Ophiopogonin D improves oxidative stress and mitochondrial dysfunction in pancreatic β cells induced by hydrogen peroxide through Keap1/Nrf2/ARE pathway in diabetes mellitus Zhang H, Kang X, Ruan J, Ma L, Peng W, Shang H, Wang B, Sun Y,

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ORIGINAL ARTICLE

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Ophiopogonin D improves oxidative stress and mitochondrial dysfunction in pancreatic β cells induced by hydrogen peroxide through Keap1/Nrf2/ARE pathway in diabetes mellitus

Hongyan Zhang¹, Xuezhi Kang², Jun Ruan³, Li Ma¹, Wenbo Peng¹, Haonan Shang¹, Bing Wang¹, Yongning Sun³
¹ Department of Traditional Chinese Medicine, Shanghai Sixth People's Hospital, Shanghai, China
² Department of Acupuncture and Traumatology, Shanghai Sixth People's Hospital, Shanghai, China
³ Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China

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Date of Submission	10-May-2023
Date of Decision	07-Jul-2023
Date of Acceptance	04-Aug-2023
Date of Web Publication	20-Nov-2023

Abstract

Diabetes mellitus (DM) is a metabolic disease characterized by high blood sugar. Due to its complex pathogenesis, no effective drugs have been found so far. Ophiopogonin D (OP-D) has anti-inflammatory, antioxidant, and anticancer activities, but its role in DM has not been studied so far. Hydrogen peroxide (H₂O₂) was used to induce INS-1 cells. INS-1 cells induced by H₂O₂ were treated with OP-D, and cell apoptosis, oxidative stress damage, and related indexes of mitochondrial function were respectively detected by cell counting kit-8, flow cytometry, western blot, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, JC-1 fluorescent probe, and related kits. Subsequently, molecular docking techniques were used to investigate the relationship between OP-D and Keap1 and to explore the regulation mechanism of OP-D on H₂O₂-induced oxidative stress and mitochondrial function in INS-1 cells. OP-D inhibited the apoptosis and oxidative stress level of H₂O₂-induced INS-1 cells, thereby inhibiting cell damage. Moreover, OP-D inhibited mitochondrial dysfunction in H₂O₂-induced INS-1 cells. At last, we found that Keap1/Nrf2 specific signaling pathway inhibitor ML385 was able to reverse the inhibitory effect of OP-D on H₂O₂-induced oxidative stress and mitochondrial dysfunction in INS-1 cells. In conclusion, OP-D improves oxidative stress and mitochondrial dysfunction in pancreatic β cells induced by H₂O₂ through activating Keap1/Nrf2/ARE pathway in DM.

Keywords: Diabetes mellitus, Keap1/Nrf2/ARE pathway, mitochondrial dysfunction, ophiopogonin D, oxidative stress

How to cite this URL:
Zhang H, Kang X, Ruan J, Ma L, Peng W, Shang H, Wang B, Sun Y. Ophiopogonin D improves oxidative stress and mitochondrial dysfunction in pancreatic β cells induced by hydrogen peroxide through Keap1/Nrf2/ARE pathway in diabetes mellitus. Chin J Physiol [Epub ahead of print] [cited 2023 Nov 30]. Available from: https://www.cjphysiology.org/preprintarticle.asp?id=389960

Hongyan Zhang, Xuezhi Kang: Equal contribution

Introduction

Diabetes mellitus (DM) is a chronic, progressive, non-infectious lifelong disease and a metabolic disease characterized by hyperglycemia.^[1] Long-term hyperglycemia can cause damage to various target organs, lead to various complications, bring heavy mental pressure and economic burden to patients, endanger human physical and mental health, and even contribute to death in severe cases.^[2] So the search for a cure for DM is urgent.

A previous study has shown that the dysfunction of pancreatic β cells is the central link in the pathogenesis of DM.^[3],[4] Oxidative stress is an important factor in the occurrence and development of DM. Long-term hyperglycemia environment causes oxidative stress in pancreatic β cells, which leads to imbalance between oxidant and antioxidant systems, and excessive release of free radicals induces apoptosis and damage of pancreatic β cells.^[5],[6],[7] Therefore, inhibition of oxidative stress and apoptosis of pancreatic β cells is an effective treatment to control the development of DM.

Ophiopogonin D (OP-D) is a steroid saponin extracted from the root tuber of Ophiopogonin, which has anti-inflammatory, antioxidant, and anticancer activities.^[8] The study has shown that OP-D inhibits reactive oxygen species (ROS) overproduction through the Wnt/β-catenin signaling pathway and improves bone integration of titanium alloy implants in diabetic conditions.^[9] OP-D improves renal function by inhibiting oxidative stress and inflammatory response in streptozotocin-induced diabetic nephropathy rats.^[10] However, the effect of OP-D on pancreatic β cells in DM has not been reported.

Therefore, in this paper, we will explore the effect of OP-D on pancreatic β cells in DM and its regulatory mechanism, to provide a solid theoretical basis for the clinical OP-D-based treatment of DM.

Materials and Methods

Cell culture

Rat pancreatic β-cells INS-1 were obtained from the BeNa Culture Collection (cat. no. BNCC337862, Henan, China). INS-1 cells were cultured in Dulbecco's modified eagle medium (Gibco) supplemented with 10% FBS (Gibco) at 37°C in a humidified atmosphere with 5% CO₂ and 95% air. INS-1 cells were incubated with 50 μM H₂O₂ for an additional 2 h and then 5 μM, 10 μM and 20 μM OP-D (Cat. no. HY-N0515, Purity: 98.59%, MedChemExpress) was used to culture the INS-1 cells for 24 h. As H₂O₂ is unstable, it was prepared locally and protected from light. To investigate the effects of OP-D on H₂O₂-induced INS-1 cells, the cells were then divided into control, H₂O₂, H₂O₂ + 5 μM OP-D, H₂O₂ + 10 μM OP-D, and H₂O₂ + 20 μM OP-D groups. In addition, INS-1 cells were treated with 10 μM ML385 (cat. no. HY-100523, MedChemExpress), a Keap1/Nrf2 specific signaling pathway inhibitor, for 24 h and cells were divided into control, H₂O₂, H₂O₂ + OP-D and H₂O₂ + OP-D + ML385 groups.

Cell counting kit-8

Cells were seeded in 96-well plates at the density of 1 × 10⁴ cells per well and subjected to indicated treatment. At the end of treatment, 10 μL cell counting kit-8 (CCK-8) reagent was added to each well for 2 h of incubation at 37°C. The absorbance value at 450 nm was measured by a microplate reader to evaluate the cell viability.

Flow cytometry analysis

Cell apoptosis was analyzed using an Annexin V-FITC/PI apoptosis detection kit according to the instructions (BD Biosciences, San Jose, CA, USA). INS-1 cells were subjected to the indicated treatment and then digested with trypsin, and resuspended in a binding buffer solution containing Annexin V-FITC and PI for 15 min at dark at room temperature. At last, INS-1 cells were analyzed by flow cytometry.

Western blot

Cell protein lysates were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (PVDF; Millipore, USA). According to the instructions, the cells were treated with primary and secondary antibodies (Abcam). The signals were visualized by using an Enhanced ECL Chemiluminescent Substrate Kit (Yeasen Biotechnology (Shanghai) Co., Ltd.), and the signal intensity was measured with ImageJ software (Bio-Rad Laboratories, Inc.).

Enzyme-linked immunosorbent assay

Nicotinamide adenine dinucleotide phosphate (NADPH), insulin and oxidative stress-related factors malonaldehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels were assayed by enzyme-linked immunosorbent assay (ELISA) kits provided by Nanjing Jiancheng Bioengineering Institute following the manufacturer's protocol.

RNA isolation and real-time quantitative polymerase chain reaction

The total RNA in cells was isolated with TRIzol reagent (Invitrogen), and then reversed and transcribed into cDNA via the PrimeScript RT reagent Kit (Takara, Dalian, China). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using SYBR Green Kit (Takara) on the ABI 7500 real-time PCR system (Applied Biosystems). The thermal cycling conditions were 5 min at 95°C, followed by 40 cycles of 15 s at 95°C and 34 s at 60°C. GAPDH was selected as an internal control gene for standardization and the expression level was calculated according to the 2^−ΔΔCt method. The sequences of the primers were as follows: Insulin 1 forward 5'-CTACACACCCAAGTCCCGTC-3' and reverse 5'-CCAAGGTCTGAAGATCCCCG-3'; Insulin 2 forward 5'-TTATCCTCTTATCCGTCCTC-3' and reverse 5'-TGTTAAGTCGAAGGGAGC-3'; GAPDH forward 5'-GCATCTTCTTGTGCAGTGCC-3' and reverse 5'-GATGGTGATGGGTTTCCCGT-3'.

For the measurement of mitochondrial DNA (mtDNA), cells were lysed and centrifuged (800 × g) at 4°C for 5 min to obtain supernatants. Then, supernatants were treated with Cell Mitochondria Isolation Kit (Beyotime) and centrifuged (15,000 × g) at 4°C for 10 min to extract the mitochondria. mtDNA was extracted from mitochondria using a Universal Genomic DNA Kit (CWBIO, Beijing, China). 10 ng of the DNA was measured by the RT-qPCR. The mitochondrial ND1 gene (mtND1) was used to measure mtDNA copy number and was normalized to the nuclear hemoglobin subunit beta gene (Hbb). The sequences of the primers were as follows: mtND1 forward 5'-TTATCCTCTTATCCGTCCTC-3' and reverse 5'-TGTTAAGTCGAAGGGAGC-3'; Hbb forward 5'-CAGTACTTTAAGTTGGAAAC-3' and reverse 5'-ATCAACATAATTGCAGAGC-3'.

Determination of mitochondrial membrane potential

JC-1 fluorescent probe was used to detect mitochondrial membrane potential (MMP) in INS-1 cells. After indicated treatment, INS-1 cells were washed with PBS and JC-1 was added to stain the cells at 37°C for 20 min. Cells were fixed with 4% paraformaldehyde for 10 min after being washed with pre-cooled diluted dye buffer. At last, an anti-fluorescence quencher was added to the coverslips (10 μL/well). JC-1 monomers (green fluorescence, 527 nm) in the cytoplasm rely on the polarity of the mitochondrial membrane to enter the mitochondria and form a polymer (JC-1 aggregate, red fluorescence, 590 nm), and the images were collected and analyzed with LSM510 META laser scanning confocal microscope (Zeiss, Thornwood, NY, USA).

ATP assay

INS-1 cells were seeded in 6-well plates at the density of 4 × 10⁵ cells per well and subjected to indicated treatment. The ATP levels were measured using an ATP assay kit. The method is based on a luciferase-luciferin reaction assay. The results were normalized by protein concentration.

Molecular docking simulation

The 2D structures of OP-D was drawn in the ChemDraw software, and then imported into OpenBabel software (v2.2.1 Open Babel development team, USA) for hydrogenation and converted into a mol2 format file. Subsequently, the structure of Keap1 (PDB ID: 1U6D) was obtained from the RCSB PDB webpage (https://www.rcsb.org/). Thereafter, the protein Keap1 file was opened in PyMOL software (v2.2.0 Schrodinger, Inc., USA) to remove the excess water molecules, delete any irrelevant small ligands originally carried, and only keep the protein structure. The original ligands were deleted and the original ligand positions were set as docking sites since the downloaded protein structure had ligands. AutoDock (v4.2) was utilized to display the specific docking energy value after running. Finally, the results were analyzed with the adoption of Protein-Ligand Interaction Profiler (PLIP; https://plip-tool.biotec.tu-dresden.de/plip-web).

Statistical analysis

Data were reported as means ± standard deviation. Statistical analysis was carried out with SPSS (version 18.0, SPSS, Inc., Chicago, IL, USA). The differences among all groups were compared with one-way analysis of variance followed by Tukey's post hoc analysis. P < 0.05 was considered statistically significant.

Results

Ophiopogonin D inhibits the apoptosis of H₂O₂-induced INS-1 cells

The chemical structure formula of OP-D is displayed in [Figure 1]a. To detect the effects of different concentrations of OP-D on the viability of INS-1 cells, CCK-8 was used to detect INS-1 cell viability, and the results showed no significant changes in cell viability [Figure 1]b. Then, we investigated the effects of OP-D on the viability and apoptosis of H₂O₂-induced INS-1 cells, and CCK-8 results showed that the cell viability in H₂O₂ group significantly decreased about 53% compared with control group. Compared with the H₂O₂ group, cell viability was significantly increased in the H₂O₂ + OP-D group, and increased in a dependent manner with the increase of OP-D concentrations [Figure 1]c. Flow cytometry showed that apoptosis was significantly increased in the H₂O₂ group compared with the control group, and OP-D could reverse apoptosis in a dose-dependent manner [Figure 1]d. Western blot detection of apoptosis-related protein expression showed that compared with the control group, Bcl-2 expression was decreased while Bax and cleaved caspase 3 expressions were increased in the H₂O₂ group. Compared with H₂O₂ group, the expressions of apoptosis-related proteins were reversed in H₂O₂ + OP-D group [Figure 1]e. These results indicated that OP-D could significantly inhibit H₂O₂-induced INS-1 cell damage.

Figure 1: Ophiopogonin D (OP-D) inhibits the apoptosis of hydrogen peroxide (H₂O₂)-induced INS-1 cells. (a) The chemical structure formula of OP-D. (b and c) cell counting kit-8 was used to detect cell viability after being given with different concentrations of OP-D or H₂O₂. N = 5. (d) Flow cytometry detected cell apoptosis. N = 3. (e) Western blot was used to detect the expressions of apoptosis-related proteins Bcl-2, Bax, cleaved caspase 3, and caspase 3. N = 3. ***P < 0.001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

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Ophiopogonin D increases insulin secretion capacity and reduces oxidative stress level in H₂O₂-induced INS-1 cells

The research on the molecular mechanism of hypoglycemia mainly plays a role in reducing insulin resistance and improving antioxidant stress ability. Subsequently, we examined the effects of OP-D on the insulin secretion capacity and oxidative stress capacity of H₂O₂-induced INS-1 cells. ELISA was used to detect insulin secretion, and the results showed that insulin secretion was significantly reduced by H₂O₂ induction compared with control group. OP-D at different concentrations promoted insulin secretion in a concentration-dependent manner [Figure 2]a. The levels of Insulin 1 and Insulin 2 were measured by RT-qPCR, and the results showed that H₂O₂ induction significantly decreased the expressions of Insulin 1 and 2, while OP-D treatment reversed the expressions of Insulin 1 and Insulin 2 [Figure 2]b. Subsequently, the levels of oxidative stress-related factors were examined with related kits, and the results showed that the level of MDA increased and the level of SOD and GSH-Px decreased after H₂O₂ induction. Compared with H₂O₂ group, the levels of MDA, SOD, and GSH-Px in H₂O₂ + OP-D group were reversed [Figure 2]c. It indicated that OP-D increases insulin secretion capacity and reduced reduces oxidative stress level in H₂O₂-induced INS-1 cells.

Figure 2: Ophiopogonin D (OP-D) increases insulin secretion capacity and reduces the oxidative stress level in hydrogen peroxide (H₂O₂)-induced INS-1 cells. (a) Enzyme-linked immunosorbent assay was used to detect insulin secretion. N = 5. (b) The levels of Insulin 1 and Insulin 2 were measured by real-time quantitative polymerase chain reaction. N = 5. (c) The levels of oxidative stress-related factors malonaldehyde, superoxide dismutase, and glutathione peroxidase were examined with related kits. N = 5. ***P < 0.001; ^##P < 0.01, ^###P < 0.001.

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Ophiopogonin D inhibits mitochondrial dysfunction in H₂O₂-induced INS-1 cells

Mitochondrial dysfunction may be involved in insulin resistance and secretion dysfunction in pancreatic β cells. Elevated glucose levels can lead to increased production of ROS in mitochondria, which can damage pancreatic β cells. Hence, the detection of mitochondrial function is very important. MMP was detected by JC-1 fluorescence probe, and the results showed that MMP in H₂O₂ group was significantly reduced compared with control group. Compared with the H₂O₂ group, the MMP in the cells was increased with the increasing doses of OP-D [Figure 3]a. ATP content was measured by the kit, and the results showed that OP-D could significantly reverse the reduction of ATP induced by H₂O₂ [Figure 3]b. NADPH level was also measured by a related kit and it was shown that OP-D significantly reversed the H₂O₂-induced increase in NADPH [Figure 3]c. The mtDNA copy number was detected by RT-qPCR, and the results showed that the mtDNA copy number in the H₂O₂ group was significantly reduced compared with the control group. Compared with the H₂O₂ group, the mtDNA copy number increased dose-dependently after OP-D administration [Figure 3]d. We concluded that OP-D inhibits mitochondrial dysfunction in H₂O₂-induced INS-1 cells.

Figure 3: Ophiopogonin D (OP-D) inhibits mitochondrial dysfunction in hydrogen peroxide-induced INS-1 cells. (a) Mitochondrial membrane potential was detected by JC-1 fluorescence probe to detect mitochondrial function. N = 3. (b) ATP content was measured by the ATP kit. N = 3. (c) The kit was used to measure nicotinamide adenine dinucleotide phosphate level. N = 5. (d) The mtDNA copy number was detected by real-time quantitative polymerase chain reaction. N = 5. ***P < 0.001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

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Ophiopogonin D regulates the Keap1/Nrf2/ARE pathway

Next, we explored the regulatory mechanism of OP-D in H₂O₂-induced INS-1 cells. Molecular docking technology was used to detect the potential targets of OP-D. The three-dimensional structure of Keap1 protein (PDB ID: 1U6D) was obtained from the PDB database (https://www.rcsb.org/), and molecular docking using Autodock (version 4.2) showed that OP-D was able to bind to Keap1 [Figure 4]a. Western blot analysis was performed to detect the expressions of Keap1/Nrf2/ARE pathway-related proteins in different groups. The results showed that compared with the control group, Keap1 expression in H₂O₂ group was significantly increased, while Nrf2, HO-1, and NQO1 expressions were significantly decreased. The expressions of Keap1, Nrf2, HO-1, and NQO1 were reversed after further administration of OP-D [Figure 4]b. All these findings indicated that OP-D regulated Keap1/Nrf2/ARE pathway.

Figure 4: Ophiopogonin D (OP-D) regulates the Keap1/Nrf2/ARE pathway. (a) Molecular docking was used to detect that OP-D was able to bind to Keap1. (b) Western blot analysis was performed to detect the expressions of Keap1/Nrf2/ARE pathway-related proteins Keap1, Nrf2, HO-1, and NQO1. N = 3. ***P < 0.001; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

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Ophiopogonin D attenuates oxidative stress and mitochondrial dysfunction in H₂O₂-induced INS-1 cells through Keap1/Nrf2/ARE pathway

Keap1/Nrf2/ARE signaling plays an important role in DM, and its components can be used as therapeutic targets in the treatment of DM and its complications. In order to further explore the regulatory mechanism of OP-D on H₂O₂-induced INS-1 cells, cells were treated by Keap1/Nrf2 specific signaling pathway inhibitor ML385. CCK8 results showed that cell viability in H₂O₂ + OP-D + ML385 group was significantly decreased compared with H₂O₂ + OP-D group [Figure 5]a. Flow cytometry showed that the apoptosis was significantly increased in H₂O₂ + OP-D + ML385 group compared with H₂O₂ + OP-D group [Figure 5]b. Western blot results of apoptosis-related proteins showed that compared with H₂O₂ + OP-D group, Bcl-2 expression decreased while Bax and cleaved caspase 3 expressions increased in H₂O₂ + OP-D + ML385 group [Figure 5]c. In addition, insulin secretion was significantly decreased in the H₂O₂ + OP-D + ML385 group compared with the H₂O₂ + OP-D group [Figure 5]d and [Figure 5]e. The expression results of oxidative stress-related factors showed that compared with H₂O₂ + OP-D group, MDA expression was increased in H₂O₂ + OP-D + ML385 group, while SOD and GSH-Px expressions decreased [Figure 5]f. JC-1 results showed that MMP in H₂O₂ + OP-D + ML385 group was significantly decreased compared with H₂O₂ + OP-D group [Figure 6]a. In addition, compared with H₂O₂ + OP-D group, ATP content in H₂O₂ + OP-D + ML385 group was also significantly decreased, NADPH level was increased, and mtDNA copy number was decreased [Figure 6]b, [Figure 6]c, [Figure 6]d. We know that ML385 was able to reverse the inhibitory effect of OP-D on H₂O₂-induced oxidative stress and mitochondrial dysfunction in INS-1 cells.

Figure 5: Ophiopogonin D (OP-D) improves oxidative stress in hydrogen peroxide-induced INS-1 cells through Keap1/Nrf2/ARE pathway. (a) Cell counting kit-8 was used to detect cell viability after a Keap1/Nrf2 specific signaling pathway inhibitor ML385 was further added. N = 5. (b) Flow cytometry detected cell apoptosis after a Keap1/Nrf2 specific signaling pathway inhibitor ML385 was further added. N = 3. (c) Western blot was used to detect the expressions of apoptosis-related proteins Bcl-2, Bax, cleaved caspase 3, and caspase 3. N = 3. (d) Enzyme-linked immunosorbent assay was used to detect insulin secretion. N = 5. (e) The levels of Insulin 1 and Insulin 2 were measured by real-time quantitative polymerase chain reaction. N = 5. (f) The levels of oxidative stress-related factors malonaldehyde, superoxide dismutase, and glutathione peroxidase were examined with related kits. N = 5. ***P < 0.001; ^##P < 0.01, ^###P < 0.001; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001.

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Figure 6: Ophiopogonin D (OP-D) improves mitochondrial dysfunction in hydrogen peroxide-induced INS-1 cells through Keap1/Nrf2/ARE pathway. (a) Mitochondrial membrane potential was detected by JC-1 fluorescence probe to detect mitochondrial function after a Keap1/Nrf2 specific signaling pathway inhibitor ML385 was further added. N = 3. (b) ATP content was measured by AKT kit. N = 3. (c) The kit was used to measure nicotinamide adenine dinucleotide phosphate level. N = 5. (d) The mtDNA copy number was detected by real-time quantitative polymerase chain reaction. N = 5. ***P < 0.001; ^##P < 0.01, ^###P < 0.001; ⁺P < 0.05, ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001.

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Discussion

The research on the molecular mechanism of hypoglycemia mainly plays a role in the inhibition of apoptosis of islet cells, the reduction of insulin resistance,^[11] the improvement of anti-oxidative stress ability, and the regulation of related signaling pathways.^[12] Oxidative stress refers to the imbalance between oxidation and antioxidant action in the body.^[13] When the body is under oxidative stress, the expression of antioxidant enzymes and lipid peroxidation in the body is completely unbalanced, leading to the apoptosis of pancreatic β cells.^[14] As a chemical inducer, H₂O₂ is frequently utilized to study oxidative damage in cells. Multiple studies have used H₂O₂-induced cellular model of DM through stimulating oxidative stress in pancreatic β cells.^[15],[16] Therefore, in our experiment, H₂O₂ was used to induce islet β cells, and we found the increased death of islet β cells and the occurrence of oxidative stress.

In addition, mitochondria are widely distributed and involved in cellular energy production, oxidative stress, Ca²⁺ homeostasis, regulation of some cellular metabolic and biosynthetic pathways, and apoptosis. Studies have found that mitochondrial dysfunction may be implicated in insulin resistance and secretion as well as dysfunction of pancreatic β cells.^[17],[18] Persistently elevated glucose levels may lead to increased production of ROS in mitochondria, which may damage islet β cells. And ZnT8 haploid dysfunction prevents islet β cell dysfunction in type 1 diabetes by increasing mitochondrial respiration.^[19] Therefore, the detection of oxidative stress and the regulation of mitochondrial function is an important way to study DM. Our experiment found that H₂O₂ induction might lead to the mitochondrial dysfunction in pancreatic β cells.

Due to the advantages of traditional Chinese medicine, such as low cytotoxicity, easy access and abundant resources, researchers have tried to find safe and effective anti-diabetic drugs. The aim of this study was to investigate the effects of OP-D on oxidative stress and mitochondrial function in H₂O₂-induced pancreatic β cells. OP-D is a potent antioxidant.^[8] Moreover, OP-D can alleviate doxorubicin-induced myocardial cell death by alleviating mitochondrial damage in vitro and in vivo.^[20] OP-D alleviates myocardial damage in DM by regulating mitochondrial dynamics.^[21] However, the regulation of OP-D on oxidative stress and mitochondrial function of pancreatic β cells in DM has not been reported. Our results showed that OP-D could significantly inhibit H₂O₂-induced apoptosis, improve insulin secretion capacity, and reduce the level of oxidative stress and mitochondrial dysfunction of INS-1 cells.

We found that OP-D could target Keap1 through molecular docking techniques. A previous study has shown that activating Keap1/Nrf2 signaling in pancreatic β cells may be a useful pharmacological strategy for clinical prevention and treatment of type 1 diabetes.^[22] In the diabetic model, the effects of cinnamon tannin D1 on the activation of antioxidant Keap1/Nrf2 signaling pathway and the improvement of inflammation, endoplasmic reticulum stress, and apoptosis were realized through the induction of the autophagy of HG/PA-treated INS-1 cells.^[23] In addition, Keap1/Nrf2/ARE signaling components have been used as therapeutic targets for the treatment of DM and its complications.^[24] Regulating Keap1/Nrf2/ARE signaling can promote high glucose-induced podocyte injury.^[25] It was reasonable to speculate that OP-D targeted Keap1 and played a role in H₂O₂-induced INS-1 cells by regulating the downstream Nrf2/ARE signaling pathway. Our results showed that after induction of H₂O₂ in INS-1 cells, Keap1/Nrf2/ARE signaling pathway was activated, and OP-D could reverse the H₂O₂-induced abnormal expressions of Keap1/Nrf2/ARE signaling pathway-related proteins Keap1, Nrf2, HO-1, and NQO1. After the pre-treatment with the specific Keap1/Nrf2 signaling pathway inhibitor ML385, it was found that ML385 could reverse the inhibitory effect of OP-D on apoptosis, oxidative stress, mitochondrial dysfunction, and the stimulatory role of OP-D in insulin secretion.

Of course, our article has its limitations. We have only carried out experiments in cells and have not verified our conclusions in animal experiments. We will further verify the regulatory role of OP-D in diabetes in animals in subsequent experiments.

Conclusion

OP-D could attenuate oxidative stress and mitochondrial dysfunction in pancreatic β cells induced by H₂O₂ through Keap1/Nrf2/ARE pathway in DM. Our article might provide strong evidence for the clinical treatment of DM with OP-D.

Data availability statement

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Financial support and sponsorship

The project was supported by a hospital-level fund project of Shanghai Sixth People's Hospital (No. ynlc201827). The project was supported by the National Natural Science Foundation of China (No. 82104649).

Conflicts of interest

There are no conflicts of interest.

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