Kurarinone activates the Nrf-2/HO-1 signaling pathway and alleviates high glucose-induced ferroptosis in HK2 cells

Abstract

To investigate the possible effects of Kurarinone on the ferroptosis and EMT of high glucose (HG)-stimulated HK2 cells, and uncover the mechanism. HK2 cells were treated with glucose to construct a DN cell model. CCK-8 and FCM assays exhibited the effects on growth as well as apoptosis of HK2 cells. DCF staining as well as Immunoblot assays exhibited the effects on ferroptosis. JC-1 staining exhibited the effects on mitochondrial function. Immunoblot assays showed the effects on the EMT process of HK2 cells. Immunoblot assays confirmed the mechanism. Kurarinone inhibited the apoptosis of HG-stimulated HK2 cells. It also blocked the ferroptosis of HG-stimulated HK2 cells. Further data showed that Kurarinone suppressed the mitochondrial damage in HG-stimulated HK2 cells, and restrained EMT process. Mechanically, Kurarinone activated the Nrf-2 pathway in HG-stimulated HK2 cells. Kurarinone activates the Nrf-2 pathway and alleviates HG-stimulated ferroptosis and EMT in HK2 cells.

Introduction

Diabetic nephropathy (DN) is one of the major causes of death in patients with chronic kidney disease, and is characterized by extracellular matrix protein deposition, renal tubulointerstitial fibrosis, mesangial matrix as well as glomerular basement membrane dilatation, and loss of waste removal over time.⁽¹⁾ The pathogenesis of DN is believed to involve multiple factors, including hyperglycemia, oxidative stress, inflammation, accumulation of advanced glycosylation end products, activation of fibroblast, mitochondrial damage, etc.⁽²⁾ To combat this disease, new and promising drugs are still needed to be developed.

In response to pathological stimuli such as urinary sugar and urinary protein, tubular epithelial cells develop EMT and lose their epithelial phenotype, ultimately leading to renal deterioration and tubular interstitial fibrosis during DN progression.⁽³⁾ During the progression of EMT, there is an excess of ROS in renal tubular epithelial cells.⁽⁴⁾ Mitochondria are the main source of production of ROS. When mitochondria are challenged by high glucose (HG), their membrane potential is affected, resulting in decreased potential and further increased ROS levels.⁽⁵⁾ Overproduction of ROS is a major cause of oxidative stress as well as apoptosis.

Kurarinone is a natural lavender acylated flavanone isolated from the herb Sophora flavescens Aiton, which has immunosuppressive and antioxidant effects.⁽⁶⁾ Kurarinone alleviates collagen-induced arthritis by inhibiting Th1/Th17 cell response and oxidative stress.⁽⁷⁾ By regulating IGF1/PI3K/Akt signal transduction, Kurarinone changes the M1/M2 polarization of microglia, thereby alleviating heme-induced neuroinflammation and microglia-mediated neurotoxicity.⁽⁸⁾ In addition, Kurarinone also has an anti-fibrotic effect, which inhibits the TGF-β signaling pathway and reduces BLM-induced pulmonary fibrosis.⁽⁹⁾ Kurarinone down-regulates the expression of TGF-β1 and Col I, inhibits the transdifferentiation of ECM, inhibits the activation as well as growth of myofibroblasts, and improves renal fibrosis.⁽¹⁰⁾ However, the role and mechanism of Kurarinone in DN are still unclear.

This study aimed at investigating the role of Kurarinone in the DN progression at cellular levels. The results showed that Kurarinone activated the Nrf-2 pathway to block ferroptosis and mitochondrial damage, and improve renal fibrosis in HG induced HK2 cells.

Materials and Methods

Cell culture and treatment

HK2 cells were purchased from ATCC (Manassas, VA), and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA) complemented with 10% FBS (Gibco, Waltham, MA) in a humidified atmosphere (Thermo Fisher Scientific) at 37°C and 5% CO₂. The HK2 cells were maintained in a medium with 30 ‍mM glucose, and normal cells were maintained in a medium with 5.6 ‍mM glucose. Kurarinone (MedChemExpress, Monmouth Junction, NJ) was added into HK2 cells at the concentration of 0, 0.25, 0.5, 1, and 2 ‍μM for 24 ‍h. Fer-1 (Sigma, St. Louis, MO) was added to HK2 cells at concentrations of 10 ‍μM for 24 ‍h. The cell nuclei were extracted using a nuclear extraction kit (NE-PER^TM Nuclear and Cytoplasmic Extraction Reagents; Thermo Fisher Scientific) following the manufacturer’s instructions.

Cell viability

HK2 cells were seeded into 96-well plates, and then CCK-8 was added to each well for cell viability detection for 4 ‍h before the measurement of OD450 value.

Cell apoptosis

HK2 cells were digested and resuspended in binding buffer containing Annexin V and PI for 5 ‍min away from light. Cell apoptosis was determined by a flow cytometer (BD Biosciences, Franklin Lakes, NJ).

JC-1 staining

Cells were incubated with 2 ‍μM JC-1 for 15 ‍min at 37°C in the dark. After rinsing in PBS, the cells were analyzed using a fluorescent microscope.

DCF staining

The HK2 cells, after the indicated treatment, were fixed as well as blocked with goat serum for 1 ‍h. Slices were further incubated with DCF/ROS detection kit (ab238535; Abcam, Cambridge, UK) according to the manufacturer’s guidelines.

FCM detection

For the FCM detection of lipid peroxidation, HK-2 cells were incubated with 1 ‍μM BODIPY C11 probe (Sigma) for 30 ‍min at 37°C. The fluorescence intensity was then measured using a flow cytometer with excitation at 488 ‍nm and emission at 530 ‍nm (for peroxidized lipids) and 585 ‍nm (for non-peroxidized lipids).

Immunoblotting

Proteins were resolved with 10% SDS-PAGE and transferred onto PVDF membranes. Then the membranes were incubated with 5% BSA followed by primary antibodies including GPX-4 (1:1,000, ab125066; Abcam), SLC7A11 (1:1,000, ab307601; Abcam), ACSL4 (1:1,000, ab155282; Abcam), PTGS2 (1:1,000, ab23672; Abcam), Histone H3 (1:1,000, ab1791; Abcam), Vimentin (1:1,000, ab92547; Abcam), α-SMA (1:2,000, ab7817; Abcam), E-cadherin (1:1,000, ab40772; Abcam), Nrf-2 antibody (1:1,000, ab62352; Abcam), HO-1 (1:1,000, ab305290; Abcam), and beta-actin (1:3,000, ab8226; Abcam). The membranes were maintained in HRP secondary antibodies at 1:1,000 for 2 ‍h. The signals were detected with ECL detection kit.

Statistical analysis

Data was represented by mean ± SD. Statistical analysis was performed using GraphPad. P<0.05 was considered as significance.

Results

Kurarinone inhibited the apoptosis of HG-stimulated HK2 cells

To reveal the possible effects of Kurarinone on DN progression, a DN cell model using HK2 cells after treatment with HG (30 ‍mM) for 24 ‍h was first constructed. Kurarinone had modest effects on the viability of HK2 cells at a low concentration (0.25, 0.5, and 1 ‍μM) for 24 ‍h, whereas high concentration (2 ‍μM) of Kurarinone suppressed the viability of HK2 cells, with the decreased OD450 value (Fig. 1A). Subsequently, the low concentration (1 ‍μM) of Kurarinone was used in the next experiments. Furthermore, CCK-8 assays indicated HG suppressed the viability of HK2 cells, whereas Kurarinone treatment promoted the viability of HG-stimulated HK2 cells (Fig. 1B). FCM assays indicated that the apoptosis of HK2 cells was stimulated after HG treatment, whereas Kurarinone treatment suppressed the apoptosis of HG-stimulated HK2 cells, with the decreased percentage of apoptosis cells (Fig. 1C). Therefore, Kurarinone inhibited the apoptosis of HG-stimulated HK2 cells.

Fig. 1.

Kurarinone inhibited apoptosis of HG-stimulated HK2 cells. (A) CCK-8 assays showed the effects of Kurarinone on the viability of HK2 cells at the concentration of 0, 0.25, 0.5, 1, and 2 ‍μM for 24 ‍h. The OD450 value was measured. ^^^p<0.001. (B) CCK-8 assays showed the effects of Kurarinone on the viability of control or HG-induced HK2 cells for 24 ‍h. The OD450 value was measured. ^^^p<0.001, HG vs control, ^aap<0.01, HG + Kur vs HG, ^bbp<0.01, HG + Kur vs Kur. (C) FCM assays showed the effects of Kurarinone on the viability of control or HG-induced HK2 cells for 24 ‍h. The percentage of apoptosis cells was calculated. ^^^p<0.001, HG vs control, ^ap<0.05, HG + Kur vs HG, ^bbp<0.01, HG + Kur vs Kur. HG, high glucose; Kur, Kurarinone.

Kurarinone blocked the ferroptosis of HG-stimulated HK2 cells

Then the effects of Kurarinone on the ferroptosis of HK2 cells were determined. DCF staining showed the ROS levels increased after the treatment of HG in HK2 cells, with the increased staining intensity (Fig. 2A). However, Kurarinone treatment suppressed the DCF staining intensity in HG-stimulated HK2 cells, suggesting the blocking of ROS (Fig. 2A). Further, the expression of ferroptosis, including GPX4 and SLC7A11, was determined using Immunoblot. It was discovered that HG suppressed the expression of GPX4 as well as SLC7A11 in HK2 cells, whereas Kurarinone increased the expression of these proteins in HG-stimulated HK2 cells (Fig. 2B). Further, FCM assays confirmed HG increased the levels of lipid peroxidation in HK2 cells, whereas Kurarinone treatment suppressed the lipid peroxidation in HK2 cells upon HG treatment (Fig. 2C). In addition, it was discovered that HG increased the expression of PTGS2 as well as ACSL4 in HK2 cells, whereas Kurarinone decreased the expression of these proteins in HG-stimulated HK2 cells (Fig. 2D). Further, the inhibitor of Ferroptosis, Fer-1, suppressed the expression of these proteins compared to HG + Kur group (Fig. 2D). Collectively, Kurarinone blocked the ferroptosis of HG-stimulated HK2 cells.

Fig. 2.

Kurarinone blocked the ferroptosis of HG-stimulated HK2 cells. (A) Immunostaining showed the degree of DCF staining in the HK2 cells upon the indicated treatment. Scale bar indicates 100 ‍μm. (B) Immunoblot showed the expression of GPX4 and SLC7A11 in HK2 cells upon the control or HG treatment or Kurarinone incubation for 24 ‍h. (C) Flow cytometry (FCM) to evaluate lipid peroxidation in HK2 cells upon the control or HG treatment or Kurarinone incubation for 24 ‍h. (D) Immunoblot showed the expression of PTGS2 and ACSL4 in HK2 cells upon the control or HG treatment or Kurarinone incubation for 24 ‍h. ^^^p<0.001, HG vs control, ^ap<0.05, ^aaap<0.001, HG + Kur vs HG, ^bbbp<0.001, HG + Kur vs Kur. HG, high glucose; Kur, Kurarinone.

Kurarinone suppressed the mitochondrial damage in HG-stimulated HK2 cells

Subsequently, the effects of Kurarinone on the mitochondrial damage of HK2 cells were explored through JC-1 staining. It was observed that HG treatment increased the intensity of JC-1 monomer in HK2 cells, and decreased the intensity of aggregates, suggesting the promotion of mitochondrial damage (Fig. 3). However, Kurarinone treatment suppressed the intensity of JC-1 monomer, and increased the intensity of aggregates of JC-1 in HG-stimulated HK2 cells (Fig. 3). Therefore, Kurarinone suppressed the mitochondrial damage in HG-stimulated HK2 cells.

Fig. 3.

Kurarinone suppressed the mitochondrial damage in HG-stimulated HK2 cells. JC-1 staining showed the mitochondrial function of HK2 cells upon the control or HG treatment or Kurarinone incubation for 24 ‍h. Green panel indicates JC-1 monomer, red panel indicates JC-1 aggregate, and scale bar indicates 100 ‍μm. HG, high glucose; Kur, Kurarinone.

Kurarinone restrained EMT process in HG-stimulated HK2 cells

The effects of Kurarinone on the EMT process of HK2 cells were further detected. Immunoblot assays showed that HG treatment increased the expression of EMT markers, including Vimentin as well as α-SMA, in HK2 cells (Fig. 4A). However, the expression of these proteins was downregulated after Kurarinone treatment in HG-stimulated HK2 cells (Fig. 4A). Similarly, HG-treatment decreased the expression of E-cadherin, whereas Kurarinone treatment reversed the decrease of E-cadherin expression in HK2 cells caused by HG treatment (Fig. 4B). In summary, Kurarinone restrained EMT process in HG-stimulated HK2 cells.

Fig. 4.

Kurarinone restrained EMT process in HG-stimulated HK2 cells. (A) Immunoblot assays showed the expression of Vimentin and α-SMA in HK2 cells upon the control or HG treatment or Kurarinone incubation for 24 ‍h. The relative expression levels were compared. (B) Immunoblot assays showed the expression of E-cadherin in HK2 cells upon the control or HG treatment or Kurarinone incubation for 24 ‍h. The relative expression levels were compared. ^^p<0.01, ^^^p<0.001, HG vs control, ^ap<0.05, ^aap<0.01, HG + Kur vs HG, ^bp<0.05, ^bbp<0.01, HG + Kur vs Kur. HG, high glucose; Kur, Kurarinone.

Kurarinone activated the Nrf-2 pathway in HG-stimulated HK2 cells

Since Kurarinone contributed to the ferroptosis and EMT of HG-stimulated HK2 cells, the underlying mechanism was determined through Immunoblot assays. HG treatment decreased both Nrf-2 and HO-1 expression in HK2 cells (Fig. 5A). However, Kurarinone treatment further increased the expression levels of these regulators in the Nrf-2/HO-1 pathway in HG-stimulated HK2 cells (Fig. 5A). We further found Kurarinone treatment further increased the expression levels of Nrf-2 in the nucleus in HG-stimulated HK2 cells (Fig. 5B). Additionally, we performed molecular docking between Kur and Nrf2 (2FLU), and the Vina score (binding energy) was −8.2 (Fig. 5C). Therefore, Kurarinone activated the Nrf-2 pathway in HG-stimulated HK2 cells.

Fig. 5.

Kurarinone activated the Nrf-2 pathway in HG-stimulated HK2 cells. (A) Immunoblot assays showed the expression of Nrf-2 and HO-1 in HK2 cells upon the control or HG treatment or Kurarinone incubation for 24 ‍h. The relative expression levels were compared. (B) Immunoblot assays showed the expression of Nrf-2 and Histone H3 in the nucleus of HK2 cells upon the control or HG treatment or Kurarinone incubation for 24 ‍h. The relative expression levels were compared. (C) Molecular docking between Kur and Nrf2 (2FLU) showed the Vina score (binding energy) was −8.2. ^^p<0.01, ^^^p<0.001, HG vs control, ^ap<0.05, ^aaap<0.001, HG + Kur vs HG, ^bbbp<0.001, HG + Kur vs Kur. HG, high glucose; Kur, Kurarinone.

Discussion

DN is one of the more serious diabetic microvascular complications, and one of the most important complications in diabetic patients.⁽¹¹⁾ The drug treatment of DN includes hypoglycemic drugs, antihypertensive drugs, and drugs regulating lipid metabolism disorders.⁽¹²⁾ To combat this disease, more drugs are still needed to develop. Here, HK2 cells were treated with 30 ‍mM glucose for 24 ‍h to construct a DN cell model. This study revealed that Kurarinone improves DN phenotype at the cellular level. Therefore, Kurarinone could serve as a promising drug for DN.

Kurarinone is a flavanone from sophora flavescens roots, which is an effective activator of large conductance of calcium-activated potassium channels.⁽¹³⁾ Kurarinone reduces the contraction of the bladder strip induced by acetylcholine, and reduces the frequency of urination in rat OAB models.⁽¹⁴⁾ Kularidone has cytotoxic and anti-inflammatory activities, and sensitizes TRAIL-induced apoptosis of tumor cells by inhibiting NF-κB dependent cFLIP expressions.⁽¹⁵⁾ Its anti-inflammatory and antioxidant effects have been widely revealed.⁽¹⁶⁾ Interestingly, in this study, its anti-inflammatory and antioxidant effects were demonstrated in a cellular model of DN.

The increase of ROS contributes to the expression of EMT proteins, leading to renal tubule interstitial injury.⁽¹⁷⁾ Excess ROS attacks the biofilm and induces lipid peroxidation, leading to many forms of cell death, such as ferroptosis.^(18,19) Iron overload triggers the Fenton reaction, leading to the accumulation of intracellular ROS and further promoting the progression of EMT.⁽²⁰⁾ Tubular EMT is known to be a key link in the development and progression of renal interstitial fibrosis in DN, but the mechanism of EMT in DN remains unclear. Interestingly, the data in this study confirmed that Kurarinone inhibits HG-induced EMT in HK2 cells. Therefore, it improves DN phenotype in HK2 cells.

At the subcellular level, the mitochondria of the proximal tubule are key organelles regulating ROS and hypoxic damage in diabetic states, and dysfunctional mitochondria accelerates the occurrence of early diabetic tubular lesions. Interestingly, this study revealed the effects of Kurarinone on the mitochondrial function of HG-stimulated HK2 cells.

This study revealed that Kurarinone activates the Nrf-2/HO-1 pathway. Nrf-2 is a member of the transcription factor leucine zipper transcription activator family, and is highly expressed in liver, kidney and other organs. Nrf-2 plays an important role in anti-oxidative stress by regulating the expression of a series of signaling proteins and enzymes. In addition, Nrf-2 is involved in regulating the expression of multiple genes related to iron storage and transport at the transcriptional level, and mice with Nrf-2 knockout showed increased iron content in the spleen and liver. Some studies have found that the protein expression of Nrf-2 in renal tubules of DN mice is reduced, and Nrf-2 knockout increases the sensitivity of HK-2 cells to ferroptosis under HG conditions.⁽²¹⁾ Under oxidative stress, Nrf-2 is transported to the nucleus, activates the transcription of antioxidant response element (ARE) dependent genes. HO-1 is a key antioxidant enzyme regulated by Nrf-2. We believe that Kurarinone can directly affect the Nrf-2 signaling pathway in the case of HG-induced conditions. HG induction itself can affect the Nrf-2 pathway, and Kurainone further inhibits cell damage caused by HG by targeting this pathway. Therefore, the activation of the Nrf-2/HO-1 pathway is a meaningful therapeutic approach to protect DN. Targeting Nrf-2 to regulate lipid peroxidation and ferroptosis is a feasible disease intervention strategy, and this study further confirmed it.

It has been previously reported in the literature that Kur activates the Nrf2 pathway, which plays a crucial role in cellular antioxidant defense mechanisms.⁽⁷⁾ This activation of Nrf2 may contribute to the protective effects of Kur against oxidative stress-related cellular damage, further supporting its potential therapeutic applications in diseases involving oxidative damage.

The previous study indicated Vitexin attenuates ferroptosis and EMT of HK-2 cells via the Nrf-2 pathway.⁽²²⁾ Vitexin ameliorated DN via suppressing GPX4-mediated ferroptosis. The previous study indicated knockdown of GPX4 by shRNA migrated the protective effect of vitexin on HG-challenged HK-2 and reversed the ferroptosis induced by Vitexin. Vitexin alleviated renal fibrosis, damage and ferroptosis in DN rat. Herein our data confirmed Kurarinone activates the Nrf-2/HO-1 pathway and therefore alleviates HG-induced ferroptosis and EMT in HK2 cells, whether it was affected through GPX5 also needs further study.

Conclusion

Kurarinone activates the Nrf-2/HO-1 pathway and therefore alleviates HG-induced ferroptosis and EMT in HK2 cells. Therefore, it could serve as a drug of DN.

Conflict of Interest

No potential conflicts of interest were disclosed.