-P groups were dramatically increased. Specifically, the 24-h urine volume and the 24-h urinary albuminuria for the DM group increased by 30 and 100 times respectively, compared with the normal mice. Although the levels of these four indicators in Fer-1 group mice were still higher than NC group, we found that all of these indicators were significantly less than DM group (Fig. 3c–f). H&E staining showed the notable morphological changes in the kidneys of DM group, including glomerular changes, degeneration of tubular epithelia with loss of brush borders and tubular lumen dilatation (Fig. 3g and j). Compared with the NC group, PAS staining showed obvious expansion of mesangial (Fig. 3h and k), and Masson staining showed aggravation of renal fibrosis in the DM group (Fig. 3i and k). Interestingly, these renal damages of diabetic mice were significantly ameliorated after being treated with Fer-1 (Fig. 3g–k). And we suggested the high-glucose ambience promoting the progression of DN through ferroptosis in Fig. 3l. 4.4. The low expression of Nrf2 enhanced the sensitivity to ferroptosis Nrf2 plays a central role in response to oxidative stress. The immu- nofluorescent analysis showed that the fluorescence intensity of renal tubules in diabetic mice was significantly weaker than that of normal mice, indicating the decreased protein expression of Nrf2 in diabetes (Fig. 4a). The western blotting (Fig. 4b and c) and qRT-PCR results (Fig. 4d) showed the protein and mRNA expression levels of Nrf2 in DM group mice was lower than NC group mice. And the low expression of Nrf2 protein (Fig. 4e and f) and mRNA (Fig. 4g) were also verified in cells being cultured in 30 mmol/L glucose for 72 h, there were no sig- nificant changes in the Mannitol group. In order to further study the relationship between Nrf2 and ferroptosis, we knockdown Nrf2 in cells. The protein expression of GPX4, SLC7A11, FTH-1 was significantly decreased and TFR-1was increased in Si-Nrf2 cells being cultured in high-glucose medium compared with Si-Ctrl cells under the same con- ditions (Fig. 4h and i), the mRNA expression of these genes presented the parallel results (Fig. S1). Higher levels of MDA (Fig. 4j) and iron (Fig. 4k) were also detected in the Si-Nrf2 group compared with the Si-Ctrl group, both of the groups being cultured in high-glucose medium. However, the changes caused by Nrf2 knockdown were ameliorated after the treatment of Fer-1 (Fig. 4h–k). The above results suggested that the suppression of Nrf2 enhanced ferroptosis sensitivity of HK-2 cells in high glucose situation. Fig. 2. High glucose trigged ferroptosis-related changes in HK-2 cells. The viability of HK-2 cells being cultured in the media with various glucose concentrations for 24, 48 and 72 h, respectively (a). The viability of HK-2 cells being cultured in 30 mmol/L glucose medium with different concentrations of Fer-1 for 48 h (b). Transmission electron microscopy was used to detect the mitochondrial morphology of cells in Ctrl, HG and Fer-1 groups. The red arrow indicates the mitochondria (c). The mRNA expression levels for GPX4 (d), SLC7A11 (e), FTH-1 (f) and TFR-1 (g) in each group of cells. The protein expression levels of GPX4, SLC7A11, FTH-1 and TFR-1 in cells of Ctrl, HG and Mannitol groups, respectively (h). The protein expression levels of above described gens in cells of HG, Fer-1 and DMSO groups (i). Semi-quantitative analysis of immunoblotting results of GPX4, SLC7A11, FTH-1 and TFR-1 in each group of cells (j and k). The production of ROS (green), nuclear staining with DAPI (blue) in each group of cells (l). The MDA (m) and iron concentrations (n) in cells. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. Ctrl group; #p < 0.05, ##p < 0.01 vs. HG group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 3. Inhibition of ferroptosis by Fer-1 improved renal pathological damages in diabetic mice. Mice blood glucose levels (a) and body weight (b) were monitored at 0, 2, 4, 8 and 12 weeks. Mice 24-h urine volume (c), 24-h urinary albumin levels (d), urine creatinine (e) and ACRs (f) were measured at 0 and 12 weeks. H&E staining of mouse kidney sections in each group (g). PAS staining (h) and Masson staining (i) of mouse kidney sections in each group. Semi-quantitative analysis of the glomerular area and tubular damage (j), mesangial scores and the degree of fibrosis (k). Activating ferroptosis promotes the development of DN (l). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05, ##p < 0.01 vs. DM group; Δ p < 0.05, ΔΔ p < 0.01 vs. baseline of each group, respectively. 4.5. Regulation of Nrf2 signaling pathway by fenofibrate Immunofluorescent staining showed the fluorescence intensity of renal tubules was stronger in Fn group mice than DM group mice, indicating the Nrf2 protein expression increased significantly (Fig. 5a). The results of western blotting (Fig. 5b and c) and qRT-PCR (Fig. 5c) also illustrated that the expression level of Nrf2 was significantly increased in the kidney tissues of Fn group compared with the diabetic mice. To further verify fenofibrate might protect the kidneys of diabetic mice by up-regulating Nrf2, fenofibrate was used to treat the Si-Nrf2 cells. The protein expression of Nrf2 was notably increased in Si-Nrf2 cells being treated with fenofibrate compared with the Si-Nrf2 group (Fig. 5e and f), and the mRNA expression of Nrf2 presented parallel results as protein (Fig. S2a). And we also found that the expression of ferroptosis-related genes at the protein level was reversed by fenofibrate. The decreased expression of GPX4, SLC7A11, FTH-1 was rescued and increased TFR-1 was inhibited by fenofibrate in Si-Nrf2 group cells (Fig. 5e and f), and the mRNA expression of these genes changed in the same trend as pro- tein (Fig. S3b-3e). Simultaneously, pretreated with fenofibrate reduced the concentrations of iron (Fig. 5g) and MDA (Fig. 5h), which implied fenofibrate could improve the situations of iron overload and lipid peroxidation caused by cellular Nrf2 knockdown. Accordingly, the above results suggested that fenofibrate promoted the expression of Nrf2 in HK-2 cells and might inhibit ferroptosis occurrence. Fig. 4. Knockdown Nrf2 improved the sensitivity of cells to ferroptosis under high glucose conditions. Immunofluorescent analysis with Nrf2 antibody (green), nuclear stained with DAPI (blue) in kidney tissue sections of NC and DM groups (a). The western blotting results of Nrf2 in mice kidney tissues of NC and DM groups (b). Semi-quantitative analysis of immunoblotting results of Nrf2 in mice kidney tissues (c). The mRNA expression of Nrf2 in the kidney tissues of the diabetic mice in NC and DM groups (d). The protein expression of Nrf2 in cells of Ctrl, HG and Mannitol groups (e). Semi-quantitative analysis of immunoblotting results of Nrf2 in each group of cells (f). The mRNA expression of Nrf2 in each group of cells (g). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. NC group; #p < 0.05, ##p < 0.01 vs. Ctrl group. The protein expression of GPX4, SLC7A11, FTH-1 and TFR-1 in cells of Si-Ctrl group, Si-Nrf2 group, Si-Nrf2+Fer-1 group and Si-Nrf2+DMSO group (h). Semi-quantitative analysis of immunoblotting results of GPX4, SLC7A11, FTH-1 and TFR-1 in each group of cells (i). The concentrations of iron (j) and MDA (k) in each group of cells. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. Si-Ctrl group; #p < 0.05, ##p < 0.01 vs. Si-Nrf2 group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 5. The Nrf2 single pathway was activated by treating with fenofibrate. Immunofluorescent analysis with Nrf2 antibody (green) and nuclear staining with DAPI (blue) in kidney tissue sections of DM and Fn groups (a). The western blotting results of Nrf2 in mice kidney tissues of DM, Fn and vehicle-G groups (b). Semi- quantitative analysis of immunoblotting results of Nrf2 in mice kidney tissues (c). The mRNA expression of Nrf2 in kidneys of DM, Fn and vehicle-G groups (d). Data are expressed as mean ± SD. #p < 0.05, ##p < 0.01 vs. DM group. The protein expression levels of Nrf2, GPX4, SLC7A11, FTH-1 and TFR-1in Si-Nrf2 group, Si- NRF2 + Fn and Si-Nrf2+DMSO group for 48 h (e). Semi-quantitative analysis of immunoblotting results of Nrf2, GPX4, SLC7A11, FTH-1 and TFR-1 in cells (f). The concentrations of iron (g) and MDA (h) in cells. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. Si-Nrf2 group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4.6. RSL-3 induced ferroptosis improved by fenofibrate Cell counting kit-8 assay was performed to assess the viability of cells treating with RSL-3. We found that the cell viability was significantly decreased as RSL-3 concentration increased. The cell viability decreased significantly when the concentration of RLS-3 was over 0.1 μM. Besides, the cell viability was less than 10% and became stable while the concentration was over 0.4 μM (Fig. 6a). Pretreated with fenofibrate rescued the decreased expression level of Nrf2 in the RSL-3 treated cells (Fig. 6b, g and 6h). Decreased expression of GPX4 (Fig. 6c, g and 6h),SLC7A11 (Fig. 6d, g and 6h) and FTH-1 (Fig. 6e, g and 6h), as well as increased expression of TFR-1 (Fig. 6f, g and 6h) caused by ferroptosis, were significantly inverted after being treated with fenofibrate. The accumulation of ROS trigged by RSL-3 showed a stronger fluorescence intensity compared with the Ctrl group, and the high degree fluores- cence was effectively inhibited by pretreatment of fenofibrate (Fig. 6i). Moreover, the accumulation of lipid peroxidation product (Fig. 6j) and high iron concentrations (Fig. 6k) under ferroptosis conditions were also reduced after fenofibrate treatment. These results implied that fenofi- brate could relieve the damages of ferroptosis to HK-2 cells by up- regulating the expression of Nrf2. Fig. 6. Cellular ferroptosis trigged by RSL-3 was inhibited via using of fenofibrate. The viability of HK-2 cells being treated with different concentrations of RSL-3 in 5.5 mmol/L glucose medium for 24 h (a). The mRNA (b–f) and protein (g) expression levels of Nrf2 (b and g), GPX4 (c and g), SLC7A11 (d and g), FTH-1 (e and g) and TFR-1 (f and g) in cells of Ctrl, RSL-3, RSL-3+Fn and DMSO groups. Semi-quantitative analysis of immunoblotting results of Nrf2, GPX4, SLC7A11, FTH-1 and TFR-1 in each group of cells (h). The production of ROS (green), nuclear staining with DAPI (blue) in each group of cells (i). The MDA (j) and iron (k) concentrations in each group cells. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. Ctrl group; #p < 0.05, ##p < 0.01 vs. RSL-3 group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4.7. Ferroptosis in DN model was mediated by fenofibrate Compared with the DM group, the protein expression levels of GPX4, SLC7A11 and FTH-1 were obviously enhanced and the protein expres- sion of TFR-1 was significantly decreased in the renal sections of Fn group mice (Fig. 7a and 7b). And the mRNA expression of the corre- sponding genes was parallel to the above described results of immuno- histochemical staining (Fig. 7c–f). In addition, we found that the iron accumulation (Fig. 7g) fell by almost 60%, and the increased GSH indicating the capacity of anti-oxidative stress in the kidneys of Fn group mice was improved (Fig. 7h). The lipid peroxidation products, MDA (Fig. 7i) and 4-HNE (Fig. 7j), were also reduced by fenofibrate. Both of them dropped by about 30%, compared with the DM group mice. The mitochondria morphological changes of cells being cultured in high- glucose were mitigated after being pretreated with fenofibrate. The mitochondrial ridge was still clearly visible and the situations of mito- chondrial shrinkage and increased membrane density were lightened in Fn group (Fig. 7k). The results indicated that the mitochondrial morphological changes induced by diabetic ferroptosis could be reversed by pretreating with fenofibrate. The diabetic ferroptosis biomarkers were detected in HK-2 cells, and a similar trend was observed in fenofibrate treated HK-2 cells (Fig. S3). Fig. 7. Fenofibrate improves ferroptosis related indicators in the DN model. Immunohistochemical staining results of GPX4, SLC7A11, FTH-1 and TFR-1 in mice kidneys (a). Semi-quantification of immunohistochemical staining (b). The mRNA expression of GPX4 (c), SLC7A11 (d), FTH-1 (e) and TFR-1 (f) in mice kidneys. The concentrations of iron (g) and GSH (h) and lipid peroxidation products [MDA (i) and 4-HNE (j)] in mice kidneys. Transmission electron microscopy was used to detect the mitochondrial morphology of cells in HG and Fn groups. The red arrow indicates the mitochondria (k). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. DM group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 4.8. Fenofibrate ameliorates renal pathological damages in diabetic mice There was no significant difference in blood glucose (Fig. 8a) and body weight (Fig. 8b) in the DM group, Fn group and vehicle-G group. Treatment with fenofibrate did not affect the levels of blood glucose and body weights in diabetic mice, which implied that fenofibrate has no effect on controlling the blood glucose of diabetes. The symptoms of polyuria in diabetic mice has been greatly improved after being treated with fenofibrate. The average 24-h urine volume of DM group was almost 14 mL, however, the average urine volume of Fn group was about 4 mL (Fig. 8c). Although these indexes of renal damages were still higher than the normal range in the Fn group, they were significantly improved compared with the untreated diabetic mice, suggesting a reduction of renal pathological injuries in diabetic mice. In Fn group, the level of 24-h urinary albuminuria (Fig. 8d) was almost 6 times lower than DM group, and the average urine creatinine (Fig. 8e) decreased from 212 to 143 μg/ mL, the ACRs (Fig. 8f) dropped by almost 60% compared with the un- treated diabetic mice. Histopathological staining demonstrated that fenofibrate improved the pathological changes in glomerular and tubular epithelial disruption (Fig. 8g and j). PAS staining revealed the reduced mesangial expansion and Masson staining showed the mitigated renal fibrosis in Fn group (Fig. 8h, i and 8k). The above results indicated that fenofibrate obviously ameliorated the renal damages in diabetic mice. And we presented the mechanism of fenofibrate improves the kidney ferroptosis in diabetes by up-regulating Nrf2 in Fig. 8l. 5. Discussion The weakened antioxidant capacity, iron overload and the accumu- lation of lipid peroxidation products were the characteristic indicators of ferroptosis, which were detected in the DN models and rescued by Fer-1. Inhibition of ferroptosis delayed the development of renal pathological in diabetic mice and improved the damaging effect of high glucose on HK-2 cells. Furthermore, we found that up-regulation of Nrf2 treated with fenofibrate reduced the negative effect of diabetes-related ferrop- tosis, benefit to delay the progression of DN. Fig. 8. Fenofibrate delayed the renal damages in diabetic mice. Mice blood glucose levels (a) and body weight (b) were monitored at 0, 2, 4, 8 and 12 weeks. Mice 24-h urine volume (c), 24-h urinary albumin levels (d), urine creatinine (e) and ACRs (f) were measured at 0 and 12 weeks. H&E staining of mouse kidney sections in each group (g). PAS staining (h) and Masson staining (i) of mouse kidney sections in each group. Semi-quantitative analysis of the glomerular area and tubu- lointerstitial damage index (j). Semi-quantitative analysis of mesangial scores and the degree of fibrosis (k). The action mechanism of fenofibrate ameliorating ferroptosis in the diabetic kidney is via upregulation of Nrf2 pathway (l). Data are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs. DM group. Iron is an indispensable element for various metabolic and physio- logical functions in living organisms. Conversely, iron overload is also biologically cytotoxic. Because the aberrant accumulation of iron pro- duce massive free radicals, leading to the damage of DNA, proteins or other biomolecules [26]. Although iron overload has a negative impact on the occurrence and development of DN, the underlying damage mechanism was still unclear. The discovery of ferroptosis provides a new explanation for the pathogenic mechanism of iron overload in diseases. Ferroptosis was a negative outcome, which exacerbated pathological injuries or promoted disease progression, such as cancer, neuro- degeneration, ischemic injury and cardiovascular disease [27–29]. However, there was no report on the link between the deterioration of DN and ferroptosis. Herein, we proposed for the first time that the cell ferroptosis aggravated kidney damages, which promotes the develop- ment of DN. ROS accumulation is the central link leading to ferroptosis. MDA and 4-HNE produced by ROS-induced lipid peroxidation of phospholipid- containing cell membranes, which directly caused cytotoxicity, then inducing ferroptosis [30]. A small amount of ROS generated by the healthy kidney can be tolerated due to the normal antioxidant capacity of the body, but excessive ROS will lead to significant injuries to the kidney [31]. The significant accumulation of ROS was observed in high glucose cultured HK-2 cells. And the enhanced concentrations of lipid peroxidation products, MDA and 4-HNE, were both detected in vivo and vitro DN models. In addition, the shrunken mitochondria with increased membrane density and the reduced or even disappeared mitochondrial ridge were observed in cells being cultured in high glucose medium. It be defined as the specific morphological changes of ferroptosis, which were different from other death modes. Above results suggested that ferrop- tosis might be closely related to the development of DN. GPX4 is one of the most important antioxidant enzymes in mammals and the only known enzyme capable of reducing phospholipid hydro- peroxide. GPX4 deficiency has been regarded as one of the biomarkers of ferroptosis, and the inducible GPX4 depletion caused massive renal tubular epithelial cells die for ferroptosis [32–35]. We observed that the expression of GPX4 was significantly decreased in the cells and kidneys of DN models, indicating that the renal damage of DN might be related to ferroptosis. Besides, the downregulated expression of SLC7A11 and decreased GSH concentration were also detected in DN models, which suggested that the antioxidant capacity of kidneys was impaired under the high glucose condition. The synthesis of intracellular GSH requires SLC7A11 to transport cystine (a precursor of GSH) into the cytoplasm, and the down-regulation of SLC7A11 also resulted in a decrease in GSH concentration. The loss of GSH is another key mechanism for ferroptosis, GSH acts as the tripeptide antioxidant and GPX4 co-factor to reduce ROS [36,37]. Therefore, the low expression of GPX4 and SLC7A11 and the absence of GSH limited the ability to resist the impairment of ROS, increasing the sensitivity of cells to ferroptosis. TFR-1 and FTH-1 play important roles in the regulation of iron metabolism, which are closely related to the occurrence of ferroptosis [38,39]. Most cells regulate the absorption of iron by modulating the expression of TFR-1 [40]. FTH-1 catalyzes the conversion of ferrous iron to the ferric iron by its oxidase activity, and promotes iron to be incor- porated into the ferritin, thereby reducing the free iron levels [41]. In the DN models, we found that the expression of TFR-1 was significantly increased, while the expression of FTH-1 was obviously reduced at both mRNA and protein levels, implying there is an imbalance of iron ho- meostasis in the case of diabetes. And the obvious iron overload has been detected in DN models, which was considered as a typical characteristic of ferroptosis [42,43]. The treatment of Fer-1 mitigated the damage of oxidative stress and iron deposition, improved the morphological changes of mitochondria and alleviated renal pathological damages in diabetic mice. Thus, these results demonstrated that ferroptosis played an important role in the progression of DN. According to the above results, we verified that ROS accumulation, antioxidant capacity reduction and iron deposition in kidneys accelerated the progression of lipid peroxidation, further trig- gered the ferroptosis of kidney, which accelerated the progression of DN, as was displayed in Fig. 3l.Nrf2, the primary transcription factor, is responsible for the upregulation of oxidative responses. However, after the long-time resistance of oxidative stress, the expression level of Nrf2 would gradually decrease, which was strongly correlated with the aggravated damage effect of oxidative stress [44,45]. The accumulation of ROS caused by hyperglycemia provides conditions for oxidative stress. The reduction of antioxidant enzyme and protein (GPX4 and GSH) further aggravated the oxidative stress response of diabetic kidney. The down-regulated expression of Nrf2 was also observed in DN models, which was consis- tent with the previous studies [46,47]. Given the regulatory role of Nrf2 in iron metabolism and anti-oxidative stress, we investigated whether the occurrence of diabetic ferroptosis could be inhibited by the up-regulation of Nrf2 expression, thereby delaying the progression of DN. First of all, we verified that the specific knockdown of Nrf2 increased the sensitivity of HK-2 cells to ferroptosis under high glucose conditions. After silencing Nrf2, the expression of its target gene FTH-1 was also decreased, while the expression of TFR-1 was increased. Considering the central role of Nrf2 in anti-oxidative stress, silencing Nrf2 severely damaged the anti-oxidation capacity of cells being cultured in high glucose medium, and the expression of GPX4 and SLC7A11 was also dropped. Knockdown of Nrf2 weakened the resistance to oxidative stress, increased the deposition of free iron in cells and accelerated the occurrence of lipid peroxidation, which provided a basis for the occurrence of ferroptosis, and further strengthened the cell damage in high glucose environment.

Subsequently, we found that the expression of Nrf2 could be up- regulated after being treated with fenofibrate, which resulted in that the expression level of GPX4, SLC7A11, FTH-1 and TFR-1 was also changed, and the situation of ferroptosis in Nrf2 knockdown cells was alleviated in this way. To further verify the effect of fenofibrate on ferroptosis, we used fenofibrate on cells induced by RSL-3 and then observed the increased expression of Nrf2 and the improvement effect on ferroptosis biomarkers. Existing researches showed that fenofibrate could delay the development of DN, but its protective mechanism has been unclear [48]. We applied fenofibrate to diabetic mice and found that the down-regulation of Nrf2 in mice was controlled, and the fer- roptosis caused by diabetes was also improved. The renal pathological damages of diabetic mice were also mitigated by using fenofibrate. And according to the above results, we hypothesized that fenofibrate increased the expression levels of GPX4 and SLC7A11 by up-regulating Nrf2, thereby improving the anti-oxidative stress ability of the kidney of diabetic mice. Moreover, the increased expression of FTH-1 and the down-regulated TFR-1 improved the iron deposition in the kidneys of diabetic mice. The amelioration of antioxidant capacity and the stabi- lization of iron metabolism mitigated ferroptosis of the kidneys in dia- betic mice. Therefore, up-regulation of Nrf2 could slow down the progression of diabetic nephropathy by inhibition of ferroptosis, as was displayed in Fig. 8l. Our research provided an explanation for the nephroprotection of fenofibrate from a new angle other than its tradi- tional lipid-lowering effect: inhibition of ferroptosis by up-regulating Nrf2.

In conclusion, our study revealed that cellular ferroptosis occupied an important position in the progression of DN, and the mitigating fer- roptosis by up-regulating Nrf2 was of great significance in alleviating the progression of DN. Collectively, the present work may provide a new approach for the prevention and treatment of DN.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81671835) and the Key Projects of Tianjin Natural Science Foundation (No. 19JCZDJC36900).

Declaration of competing interest

None.

Acknowledgements

We would like to thank the team of professor Baocheng Chang (Tianjin Medical University Chu Hsien-I Memorial Hospital) for donating HK-2 cells.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.freeradbiomed.2020.10.323.

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