Cytoprotective agent against ferroptosis

Abstract

[Problem] The purpose of the present invention is to provide, as each useful agent that contains DHMBA as an active ingredient, a cytoprotective agent against ferroptosis, the cytoprotective agent containing DHMBA as an active ingredient and having a cytoprotective action against ferroptosis by verifying the effect of suppressing ferroptosis cell death of DHMBA using a DN model mouse and a human renal tubular cell HK-2. [Solution] The present invention provides a cell agent against ferroptosis, the cell agent containing 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient and having a cytoprotective action against ferroptosis.

Description

Cell protectant against ferroptosis

[0001] The present invention relates to a cell protectant against ferroptosis.

[0002] The oxidative stress state refers to a state in which excessive reactive oxygen species (ROS) accumulate in cells and the balance between oxidation and antioxidation is excessively tilted towards the oxidation side. Mitochondria are the largest energy-producing organs in cells but also the largest source of ROS as a by-product. ROS attacks the factors constituting cells and causes human aging and various oxidative stress-related diseases (Ott M, et al., Apoptosis, 12: 913-922, 2007; Giorgi C, et al., Int Rev Cell Mol Biol, 340: 209-344, 2018). Proximal tubular cells in the kidney are rich in mitochondria and supply the energy required for reabsorbing nutrients such as sugars, amino acids, and fatty acids (Bhargava P and Schnellmann RG, Nat Rev Nephrol, 13: 629-646, 2017). The ROS generated during this process may lead to cell death in tubular epithelial cells. In recent research, a new form of cell death called ferroptosis has attracted attention (Dixon SJ, et al., Cell, 149: 1060-1072, 2012). Ferroptosis is cell death caused by iron-dependent lipid peroxidation and is closely related to both ROS and mitochondrial function (Xie Y, et al., Cell Death Differ, 23: 369-379, 2016; Galluzzi L, et al., Cell Death Differ, 25: 486-541, 2018; Wang H, et al., Eur J Cell Biol, 99: 151058, 2020).

[0003] In the proximal tubular cells of the kidney, the large amount of ROS generated by mitochondria to reabsorb excess nutrients may be a factor in inducing ferroptosis. Clarifying the relationship between cell death and ferroptosis in proximal tubular cells of the kidney and finding ways to control it is expected to contribute to the prevention and management of various kidney diseases. Here, intracellular lipid ROS (reactive oxygen species) refers to substances that react with lipids and proteins in the body, causing oxidative stress and damaging cells and tissues. ROS are a group of highly reactive molecules derived from oxygen molecules (O2) and are mainly produced by mitochondria. They are generated in the body by exposure to environmental factors such as aerobic respiration, air pollutants, and cigarette smoke. In other words, ROS are the main causative agents of lipid peroxidation (lipid peroxides), primarily targeting membrane-bound polyunsaturated fatty acids such as arachidonic acid and linoleic acid. As a byproduct of lipid peroxidation, cell membranes are directly damaged, or protein adducts are formed, damaging cells and tissues. Furthermore, ROS are known to react with biomolecules such as DNA and enzymes in living organisms, causing DNA mutations, protein denaturation, and enzyme inactivation. The Pacific oyster is a bivalve mollusk belonging to the family Ostreidae in the order Ostreida, and is a highly nutritious food source containing large amounts of glycogen, protein, and minerals such as calcium and zinc. The inventors of this study searched for and researched physiologically active substances from the Pacific oyster, and as a result of searching for substances with antioxidant properties from the oyster extract, they discovered the groundbreaking antioxidant 3,5-dihydroxy-4-methoxybenzyl alcohol (hereinafter referred to as DHMBA), and invented many useful agents using it. To date, DHMBA has been shown to have radical scavenging capabilities and activate the Keap1-Nrf2 pathway, leading to the induction of antioxidant gene expression (such as HO-1 and NQO1) (Fuda H, et al., Food Chem, 176: 226-33, 2015; Joko S, et al., J Funct Foods, 35: 245-255, 2017).Furthermore, DHMBA has been shown to protect mitochondrial function in intestinal and renal tubular cells by reducing oxidative stress and improving ATP production (Fukai M, et al., J Clin Med., 10: 1972, 2021; Ho HJ, et al., Int J Mol Sci, 24:10061, 2023). These findings suggest its potential as a therapeutic approach for conditions involving oxidative stress and mitochondrial dysfunction.

[0004] Japanese Patent Publication No. 2024-104803

[0005] The present invention aims to provide a cytoprotective agent against ferroptosis that contains DHMBA as an active ingredient, based on the inventions of various useful agents already obtained by the inventors, which contain DHMBA as an active ingredient. This was done by verifying the effect of DHMBA on suppressing ferroptotic cell death using diabetic nephropathy (DN) model mice and human renal tubular cells HK-2.

[0006] The present invention is a cytoprotective agent against ferroptosis characterized by comprising 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, or characterized in that the cytoprotection is carried out by suppressing the accumulation of iron in the cell, or characterized in that the cytoprotection is carried out by suppressing the accumulation of the lipid ROS in the cell, or characterized in that the cell is a renal tubular cell. Furthermore, it is characterized by having a cytoprotective effect against ferroptosis with 3,5-dihydroxy-4-methoxybenzyl alcohol as the active ingredient, or having an inhibitory effect on intracellular iron accumulation in ferroptosis with 3,5-dihydroxy-4-methoxybenzyl alcohol as the active ingredient, or having an inhibitory effect on intracellular lipid ROS accumulation in ferroptosis with 3,5-dihydroxy-4-methoxybenzyl alcohol as the active ingredient, or having an inhibitory effect on intracellular iron accumulation in human renal tubular HK-2 cells in ferroptosis with 3,5-dihydroxy-4-methoxybenzyl alcohol as the active ingredient, or having an inhibitory effect on intracellular lipid ROS accumulation in human renal tubular HK-2 cells in ferroptosis with 3,5-dihydroxy-4-methoxybenzyl alcohol as the active ingredient, or The active ingredient is 3,5-dihydroxy-4-methoxybenzyl alcohol, which is characterized by having the effect of suppressing ferroptosis and improving the cell viability reduced by glucotoxicity in renal tubular cells, or 3,5-dihydroxy-4-methoxybenzyl alcohol, which is characterized by having the effect of suppressing ferroptosis and improving the cytotoxicity increased by lipid toxicity in renal tubular cells.

[0007] According to the present invention, the effects of each useful agent containing DHMBA as an active ingredient can be verified using diabetic nephropathy (DN) model mice and human renal tubular cells HK-2, thereby providing an excellent effect of providing a cytoprotective agent against ferroptosis that contains DHMBA as an active ingredient and has a cell-protective effect against ferroptosis.

[0008] This is an explanatory diagram illustrating the flow of animal experiments in the present invention. This is an explanatory diagram showing the sequences of primers used in the present invention as Table 1. This is an explanatory diagram illustrating the changes in the expression levels of kidney injury marker-related genes in DN mice. Renal Ngal expression level (A). Renal Kim-1 expression level (B). Renal Type 1 expression level (C). DN mice were administered DHMBA at 2 and 20 mg / kgBW for 4 weeks, then dissected, kidney mRNA was collected, cDNA was synthesized, and the expression levels of kidney injury marker-related genes were measured by qPCR. Mean ± SD (n = 4-6). This is an explanatory diagram illustrating the plasma iron concentration in DN mice. DN mice were administered DHMBA at 2 and 20 mg / kgBW for 4 weeks, then dissected, plasma was collected, and the plasma iron concentration was measured using the Iron Assay Kit. Mean ± SD (n = 5-6). This is an explanatory diagram illustrating the kidney MDA concentration in DN mice. DN mice were administered DHMBA at 2.20 mg / kgBW for 4 weeks, then dissected and their kidneys were recovered. Kidney MDA concentrations were measured using the TBARS Assay Kit after homogenization with PBS. Mean ± SD (n = 5-6). This is an explanatory diagram illustrating the cytoprotective effect of DHMBA under Era and FAS stimulation. HK-2 cells were cultured with Erastin at 0.5 μM and DHMBA at 62.5-500 μM, and cytoprotective tests were performed by measuring cell viability (A) and cytotoxicity (B). Similarly, cells were cultured with FAS at 100 μM and DHMBA at 62.5-500 μM, and cytoprotective tests were performed by measuring cell viability (C) and cytotoxicity (D). Mean ± SD (n = 6). This is an explanatory diagram illustrating the inhibitory effect of DHMBA on the accumulation of intracellular iron and lipid ROS under Era stimulation. HK-2 cells were cultured with 0.5 μM Erastin and 250 and 500 μM DHMBA, and then fluorescently stained for intracellular iron (A) and lipid ROS (B). This is an explanatory diagram illustrating the cytoprotective effect of DHMBA upon addition of ML162.HK-2 cells were cultured with 1 μM ML162, 250 and 500 μM DHMBA, and 1 μM Fer-1, and then cytoprotection tests were performed using cell viability (A) and cytotoxicity (B). Mean ± SD (n = 6). This is an explanatory diagram illustrating the sequence of primers used in the second experiment. This is an explanatory diagram illustrating the reduction of cytotoxicity and improvement of cell viability by adding DHMBA. (A) is an explanatory diagram illustrating the expression levels of ferroptosis-related genes in HK-2 cells under the glucotoxicity and lipid toxicity of GPX4, and (B) is an explanatory diagram illustrating the expression levels of ferroptosis-related genes in HK-2 cells under the glucotoxicity and lipid toxicity of NCOA4. (A) is an explanatory diagram illustrating the protein expression levels in the expression levels of ferroptosis-related proteins in HK-2 cells under the glucotoxicity and lipid toxicity, and (B) is an explanatory diagram illustrating the results of protein quantification in the expression levels of ferroptosis-related proteins in HK-2 cells under the glucotoxicity and lipid toxicity.

[0009] To verify the effects of the present invention, animal experiments were conducted, and the details are described below. (Experimental Method) 1. Animal Rearing Experiments were conducted using 7-week-old male C57BL / 6NJcl mice (CREA Japan). The mice were reared in an environment with a temperature of 22 ± 2℃, humidity of 50 ± 10%, and a 12-hour light-dark cycle (8:00-20:00 light period). The animal experiments in this study were conducted appropriately with the approval of the Animal Experiment Ethics Committee of Hokkaido University, in compliance with the Animal Welfare and Management Act and the "Regulations Concerning Animal Experiments of Hokkaido University, a National University Corporation," and in compliance with ethical principles based on the 3Rs (Replacement, Reduction, Refinement), which are the basic concepts of animal welfare. Animal Ethics Number: 23-0048. The flow of the experiment is shown in Figure 1. Specifically, diabetic nephropathy (DN) was induced in mice by feeding them a high-fat diet (HFD32, CLEA Japan) for 8 weeks, followed by 4 weeks of high-fat dieting along with 5 intraperitoneal injections of 35 mg / kgBW streptozotocin (Fang JY, et al. Nutrients, 11: 530, 2019; Furman BL., Curr Protoc Pharmacol. 70: 5.47.1-5.47.20., 2015). These DN mice were then orally administered 2 mg / kgBW and 20 mg / kgBW DHMBA for 4 weeks. Dissection was performed at week 13, and plasma and kidney tissue were collected and evaluated.

[0010] The mice were divided into the following groups: (1) Standard diet group (Control): CE-2 diet (Claire Japan) (2) Diabetic nephropathy group (DN): HFD32 diet + intraperitoneal streptozotocin injection (35 mg / kgBW) (3) Diabetic nephropathy + 2 mg / kgBW DHMBA administration group (DN+D2): HFD32 diet + intraperitoneal streptozotocin injection (35 mg / kgBW) + oral DHMBA (2 mg / kgBW) (4) Diabetic nephropathy + 20 mg / kgBW DHMBA administration group (DN+D20): HFD32 diet + intraperitoneal streptozotocin injection (35 mg / kgBW) + oral DHMBA (20 mg / kgBW) 2. Measurement of gene expression levels of renal damage markers It has been reported that the expression of the renal Ngal and Kim-1 genes is significantly increased in diabetic nephropathy model mice (Liu F, et al.) (al., Adv Med Sci, 60:133-138, 2015). In addition, increased expression of Type1 was confirmed in renal tubular cells of diabetic patients and model mice (Liu L, et al., Redox Biol, 52:102302, 2022). Overexpression of these genes is associated with damage to renal tubules and decreased renal function, and is thought to contribute to the progression of diabetic nephropathy. To evaluate the effect of DHMBA on renal function in diabetic nephropathy model mice, total RNA from the kidneys of mice was recovered using the NucleoSpin® RNA Kit (MACHEREY-NAGEL), and the expression levels of the Ngal and Kim-1 genes were measured. Specifically, the concentration of purified RNA was quantified using Nanodrop (Invitrogen). cDNA was synthesized from 1.0 μg of total RNA according to the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO) protocol. Gene expression levels were measured according to the THUNDERBIRD® SYBR® qPCR Mix (TOYOBO) protocol. Measurements were performed using the CFX Connect™ Real-time PCR Analysis System (BioRad), and analysis was performed using the 2-(ΔΔCT) method.The gene expression levels of the control group were set to 1 for comparison. Each expression level was corrected for the expression level of the housekeeping gene Actin. The primer sequences used are shown in Table 1 and Figure 2.

[0011] 3. Iron Concentration in Mouse Plasma To investigate the iron concentration in the blood of DN mice, the Iron Assay Kit (Ab83366, Colorimetric) (Abcam) was used to measure the iron concentration in mouse plasma. Standard samples and 10-fold diluted plasma were added to a 96-well plate, and 5 μl of Iron Reducer was added and mixed thoroughly. The mixture was incubated at 37 °C for 30 minutes. Then, 100 μl of Iron Probe was added and mixed thoroughly, and the mixture was incubated at 37 °C for 60 minutes. Finally, the absorbance at 593 nm was measured using a plate reader (Wallac 1420 ARVO Mx plate reader, PerkinElmer) to evaluate the iron concentration in the plasma.

[0012] 4. Mouse Kidney MDA (Malondialdehyde) Concentration MDA (malondialdehyde) is a compound produced by lipid peroxidation and is valued as an indicator of oxidative stress (Tsikas D. Anal Biochem, 524:13-30, 2017). DN mouse kidneys were homogenized with PBS, centrifuged, and the supernatant was collected. The MDA concentration of the collected samples was measured according to the TBARS Assay Kit (Cayman CHEMICAL) protocol. Specifically, a color reagent was prepared by mixing 17.5 mL TBA Acetic Acid, 185.5 mg TBA, and 17.5 mL TBA Sodium Hydroxide. 25 μL of SDS Solution, 25 μL of diluted standard solution or sample, and 1 mL of color reagent were added to a 1.5 mL tube in that order, and the mixture was heated at 100 °C for 1 hour. After standing on ice for 10 minutes, the samples were centrifuged at 12,000 rpm for 10 minutes. The supernatant was measured for fluorescence at excitation wavelength 530 nm and emission wavelength 550 nm using a fluorescence spectrophotometer (FP-6500, M-Technique). The obtained MDA amount was corrected for the total protein amount. The total protein amount was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher). Regent A and Regent B were mixed in a 50:1 ratio to prepare the BCA Working Regent. 5 μL of standard solution or sample and 100 μL of BCA Working Regent were added to a 96-well plate, incubated at 37 °C for 30 minutes, and the absorbance at 562 nm was measured using a plate reader (Wallac 1420 ARVO Mx plate reader).

[0013] 5. Cell Culture Human renal tubular HK-2 cells were subcultured in Dulbecco's Modified Eagle's Medium (DMEM, Nacalai Tesque) medium containing 10% fetal bovine serum and 1% penicillin-streptmycin at 37°C in a 5% CO2 incubator. 6. Cell Viability and Toxicity Tests To confirm the cytoprotective effect of DHMBA against ferroptosis cell death, cell toxicity and viability were evaluated using the ferroptosis inducer erastin (Era) and ammonium iron(II) sulfate hexahydrate (FAS, Fujifilm Wako Pure Chemical Industries). HK-2 cells were seeded in a 96-well plate at a concentration of 6.0 × 10³ cells / well and cultured for 24 hours. Next, Era was added simultaneously to a final concentration of 0.5 μM and DHMBA to a final concentration of 62.5–500 μM. After 24 hours of incubation, 50 μL of the supernatant was taken and transferred to a new 96-well plate. A mixture of the catalyst and dye solution (45:1) from the LDH Cytotoxicity Detection Kit (Takara Bio Inc.) was added at 50 μL / well. After standing at room temperature for 30 minutes, the absorbance at 490 nm was measured using a plate reader (Wallac 1420 ARVO Mx plate reader, PerkinElmer) to measure cytotoxicity. Additionally, 10 μL / well of CCK-8 reagent (Dojin Chemical Laboratories Co., Ltd.) was added to the plate in which the cells had been cultured. After incubation at 37 °C for 2 hours, the absorbance at 450 nm was measured using a plate reader (Wallac 1420 ARVO Mx plate reader) to evaluate cell viability. Cell toxicity and viability were calculated from the absorbance values ​​relative to the control (0 μg / mL) (n = 6 for each group).

[0014] 7. Fluorescence staining of intracellular iron and lipid ROS. Since cell death induced by Era has been reported to be related to the accumulation of intracellular iron and lipid ROS (Zhao J, et al. Mol Med Rep. 24:713, 2021), the amount of intracellular iron accumulation was evaluated. For iron staining, HK-2 cells were seeded at 1.0 × 10⁵ in a 35 mm dish and cultured for 24 hours, after which Era (0.5 μM) and DHMBA (250, 500 μM) were simultaneously added. After 16 hours of culture, Hank's Balanced Salt Solution (HBSS, Fujifilm Wako Pure Chemical Industries) containing 1 μM FerroOrange (Dojin Chemical Laboratories) was added, and the cells were incubated at 37 °C for 30 minutes before being observed with a fluorescence microscope BZ-9000 (Keyence). Furthermore, in intracellular lipid ROS staining, HK-2 cells were seeded at a density of 1.0 × 10⁵ in a 35 mm dish and cultured for 24 hours. Era (0.5 μM) and DHMBA (250, 500 μM) were added simultaneously. After 16 hours of culture, HBSS containing 2 μM C11-BODIPY581 / 591 (Dojin Chemical Research Institute) was added, and the cells were incubated at 37 °C for 20 minutes before being observed with a fluorescence microscope BZ-9000 (Keyence). 8. Evaluation of the GPx4 antioxidant pathway GPx4 is an enzyme that suppresses lipid peroxidation and plays an important role in inhibiting ferroptosis, and may be a promising therapeutic target in the treatment of ferroptosis-related diseases. The GPx4 inhibitor ML162 has been used in studies of pathways involving GPx4 (Weiwer M, et al, Bioorg Med Chem Lett. 22: 1822-1826, 2012). Therefore, we evaluated the cytoprotective effect of DHMBA using the GPx4 inhibitor ML162. HK-2 cells were seeded at a rate of 6.0 × 10³ cells / well in a 96-well plate and cultured for 24 hours. Then, the GPx4 inhibitor ML162 (1 μM), DHMBA (250, 500 μM), and the anti-ferroptizing agent Fer-1 (1 μM) were added simultaneously.After 24 hours of incubation, 50 μL of the supernatant was taken and transferred to a new 96-well plate. A mixture of the catalyst and dye solution (45:1) from the LDH Cytotoxicity Detection Kit (Takara Bio Inc.) was added at 50 μL / well. After standing at room temperature for 30 minutes, the absorbance at 490 nm was measured using a plate reader (Wallac 1420 ARVO Mx plate reader, PerkinElmer) to measure cytotoxicity. Additionally, 10 μL / well of CCK-8 reagent (Dojin Chemical Laboratories Co., Ltd.) was added to the plate in which the cells had been cultured. After incubation at 37 °C for 2 hours, the absorbance at 450 nm was measured using a plate reader (Wallac 1420 ARVO Mx plate reader) to evaluate cell viability. Cell toxicity and viability were calculated from the absorbance values ​​relative to the control (0 μg / mL) (n = 6 for each group).

[0015] 9. Statistical Analysis The obtained data were expressed as mean ± standard deviation (SD) and analyzed using Prism 10.2.3 (GraphPad Software). Dunnett's test was used for multiple comparisons between groups, with p<0.05 being the statistical significance level. (Results) 1. Changes in Renal Damage Marker Genes in DN Mice In the kidneys of DN mice (DN group), the expression levels of Ngal, Kim-1, and Type1, which are biomarkers for renal damage and DN, were significantly increased compared to mice on a normal diet (Control group), but decreased with the administration of DHMBA (DN+D2 group and DN+D20 group) (Figure 3). This result suggests that DHMBA improved the increased renal tubular damage and decreased renal function caused by DN, and suppressed the progression of DN.

[0016] 2. Plasma iron concentration in DN mice: Compared to the plasma iron concentration of mice on a normal diet (Control group), the plasma iron concentration in DN mice (DN group) tended to be elevated, but decreased with the administration of DHMBA (DN+D2 group and DN+D20 group) (Figure 4). This result indicates that DHMBA suppressed the elevated blood iron concentration caused by DN. 3. Renal MDA concentration in DN mice: Compared to the renal MDA concentration of mice on a normal diet (Control group), the renal MDA concentration in DN mice (DN group) was elevated, but decreased with the administration of DHMBA (DN+D2 group and DN+D20 group) (Figure 5). This result indicates that DHMBA suppressed the increased lipid peroxide products caused by DN.

[0017] 4. Cell Viability and Cytotoxicity When HK-2 cells were treated with 0.5 μM Era (0.5 μM Era + 0 μM DHMBA group), low cell viability (Figure 6A) and high cytotoxicity (Figure 6B) were observed. Next, co-treatment with 0.5 μM Era and DHMBA (62.5-500 μM) resulted in increased cell viability (Figure 6A) and suppression of cytotoxicity (Figure 6B). Furthermore, HK-2 cells treated with 100 μM FAS (100 μM FAS + 0 μM DHMBA group) also showed low cell viability (Figure 6C) and high cytotoxicity (Figure 6D). Co-treatment with 100 μM FAS and DHMBA (62.5-500 μM) resulted in increased cell viability (Figure 6C) and suppression of cytotoxicity (Figure 6D). DHMBA was shown to protect HK-2 from Era and FAS-induced ferroptosis. 5. Fluorescence staining of intracellular iron and lipid ROS In staining of intracellular iron using FerroOrange reagent, the addition of 0.5 μM Era (0.5 μM Era + 0 μM DHMBA group) to HK-2 cells resulted in stronger fluorescence than the control group, but the addition of DHMBA (0.5 μM Era + 250, 500 μM DHMBA group) resulted in a decrease in fluorescence (Figure 7A). Furthermore, when staining lipid ROS using the C11-BODIPY581 / 591 reagent, stronger fluorescence was observed in HK-2 cells when 0.5 μM Era (0.5 μM Era + 0 μM DHMBA group) was added compared to the control group. However, the addition of DHMBA (0.5 μM Era + 250, 500 μM DHMBA groups) resulted in a decrease in fluorescence (Figure 7B). These results suggest that DHMBA suppresses the accumulation of intracellular iron and lipid ROS induced by Era addition.

[0018] 6. Cell viability and cytotoxicity due to inhibition of the GPx4 antioxidant pathway. ML162 is an inhibitor of the antioxidant enzyme GPx4, and cell viability in the ML162-supplemented group (1 μM ML162 + 0 μM DHMBA group) was significantly reduced compared to the control group (Figure 8A). Co-addition of the anti-ferroptotic agent Fer-1 (1 μM ML162 + 1 μM Fer-1 group) restored cell viability, while co-addition of DHMBA (1 μM ML162 + 250, 500 μM DHMBA groups) hardly restored it. Furthermore, cytotoxicity in the ML162-supplemented group was significantly increased compared to the control group, but Fer-1 improved it to the level of the control group, while co-addition of DHMBA resulted in high cytotoxicity (Figure 8B). These results indicate that DHMBA showed little cytoprotective effect against ML162-induced ferroptosis. Therefore, it is possible that the GPX4-related pathway is involved in the protective effect of DHMBA on ferroptotic cell death.

[0019] (Conclusion) In ferroptosis, excess iron accumulated in cells amplifies intracellular ROS through the Fenton reaction, leading to lipid oxidation-dependent cell death. Therefore, it is thought that removing intracellular iron and intracellular ROS, and suppressing the accumulation of lipid peroxides, can protect cells from ferroptosis.

[0020] In this invention, we confirmed the protective effect of DHMBA against ferroptosis in DN mouse and HK-2 tubular cells. Elevated gene expression levels, plasma iron concentration, and lipid peroxide MDA levels related to renal dysfunction were observed in DN mice, and these changes improved after 4 weeks of DHMBA administration. Furthermore, in HK-2 cells, cell viability, cytotoxicity tests, and cell observations by fluorescence staining suggested that the cytoprotective effect of DHMBA against ferroptosis was due to the suppression of intracellular iron and lipid ROS accumulation. On the other hand, DHMBA did not show a cytoprotective effect against ferroptosis when GPx4 was inhibited, suggesting that the GPx4 antioxidant pathway may be involved in the cytoprotective mechanism of DHMBA against ferroptosis. These results indicate that DHMBA possesses physiological activity to suppress ferroptosis in the kidneys and renal tubular cells of DN mice.

[0021] "Second Experiment" The inventors of this study conducted a second experiment, and the details of that experiment are described below. (Introduction) Ferroptosis is characterized by the accumulation of iron ion-dependent lipid peroxides and refers to programmed cell death caused by the production of reactive oxygen species that lead to oxidative stress. Previous studies have shown that diabetes increases iron accumulation and that the expression of ferroptosis markers is confirmed in renal tubular cells. Furthermore, ferroptosis has been shown to be involved in the progression of various diseases, including diabetic nephropathy.

[0022] Diabetic nephropathy, a complication of diabetes with a relatively high mortality rate, is known to be linked to hyperglycemia and oxidative stress, which promote ferroptosis and contribute to its progression. Furthermore, diabetic nephropathy has been reported to be involved in glucotoxicity and lipid toxicity in renal cells. On the other hand, the food-derived antioxidant DHMBA has been shown to reduce lipid peroxide markers and inhibit apoptosis and necrosis in hepatocytes. It has also reduced oxidative stress in tubular cells and shown to improve mitochondrial morphology and function. Therefore, this study aimed to clarify the effects of DHMBA on ferroptosis in diabetic nephropathy.

[0023] (Subjects and experimental method of the second experiment) 1. Cell culture Human renal tubule HK-2 cells were cultured in DMEM medium in the presence of 5% CO2 in a 37°C incubator. 2. Cell viability and cytotoxicity of HK-2 cells HK-2 cells were placed in a 96-well plate in a 3.0 × 10⁶ format. 3 Cells were seeded at a rate of cells / well, and glucose (5.5–40 mM), palmitic acid (PA, 100 μM), and DHMBA (500 μM) were added simultaneously. After 24 hours of incubation, 50 μL of the supernatant was taken and transferred to a new 96-well plate, and 50 μL / well of LDH reagent was added. After standing at room temperature for 30 minutes, the absorbance at 490 nm was measured to evaluate cytotoxicity. Additionally, 10 μL / well of Cell Counting Kit-8 (CCK-8) was added to the cell-containing plate, incubated at 37°C for 90 minutes, and the absorbance at 450 nm was measured to evaluate cell viability.

[0024] 3. Measurement of gene expression levels in HK-2 cells. HK-2 cells were placed in a 96-well plate in a 3.0 × 10⁶ well. 3Cells were seeded at a rate of cells / well, and glucose (5.5-40 mM), PA, and DHMBA were added simultaneously. After 16 hours of culture, cells were harvested, mRNA was extracted, and cDNA was synthesized by reverse transcription. The expression levels of the ferroptosis-related genes GPX4 and NCOA4 were then measured by qRT-PCR. The expression levels of each gene were corrected for the housekeeping gene β-actin. The sequences of the primers used are shown in Table 2 in Figure 9. 4. Detection of ferroptosis-related proteins in HK-2 cells HK-2 cells were placed in a 96-well plate at a rate of 3.0 × 10⁶ 3 Cells were seeded in a cell / well, and PA and DHMBA were simultaneously added to the Normal glucose group (Control, 5.5 mM) and the High glucose group (HG, 40 mM). After 16 hours of culture, cells were harvested and proteins were extracted. The expression levels of anti-ferroptosis proteins xCT, CD98, and FSP1, and ferroptosis-promoting proteins DMT1 and ACSL4 were measured by Western blotting.

[0025] 5. Statistical Analysis All data were expressed as mean ± standard deviation, and the Tukey-Kramer test and Dunnett test were used to test for significance of the mean. In both cases, *p < 0.05, **p < 0.01, and ***p < 0.001 were used.

[0026] (Results) 1. Cell viability and cytotoxicity of HK-2 cells: Cell toxicity increased with the addition of PA, but decreased with the addition of DHMBA (Figure 10A). On the other hand, cell viability decreased significantly with the addition of PA, depending on the glucose concentration, but improved with the addition of DHMBA (Figure 10B). 2. Detection of ferroptosis-related genes in HK-2 cells: GPX4 expression increased with stimulation of high glucose (40 mM) and high glucose + PA, but decreased with the addition of DHMBA (Figure 11A). NCOA4 expression also increased with the same stimulation, and increased further with the addition of DHMBA (Figure 11B).

[0027] 3. Detection of ferroptosis-related proteins in HK-2 cells: Addition of high glucose (HG, 40 mM) and PA decreased the expression levels of anti-ferroptosis proteins xCT, CD98, and FSP1, while increasing the expression levels of ferroptosis-promoting proteins DMT1 and ACSL4. Addition of DHMBA increased the expression levels of CD98 and FSP1, while decreasing the expression levels of DMT1 and ACSL4 (Figure 12).

[0028] (Discussion) In the first experiment, it was confirmed that ferroptosis is involved in DN. In addition, increases in Ngal, a marker of renal damage, and Tgfb1, a marker involved in the promotion of epithelial-mesenchymal transition, were observed in the tubular cells of DN patients and DN model animals. Gpx4 plays an important role in suppressing ferroptosis, and it has been reported that Gpx4 expression increases in diabetic nephropathy to counteract increased lipid peroxides, etc. Based on the results from the DN model mouse, it is thought that it is also involved in glucotoxicity and lipid toxicity in renal tubular cells in diabetic nephropathy. It is thought that the expression level of the gene GPX4, which plays an important role in suppressing ferroptosis, increased as a compensatory effect due to glucotoxicity and lipid toxicity. Furthermore, NCOA4 is an important gene that regulates intracellular iron utilization, and its expression level increased further by the addition of DHMBA, suggesting that DHMBA is involved in regulating intracellular iron concentration. In addition, xCT, CD98, and FSP1 are anti-ferroptosis proteins that maintain cellular function by suppressing lipid peroxidation. Furthermore, DMT1 and ACSL4 are ferroptosis-promoting proteins that promote iron accumulation and lipid peroxidation, inducing cell death. In the first experiment, it was shown that stimulation with high concentrations of glucose and lipids reduces the cell viability of renal tubular cells. It has also been shown that ferroptosis occurs in renal tubular cells in diabetic nephropathy, contributing to damage to renal tissue.

[0029] Furthermore, the results of the second experiment confirmed that DHMBA suppressed ferroptosis and improved the cell viability and cytotoxicity of HK-2 cells that had been exacerbated by glucotoxicity and lipid toxicity. This experiment suggests that DHMBA, by suppressing ferroptosis, improves the decreased cell viability and increased cytotoxicity in renal tubular cells caused by glucotoxicity and lipid toxicity. In the future, elucidating the effects of DHMBA on lipid oxidation, oxidative stress, and mitochondrial function involved in ferroptosis is expected to lead to the development of effective treatments for related diseases.

Claims

1. A cytoprotective agent against ferroptosis, characterized by comprising 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient.

2. The cytoprotective agent according to claim 1, characterized in that the cell protection is achieved by suppressing the accumulation of iron in the cell.

3. The cytoprotective agent according to claim 1, characterized in that the cell protection is achieved by suppressing the accumulation of intracellular lipid ROS.

4. The cytoprotective agent according to claim 1, 2, or 3, characterized in that the cells are renal tubular cells.

5. A cell viability improving agent comprising 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, characterized by having the effect of suppressing ferroptosis and improving the cell viability rate of renal tubular cells that has been reduced by glucotoxicity.

6. A cytotoxicity improving agent comprising 3,5-dihydroxy-4-methoxybenzyl alcohol as an active ingredient, characterized by having the effect of suppressing ferroptosis and improving cytotoxicity increased by lipid toxicity in renal tubular cells.

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