Abstract
Diabetic encephalopathy (DE) is a severe complication of diabetes, but its pathogenesis remains unclear. This study aimed to investigate the roles and underlying mechanisms of high glucose (HG)- and advanced glycosylation end product (AGE)-induced oxidative stress (OS) in the cognitive decline in DE. The DE mouse model was established using a high-fat diet and streptozotocin, and its cognitive functions were evaluated using the Morris Water Maze, novel object recognition, and Y-maze test. The results revealed increased reactive oxygen species (ROS) generation, mitophagy inhibition, and decreased prohibitin 2 (PHB2) expression in the hippocampal neurons of DE mice and HG- or AGE-treated HT-22 cells. However, overexpression of PHB2 reduced ROS generation, reversed mitophagy inhibition, and improved mitochondrial function in the HG- or AGE-treated HT-22 cells and ameliorated cognitive decline, improved mitochondrial structural damage, and reversed mitophagy inhibition of hippocampal neurons in DE mice. Further analysis revealed that the Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid 2-related factor 2 (Nrf2) pathway was involved in the HG- or AGE-mediated downregulation of PHB2 in HT-22 cells. These results demonstrate that HG- or AGE-induced OS inhibits the mitophagy of hippocampal neurons via the Keap1–Nrf2–PHB2 pathway, thereby contributing to the cognitive decline in DE.
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Introduction
Diabetes mellitus (DM), a highly prevalent metabolic disease, is characterized by a persistent state of hyperglycemia caused by insufficient insulin secretion or reduced insulin sensitivity of peripheral tissues and organs1. DM is often accompanied by multiple complications, such as retinopathy, nephropathy, peripheral neuropathy, coronary heart disease, and diabetic encephalopathy (DE), which reduce the quality of life and survival status of patients2. DE is characterized by behavioral defects and cognitive dysfunction, including a decline in learning, memory, attention, language expression, and comprehension, which occur due to damage to the structure and function of the central nervous system3. Some reports have shown that oxidative stress (OS) induced by high glucose (HG) and advanced glycosylation end products (AGEs), which are typical of DM, is involved in DE pathogenesis4,5,6, but its underlying mechanism remains unclear.
AGEs are stable covalent compounds produced by non-enzymatic reactions of glucose with amino residues on proteins and lipids. AGE accumulation has been found to occur during the normal aging process, as well as in certain pathological conditions, such as Alzheimer’s disease (AD) and DM7. AGEs bind to proteins, lipids, and nucleic acids and alter their structural integrity and function8. Additionally, AGEs bind to AGE receptors and induce OS and chronic inflammatory responses, further amplifying cellular dysfunction9. Therefore, AGEs play a key role in the pathological processes of DM-induced complications, such as the cognitive decline in DE, by inducing OS.
Prohibitin 2 (PHB2), a widely expressed multifunctional protein, is predominantly localized in mitochondria but is also present in the cytoplasm, nucleus, and plasma membrane10. In addition to playing a crucial role in the normal growth and development of mitochondria, PHB2 is involved in nuclear transcriptional regulation, cell proliferation, apoptosis, cell cycle regulation, and senescence11. Furthermore, a recent study found that PHB2 acts as a mitophagy receptor in the inner mitochondrial membrane12. After the rupture of the mitochondrial outer membrane, PHB2 interacts with the microtubule-associated protein 1 light chain 3 (LC3) and mediates PTEN-induced kinase 1–parkin-dependent mitochondrial autophagy13. Moreover, a recent study found that PHB2 acts as a positive regulator of mitochondrial function in PC12 cells under OS14. PHB2 is expressed in pancreatic islet β-cells, and its knockout in mice disrupts mitochondrial function, insulin secretion, and blood glucose homeostasis, leading to DM pathogenesis15. Although the downregulation of PHB2 is closely associated with DM, its role in the inhibitory effects of HG- or AGE-induced OS on hippocampal neuronal mitophagy in DE remains to be clarified.
In this study, we explored the changes in PHB2 expression and mitophagy in the hippocampus of DE mice and hippocampal neurons under OS. Thereafter, we determined the role of PHB2 in hippocampal neuronal mitophagy, mitochondrial dysfunction, and cognitive deficit in DE and investigated its upstream pathway by in vitro and in vivo analyses. The results of this study can provide evidence to elucidate the underlying mechanisms of HG- or AGE-induced OS in DE pathogenesis and discern potential therapeutic strategies for DE.
Materials and methods
Animals and cells
Male C57BL/6 mice (aged 6–8 weeks) were purchased from EX InVivo Biotechnology Co., Ltd. (Hebei, Shijiazhuang, China). The mice were housed under specific pathogen-free conditions and maintained at constant temperature and humidity and a 12:12 h light/dark cycle. All animal experiments were performed in compliance with ARRIVE guidelines. The animal care and experimental procedures were approved by the Animal Care and Management Committee of the First Hospital of Hebei Medical University, Shijiazhuang, China (No. 20200349). All methods were performed in accordance with the relevant guidelines and regulations. HT-22 cells were purchased from Life Technologies (Waltham, MA, USA) and cultured in Dulbecco’s Modification of Eagle’s Medium (DMEM, Gibco, Invitrogen GmbH, Karlsruhe, Germany) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin.
Establishment of DE mouse model
A total of 48 male mice were fed a high-fat diet (60% kcal fat; D12492 pellets; Research Diets, Inc., Beijing, China) or a control diet (10% kcal fat; D12450B pellets; Research Diets, Inc.) for 14 weeks to establish a DM group and a negative control group, respectively. Thereafter, the mice were randomly divided and intraperitoneally injected with streptozocin (STZ; 150 mg/kg body weight, BW; Sigma-Aldrich, St. Louis, MO, USA; dissolved in cold 0.1 M citrate buffer, pH 4.5) or an equal volume of 0.1 M citrate buffer to establish a DE group and a control group (CON), respectively. Mice with persistently higher random blood glucose (BG) levels (> 16.7 mmol/L) for 1 week were considered to have DE. The BW and BG levels of the mice were measured after 1, 2, 4, 8, and 12 weeks post-injection (wpi), and the behavioral tests were conducted after 10 wpi.
In vivo experimental design
The DE mice were randomly divided into DE (model), DE + PHB2 (treatment), and DE + Mitoquinone (MitoQ; positive control) groups (n = 12/group). An animal was anesthetized with isoflurane (2%) under a flow of oxygen for 10 min. The animal is allowed to remain in deep anesthesia for approximately 10–20 s before commencing the procedure. The mice in the DE + PHB2 group were administered recombinant adeno-associated virus (AAV) vectors carrying Phb2 (AAV-Phb2; GenePharma Co., Ltd, Shanghai, China) via intracerebroventricular injection for 5 min (4 μL, 1 μL/min). The mice in the DE + MitoQ group were intraperitoneally injected with MitoQ (10 mg/kg; MedChemExpress, NJ, USA). The behavioral tests were conducted 14 d post-treatment.
Behavioral tests
Y-maze spontaneous alternation test
The mice were placed at the end of any one arm of the Y-maze and allowed to explore the maze for 10 min. The total number of entries, number of animals entering the maze arm, sequence of arm entries, total number of alternations (the number of consecutive entries into three arms of the maze, e.g., 1, 2, and 3 or 1, 3, and 2), and maximum number of alternations (total number of arms advances—2) were recorded. The rate of spontaneous alternation was calculated as follows:
Morris water maze (MWM) test
The MWM test is commonly used to evaluate cognitive functions associated with memory. For the MWM test, a circular chamber was divided into four equal quadrants and filled with opaque water (22 ± 2 °C). A circular platform was placed 1 cm underwater in the target quadrant. Markers of different shapes and colors were positioned around the pool as visual cues to help the mice locate the platform. The mice were then gently released into one of four quadrants and trained to locate the platform for 60 s/quadrant/day with an interval of 30 min for 5 consecutive days. If the mice did not find the platform, they were gently guided to the platform and placed on the platform for 10 s. On day 6, the platform was removed, and the mice were allowed to swim freely for 60 s. The time to reach the platform (escape latency), the distance of the swimming paths, and the number of crossings over the “platform” zone were automatically recorded by the Ethovision Color-Pro video tracking system (Super Maze Version, Xinruan Information Technology Co., Ltd., Shanghai, China).
Novel object recognition (NOR) test
The NOR test, which included the adaptation, training, and testing periods, was performed over 3 d. In the adaptation period (day 1), the mice were allowed to move freely in the absence of objects for 5 min. During the training period (day 2), two identical objects were placed symmetrically in the left and right corners of the test box, and the mice were allowed to explore the objects for 5 min. Lastly, during the test period (day 3), the mice were placed in the box containing one familiar and one novel object and allowed to explore the objects for 5 min. Exploratory behavior was defined as being within 2–3 cm of the object or in direct contact with the object. After each test, the box and objects were wiped with 75% ethanol to eliminate residual odors. The cognitive function was determined based on the discrimination index (DI), which was calculated as follows:
Open field (OF) test
The OF test is commonly used to evaluate anxiety and exploratory activity of mice. During the OF test, each mouse was placed in the middle of the OF box and recorded with a computerized video system for 5 min. The time spent in the central squares and the number of square crossings were used to assess the degree of anxiety. After each test, the box was thoroughly cleaned with 75% ethanol to eliminate residual odors.
Establishment of OS cell model and in vitro experimental design
The mice hippocampal neuron cell line, HT-22, was purchased from the Shanghai Cell Bank of Chinese Academy of Science (Shanghai, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (containing 25 mM glucose) supplemented with 1% penicillin–streptomycin (Solarbio, Beijing, China) and 10% fetal bovine serum in a humidified incubator at 5% CO2 and 37 °C. The cells were stimulated with glucose or AGEs at different concentrations to establish the OS cell model. The Phb2-shRNA (KD) plasmid was kindly gifted by Yao Xu (Shanghai Jiaotong University) and the Phb2-overexpression (OE) plasmid was purchased from VectorBuilder (Guangzhou, China). The HT-22 cells were cultured in a 6-well plate for 24 h and transfected with Phb2-KD or Phb2-OE plasmid using Effectene Transfection Reagent (Qiagen, Shanghai, China), according to the manufacturer’s instructions.
Cell counting kit-8 (CCK-8) assay
HT-22 cells (n = 4000) were seeded into 96-well plates for 12 h and treated with different concentrations of HG (25, 50, 75, 100, or 200 mM), mannitol, or AGEs (100, 200, 400, 600, or 800 μg/mL) for 24 or 48 h. Thereafter, the cells were incubated with CCK-8 reagent (10 μL/well; Dojindo, Japan) for 2 h. Cell viability was determined at 450 nm using a microplate reader (Tecan, Männedorf, Switzerland).
Real-time polymerase chain reaction (RT-PCR)
Total RNA from HT-22 cells or mice hippocampal tissues was extracted using TRNzol Universal Reagent (Tiangen Biotech, Beijing, China) and reverse transcribed using the FastQuant RT Kit (Tiangen Biotech). RT-PCR was performed using the SuperReal PreMix Plus (SYBR Green; Tiangen Biotech) on the LightCycler 480 (Roche, Basel, Swiss). The relative expression was calculated using the 2−ΔΔCt method. The primers used for the RT-PCR were as follows—Phb2 forward: 5'-CAAGAACCCCACCACCAGAGAA-3' and Phb2 reverse: 5'-TCCAAGAGGGCAGATAVAGAAAAG-3' and β-actin forward: 5'-ATGGAGGGGAATACAGCCC-3' and β-actin reverse: 5'-TTCTTTGCAGCTCCTTCGTT-3'.
Western blot (WB) analysis
HT-22 cells or mice hippocampal tissues were lysed on ice for 30 min using radioimmunoprecipitation assay buffer (Solarbio, Beijing, China) supplemented with a complete protease inhibitor. Equal amounts of cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes (Millipore, MA, United States). The membranes were blocked with 5% milk for 1 h and incubated overnight with anti-PHB2 (1:1000, Proteintech, Wuhan, China), anti-LC3 isoform II (LC3II; 1:1000, Abcam, Cambridge, UK), anti-nuclear factor erythroid 2-related factor 2 (Nrf2; 1:500, Abcam), anti-Beclin 1 (1:500, Bioworld, MN, USA), anti-autophagy protein 5 (ATG5; 1:500, Bioworld), anti-translocase of outer membrane 20 (TOM20; 1:2000, Proteintech), and anti-Kelch-like ECH-associated protein 1 (Keap1; 1:1000, Abcam) antibodies at 4 °C. Subsequently, the membranes were washed thrice with Tris-buffered saline with 0.1% Tween 20 and incubated with secondary antibodies for 1 h at room temperature. Band intensity was detected using an Odyssey infrared scanner (LI-COR Biosciences, Lincoln, NE, USA), and the results were analyzed using the ImageJ software.
Co-localization analysis
The HT-22 cells were treated with Mito-Tracker Green (200 nM; Beyotime Biotechnology, Shanghai, China) and Lyso-Tracker (50 nM; Beyotime Biotechnology) for 30 min and observed under a fluorescent microscope.
The hippocampal tissues of DE mice were fixed in 4% paraformaldehyde for 24 h, incubated with 30% sucrose, snap frozen in Optimal Cutting Temperature compound, and sectioned using (coronal sections, 15 μm) a cryostat. The tissue sections were blocked with 10% donkey serum for 1 h and incubated overnight with anti-TOM20 (1:200) and anti-lysosomal associated membrane protein 2 (LAMP2; 1:200, Proteintech) antibodies at 4 °C. Subsequently, the sections were rinsed with phosphate-buffered saline (PBS) and incubated with goat anti-rabbit or mouse IgG antibodies for 1 h at room temperature in the dark. The sections were then rinsed with PBS, counter-stained with DAPI, and observed and photographed using a laser scanning confocal microscope (Zeiss LSM 880, Carl Zeiss, Oberkochen, Germany). Co-localization analysis was performed using the ImageJ plugin Coloc 2, and Pearson’s correlation coefficient was used to evaluate the co-localization.
JC-1 staining
The HT-22 cells were treated with glucose for 48 h and incubated with JC-1 fluorescence dye (Beyotime Biotechnology, Shanghai, China) for 20 min at 37 °C. The cells were washed twice and observed under the confocal microscope (Carl Zeiss) to detect mitochondrial transmembrane potential.
Oxygen consumption assay
Mitochondrial respiration in the HT-22 cells was measured using an Oxygraph-2 K (Oroboros Instruments, Innsbruck, Austria), according to the mitochondrial substrate-uncoupler-inhibitor titration protocols, as described previously16.
Detection of ROS
The HT-22 cells were seeded into 6-well plates and incubated with 10 µM DCFH-DA or MitoSOX Red Mitochondrial Superoxide Indicator (ThermoFisher Scientific, MA, USA).) for 30 min at 37 °C in the dark to detect cellular or mitochondrial ROS generation, respectively. The cells were observed under a fluorescence microscope (Carl Zeiss) and the fluorescence intensity was measured.
The mice hippocampal tissues (50 mg) were homogenized with 1 mL of buffer and centrifuged at 4 °C for 3 min. The supernatant was added to a 96-well plate and incubated with 2 μL of BBoxiProbe (BestBio, China) at 37 °C for 30 min in the dark. The ROS levels were quantified using the fluorescence microplate reader (Tecan) at excitation and emission wavelengths of 510 nm and 610 nm, respectively.
Transmission electron microscopy (TEM)
The mice hippocampal tissues were fixed with 4% glutaraldehyde and 1% osmium tetroxide, dehydrated in ethanol, and embedded in Epon 812. The ultramicrostructure of the hippocampal neurons was then observed under a TEM (Hitachi, Tokyo, Japan).
Statistical analysis
The normal distribution of data was evaluated with the Kolmogorove-Smirnov test or the Shapiro Wilk test. All the results are presented as the mean ± standard deviation (SD). Statistical analysis was performed using the GraphPad Prism software (v8.0.1, USA). Differences between multiple groups were determined using one-way analysis of variance followed by least significant difference post-hoc tests. Statistical significance was considered at P < 0.05.
Ethical approval
All the experimental procedures were approved by the Animal Care and Management Committee of the First Hospital of Hebei Medical University.
Results
ROS generation, PHB2 downregulation, and mitophagy inhibition are involved in DE pathogenesis in mice
As reported previously, a high-fat diet and STZ injection were used to establish the DE mice model17. The results showed that the average BG levels were significantly increased and the average BW was significantly decreased in the DM group compared with the CON group after 1 wpi (P < 0.001; Fig. 1a,b). Behavioral tests were conducted after 10 wpi to evaluate the cognitive function of the DE mice. In the MWM test, the DE and CON groups showed a progressive decline in latency during the training period. However, compared with the CON group, the DE group had longer escape latency, longer movement trajectory and decreased number of platform crossings (Fig. 1c–e). In the NOR test, the DE group showed significantly shorter total exploration time and significantly lower DI compared to the CON group (Fig. 1f,g). In the Y-maze spontaneous alternation test, the DE group showed fewer arm entries and reduced spontaneous alternation rate compared with the CON group (P < 0.05) (Fig. 1h,i). The DE group showed increased ROS generation in the hippocampal tissues compared with the CON group (Fig. 1j). Co-immunofluorescence staining of TOM20 and LAMP2 showed reduced mitophagy in the Cornu Ammonis (CA)-3 and dentate gyrus (DG) regions of the hippocampal tissues of the DE mice compared with the CON mice (Fig. 1k,l). In addition, RT-PCR and WB analyses showed significantly decreased PHB2 mRNA (Fig. 1m) and protein levels (Fig. 1n) in the hippocampal tissues of the DE mice compared with the CON mice. Altogether, these results indicate that ROS generation, PHB2 downregulation, and mitophagy inhibition are involved in DE pathogenesis in mice.
ROS generation, mitophagy level, and PHB2 expression in the hippocampus of DE mice. (a, b) The average BW (a) and BG levels (b) in the DE and CON groups (n = 14, 13). (c–e) The representative swimming paths (c), escape latencies during the training period (d), and platform crossing times (e) of the DE or CON groups in the MWM test (n = 12). (f, g) The DI (f) and total exploration time (g) of the DE and CON groups in the NOR test (n = 12). (h, i) The spontaneous alternation rate (h) and total number of arm entries (i) of the DE and CON groups in the Y-maze test (n = 9). (j) The ROS intensity in the hippocampus of the DE or CON groups was detected using BBoxiProbe ROS probe (n = 8, 6). (k, l) Representative immunofluorescence images (k) and relative co-locations (l) of TOM20 (green) and LAMP2 (red) in the CA3 and DG hippocampus regions of the CON and DE groups (scale bar = 50 μm, n = 3). (m, n) RT-PCR (m) and WB analysis (n) of PHB2 expression in the hippocampus of DE and CON groups (n = 3). The values are shown as the means ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs the CON group.
HG and AGE-induced ROS generation, PHB2 downregulation, and mitophagy inhibition in HT-22 cells
High glucose and AGEs, which are characteristic of DM, have been reported to induce OS injury in brain tissues. To investigate the role of PHB2 in the induction of OS injury in DM, HT-22 cells were treated with different concentrations of glucose (25–200 mM) or AGEs (0–800 μg/mL) for 24 or 48 h. The CCK-8 assay showed that HT-22 cells incubated with 100 and 200 mM glucose showed significantly decreased cell viability (Fig. 2a,b). Additionally, the results showed that HT-22 cells incubated with < 400 μg/mL AGEs for 24 h showed no change in cell viability (Fig. 2c), while those incubated with > 400 μg/mL AGEs for 48 h showed decreased cell viability (Fig. 2d). Moreover, the results showed that mannitol (isotonic control) had no effect on the viability of HT-22 cells. To determine the role of mitophagy in the HG- or AGE-induced hippocampal neuronal injury, HT-22 cells were treated with HG or AGEs for 48 h and subjected to co-localization analysis. The results showed decreased mitophagy levels in the HG- or AGE-treated HT-22 cells (Fig. 2e–h), which were further validated by WB assay (Fig. 2i–p). Furthermore, the WB assay showed that compared with the untreated cells, HT-22 cells treated with HG (100 mM) or AGEs (400 μg/mL) showed reduced expression of LC3II, ATG5, Beclin 1, and TOM20 (Fig. 2i–p). DCFH-DA staining was conducted to determine the effects of HG or AGEs on the level of OS, and the results showed that ROS generation was increased in HT-22 cells treated with HG (100 mM) or AGEs (400 μg/mL) for 48 h (Fig. 2q,r). Meanwhile, HG- or AGE-treated HT-22 cells showed significantly decreased Phb2 mRNA levels (P < 0.05 and P < 0.01, respectively; Fig. 2s,t). Altogether, these results suggest that HG- or AGE-induced OS leads to Phb2 downregulation and mitophagy inhibition in HT-22 cells. Therefore, HT-22 cells treated with 100 mM HG or 400 μg/mL AGEs for 48 h were used for subsequent analyses.
ROS generation, mitophagy level, and PHB2 expression in HG- or AGE-treated HT-22 cells. (a–d) Cell viability of HT-22 cells treated with different concentrations of glucose and mannitol (a, b) or AGEs (c, d) for 24 or 48 h, as measured by CCK-8 assay; *P < 0.05, **P < 0.01, and ***P < 0.001 vs 25 mM glucose or 0 μg/mL AGE group (n = 3). (e–h) Representative fluorescence images of Mito-Tracker (green) and Lyso-Tracker (red) and their relative co-locations in HT-22 cells treated with HG (e, f) or AGEs (g, h) for 48 h (scale bar = 20 μm). (i–p) Representative WB images (i, m) and quantitative analysis of ATG5/Beclin 1 (j, o), LC3II/LC3I (k, n), and TOM20 (l, p) proteins in HT-22 cells treated with different concentrations of glucose (i–l) or AGEs (m–p); *P < 0.05, **P < 0.01, and ***P < 0.001 vs 25 mM glucose or 0 μg/mL AGE group (n = 3). (q, r) Representative fluorescence images (q) and relative ROS levels (r) in HT-22 cells treated with glucose (100 mM) or AGEs (400 μg/mL) for 48 h (scale bar = 20 μm); **P < 0.01 and ***P < 0.001 vs CON group. (s, t) RT-PCR analysis of PHB2 expression in the HT-22 cells treated with different concentrations of glucose (s) or AGEs (t). β-actin was used as the internal control; *P < 0.05 and **P < 0.01 vs 25 mM glucose or 0 μg/mL AGE group. The values are shown as the means ± SD (n = 3).
PHB2 played a critical role in the HG or AGE-induced inhibition of mitophagy in HT-22 cells
To evaluate the role of PHB2 in the HG- or AGE-induced mitophagy, PHB2 was overexpressed or knocked down in HT-22 cells via transfection with Phb2-OE or Phb2-KD plasmids, respectively. Transfection efficiency was validated by RT-PCR and WB assays (Figs. 3a,b and S1a, b). Co-localization analysis showed that Phb2 OE significantly ameliorated mitophagy inhibition in HG- or AGE-treated HT-22 cells (Fig. 3c–f). Furthermore, Phb2 OE significantly increased the levels of LC3II, ATG5, Beclin 1, and TOM20 in HG- or AGE-treated HT-22 cells, as indicated by WB analysis (Fig. 3g–n). In contrast, Phb2 KD aggravated mitophagy inhibition in HG- or AGE-treated HT-22 cells, as indicated by the co-localization analysis (Fig. S1c–f) and the decrease in LC3II, Beclin 1, and TOM20 in WB analysis (Fig. S1g–n). Altogether, these results demonstrate that PHB2 plays a critical role in HG or AGE-induced mitophagy disorder in HT-22 cells.
Effects of Phb2 OE on the HG- or AGE-induced mitophagy inhibition in HT-22 cells. (a, b) Phb2 OE in HT-22 cells validated by RT-PCR (a) and WB assay (b); *P < 0.05 and **P < 0.01 vs the NC group. (c–f) Representative fluorescence images of Mito-Tracker (green) and Lyso-Tracker (red) (c, e) and their relative co-locations (d, f) in the indicated HT-22 cells (scale bar = 20 μm); **P < 0.01 and ***P < 0.001 vs NC group and †P < 0.05 and ††P < 0.01 vs NC + HG or NC + AGEs group. (g–n) Representative WB images (g, k) and quantitative analysis of LC3II/LC3I, ATG5/Beclin 1, and TOMM20 in the indicated HT-22 cells. *P < 0.05 and **P < 0.01 vs NC group and †P < 0.05 vs NC + HG or NC + AGEs group. The values are shown as the means ± SD (n = 3).
Phb2 OE ameliorated the HG or AGE-induced mitochondrial dysfunction in HT-22 cells
To determine the effects of Phb2 OE on the HG or AGE-induced mitochondrial dysfunction mitochondrial membrane potential (MMP), and cellular and mitochondrial ROS generation were detected using JC-1, DCFH-DA, and mitoSOX staining. Compared with the NC cells, HG- or AGE-treated HT-22 cells showed weaker red fluorescence and brighter green fluorescence in the JC-1 staining assay, indicating a significant decrease in MMP. However, Phb2 OE increased the MMP of HG- or AGE-treated HT-22 cells (Figs. 4a,b and 5a,b). In addition, HG and AGE treatments increased cellular and mitochondrial ROS generation in HT-22 cells compared with the NC cells; however, Phb2 OE significantly reversed the HG- or AGE-induced increase in ROS production in HT-22 cells (Figs. 4c–f and 5c–f). Furthermore, HG and AGE treatments decreased the intracellular ATP levels in HT-22 cells compared to the NC cells; however, this effect was reversed by Phb2 OE (Fig. 4g and 5g). To study the effect of PHB2 on mitochondrial metabolic functions, we performed a high-resolution respirometry analysis of HT-22 cells. The results showed that Phb2 OE improved the HG- or AGE-induced decrease in leak respiration and oxygen consumption of complex II (Fig. 4h,i and 5h,i). In contrast, Phb2 KD aggravated MMP, cellular and mitochondrial ROS generation, intracellular ATP levels, leak respiration, and oxygen consumption of complex II induced by HG or AGE treatments (Figs. S2 and S3). Altogether, these results indicate that Phb2 OE improves the HG- or AGE-induced mitochondrial damage and dysfunction in HT-22 cells.
Effects of Phb2 OE on the HG-induced mitochondrial dysfunction in HT-22 cells. The HT-22 cells were transfected with Phb2 OE plasmid and treated with HG for 48 h. (a, b) The MMP was monitored using JC-1 staining in the indicated HT-22 cells. Red fluorescence indicated normal MMP, while green fluorescence indicated a decrease or loss of MMP (scale bar = 20 μm). (c–f) The intracellular or mitochondrial ROS levels detected by DCFH-DA (c, d) (scale bar = 20 μm) or MitoSOX (e, f) (scale bar = 50 μm) staining in the indicated HT-22 cells. (g) The intracellular ATP levels in the indicated HT-22 cells were detected using the ATP Bioluminescence Assay Kit. (h, i) Leak respiration (h) and CII-linked OXPHOS (i) of the indicated HT-22 cells were measured using a high-resolution respirometry. *P < 0.05, **P < 0.01, and ***P < 0.001 vs NC group and †P < 0.05 and ††P < 0.01 vs NC + HG group. The values are the mean ± SD (n = 3).
Effects of Phb2 OE on the AGE-induced mitochondrial dysfunction in HT-22 cells. The HT-22 cells were transfected with Phb2 OE plasmid and treated with AGEs for 48 h. (a, b) The MMP was monitored using JC-1 staining in the indicated HT-22 cells. Red fluorescence indicated normal MMP, while green fluorescence indicated a decrease or loss of MMP (scale bar = 20 μm). (c–f) The intracellular or mitochondrial ROS levels detected by DCFH-DA (c, d) (scale bar = 20 μm) or MitoSOX (e, f) (scale bar = 50 μm) staining in the indicated HT-22 cells. (g) The intracellular ATP levels in the indicated HT-22 cells were detected using the ATP Bioluminescence Assay Kit. (h, i) Leak respiration (h) and CII-linked OXPHOS (i) of the indicated HT-22 cells measured using a high-resolution respirometry. **P < 0.01 and ***P < 0.001 vs NC group and †P < 0.05, ††P < 0.01, and †††P < 0.001 vs NC + AGEs group. The values are shown as the mean ± SD (n = 3).
Upregulation of Phb2 ameliorated the cognitive decline in DE mice
To investigate the functional role of PHB2 in DE, the DE mice were injected with AAV-Phb2, and PHB2 expression was detected in the mice hippocampal tissues after 14 dpi. MitoQ, a mitochondria-targeting antioxidant, was used as the positive control. Co-immunofluorescence staining and WB analyses showed that the PHB2 levels in the hippocampal CA3 and DG regions were decreased in the DE group compared with the CON group. However, treatment with AAV-Phb2 or MitoQ significantly increased the PHB2 levels in the hippocampal CA3 and DG regions of DE mice (Fig. 6a,b).
Effects of PHB2 upregulation on the cognitive decline in DE mice. (a) Representative images of PHB2 protein levels in the CA3 and DG hippocampal regions of the indicated groups, as detected by immunohistochemical staining (scale bar = 50 μm; n = 6). (b) WB analysis of PHB2 protein levels in the hippocampus of the indicated groups; **P < 0.01 vs CON and †P < 0.05 and ††P < 0.01 vs DE group (n = 3). (c–f) The swimming speed (c), representative swimming path (d), escape latencies during training trails (e), and the number of platform crossing (f) of the indicated groups in the MWM test; *P < 0.05, **P < 0.01, and ***P < 0.001 vs CON group and †P < 0.05 and ††P < 0.01 vs DE group (n = 6 or 12). (g, h) The DI (g) and total exploration time (h) of the indicated groups in the NOR test; *P < 0.05 and **P < 0.01 vs CON group and †P < 0.05, ††P < 0.01, and †††P < 0.001 vs DE group (n = 10). (i, j) The number of square crossings (i) and total time in central squares (j) of the indicated groups in the OF test; **P < 0.01 vs CON group and †P < 0.05 and †††P < 0.001 vs DE group (n = 10). The values are shown as the mean ± SD.
Behavioral tests were performed to determine if PHB2 upregulation can ameliorate the cognitive impairment in DE mice (Fig. 6c–j). The MWM test showed no significant difference in the swimming speed among the four groups (Fig. 6c) but showed a progressive decline in their latency during the training period (Fig. 6e). The DE group had longer escape latency and longer movement trajectory compared with the CON group, while the DE + PHB2 and DE + MitoQ groups had shorter escape latency and shorter movement trajectory compared with the DE group (Fig. 6d–f). These results indicate that PHB2 upregulation significantly increased the number of platform crossings in DE mice. Similarly, in the NOR test, the DE group showed significantly lower DI compared to the CON group, while the DE + PHB2 and DE + MitoQ groups showed improved DIs and increased total exploration time compared to the DE group (Fig. 6g,h). The OF test showed no significant difference in the number of squares crossed among the four groups but found that the DE mice spent less time in the central area than the CON mice, while the DE + PHB2 and DE + MitoQ mice spent more time in the central area than the DE group (Fig. 6i,j). These results demonstrate that the upregulation of PHB2 could ameliorate the cognitive deficits in DE mice.
Upregulation of Phb2 improved mitochondrial structure and reversed mitophagy inhibition in the hippocampus of DE mice
To further investigate the effects of PHB2 upregulation on the mitochondrial structural damage and mitophagy in DE mice, the mice hippocampal neurons were observed by TEM. The results showed that the hippocampal neurons of the CON group had clear mitochondrial crests and intact mitochondrial membrane, while the hippocampal neurons of the DE group had fewer mitochondrial crests, resulting in mitochondrial swelling and vacuolar mitochondria. However, the hippocampal neurons of the DE + PHB2 and DE + MitoQ groups showed improved mitochondrial structure compared to the DE group (Fig. 7a). Quantitative mitochondrial classification (Fig. 7b) revealed that the CON group had ≤ 79.3% Class I mitochondria (filled with densely arranged and regularly distributed cristae) and relatively lower Class II (mitochondrial crest fracture and decreased matrix density) and Class III (mitochondrial swelling and membrane destruction) mitochondria. Additionally, the results showed that the DE group had the highest proportion of Class III mitochondria and the lowest proportion of Class I mitochondria. In contrast, the DE + PHB2 and DE + MitoQ groups had a higher proportion of Class I mitochondria compared to the DE group (Fig. 7c). TEM results showed that the number of mitochondrial autophagosomes was significantly reduced in the DE group compared to the CON group but increased in the DE + PHB2 and DE + MitoQ groups compared to the DE group (Fig. 7d,e). Co-immunofluorescence staining showed that mitophagy was decreased in the DE group compared with the CON group and increased in the DE + MitoQ or DE + PHB2 groups compared with the DE group (Fig. 7f). A similar trend was observed for the four treatment groups in the WB analysis (Fig. 7g–j). Altogether, these results indicate that upregulation of PHB2 could ameliorate the mitochondrial structural damage and mitophagy of hippocampal neurons in DE.
Effects of PHB2 upregulation on the mitochondrial structural damage and mitophagy inhibition in the hippocampus of DE mice. (a) Representative TEM images of the ultrastructure of mitochondria in the hippocampal neurons of indicated groups (scale bar = 0.5 μm). (b, c) Representative images of Class I–III mitochondria (b) and their quantitative distribution (c) in the hippocampal neurons of the indicated groups. (d, e) Representative images of mitophagy (d) and the quantitative graphs of the number density of mitophagy (e) in the hippocampal neurons of the indicated groups; ***P < 0.001 vs CON group and †P < 0.05 and ††P < 0.01 vs DE group (scale bar = 0.5 μm; n = 5). (f) Representative immunofluorescence images of TOM20 (green) and LAMP2 (red) in the DG region of the hippocampus of the indicated groups (scale bar = 50 μm). (g–j) Representative WB images (g) and quantitative analysis of LC3II/LC3I (h), Beclin 1 (i), and TOM20 (j) in the hippocampus of the indicated groups; *P < 0.05 and **P < 0.01 vs CON group and †P < 0.05 vs DE group. The values are shown as the means ± SD, n = 3.
HG or AGE-induced Phb2 downregulation in HT-22 cells via the Keap1–Nrf2 pathway
The results of the WB assay revealed that the HG- or AGE-treated group had significantly decreased Nrf2 and PHB2 expression and increased Keap1 expression compared with the Con group. Additionally, the ML385-treated group (Nrf2 inhibition) showed decreased Nrf2 and PHB2 expression and increased Keap1 expression, while the dimethyl fumarate (DMF)-treated group (Nrf2 activation) showed decreased Keap1 expression and increased Nrf2 and PHB2 expression compared with the Con group (Fig. 8a–h). These results suggest that the HG- or AGE-induced OS in DE downregulated PHB2 by inhibiting the Keap1–Nrf2 pathway.
The role of the Keap1–Nrf2 pathway in the HG- or AGE-induced downregulation of PHB2 in HT-22 cells. (a–h) Representative WB images (a, e) and quantitative analysis of Nrf2 (b, f), Keap1 (c, g), and PHB2 (d, h) in the indicated HT-22 cells treated with HG (a–d) or AGEs (e–h). The HT-22 cells were incubated with ML385 (5 μM) or DMF (20 μM) and treated with HG or AGEs for 48 h. *P < 0.05 vs Con group. The values are shown as the means ± SD, n = 3.
Discussion
DE, a common complication of DM, is difficult to prevent and treat and brings a heavy burden to the patients, family, and society. Previous research on DE pathogenesis was focused on insulin resistance, OS, neuroinflammation, amyloid deposition, and tau hyperphosphorylation18,19,20. However, the exact mechanisms of DE remain unknown, leading to a lack of effective treatment strategies for DE. In this study, we focused on the roles and underlying mechanisms of HG- or AGE-induced OS injury in DE pathogenesis. Our results demonstrate that HG- or AGE-induced OS inhibited the hippocampal neuronal mitophagy via the Keap1–Nrf2–PHB2 pathway, leading to cognitive impairment in DE. Therefore, targeting PHB2 may serve as a potential therapeutic strategy for DE management.
Increased OS may contribute to the development of several DM-related complications, including DE. ROS may activate aldose reductase and protein kinase C, resulting in the formation of AGEs and diacylglycerol21. In this study, we found that ROS production increased significantly in the HG- or AGE-treated HT-22 cells and hippocampus of DE mice, suggesting the potential roles of HG and AGE in inducing OS in DE. Additionally, we found that PHB2 was downregulated in the HG- or AGE-treated HT-22 cells and hippocampus of DE mice, suggesting the potential role of PHB2 in the HG- or AGE-induced OS and DE pathogenesis; however, the upstream regulatory pathways and downstream molecular mechanisms of PHB2 in DE remain elusive.
ROS-induced mitochondrial injury may be a vital activator of mitophagy; however, the effects and mechanism of ROS on the mitophagy of hippocampal neurons under OS are still unclear. In this study, we found that PHB2-mediated mitophagy was decreased in the HG- or AGE-treated HT-22 cells and hippocampus of DE mice. TOM20 is a receptor subunit of the TOM complex, located on the outer membrane of mitochondria, and plays an important role in the transport of mitochondrial proteins. Increase of TOM20 may indicate abnormal assembly or function of the TOM complex, which in turn affects the normal function of mitochondria. In this study, we found the levels of TOM20 were decreased significantly in HT-22 cells treated with 200 or 400 μg/mL AGEs, but not decreased in HT-22 cells treated with 600 μg/mL AGEs. We think these results might be due to the mitochondrial dysfunction caused by cytotoxic effects. Mitophagy is involved in many human diseases, such as Parkinson’s disease, AD, cancer, and DM and its complications. However, the role of mitophagy in DM and its complications has not been fully elucidated. Recent research found that ROS produced by mitochondrial respiratory activity under the HG environment promotes DM-related complications. Mitophagy plays an important role in alleviating DM-related complications by removing damaged mitochondria and inhibiting OS. Previous studies found that mitophagy plays an important role in DM-related complications, such as nephropathy22,23, retinopathy24,25, and cardiomyopathy26,27, but its underlying mechanisms are unknown. Currently, there are no reports on the association between mitophagy disorder and DE incidence, but a study found that mitophagy disorder plays a crucial role in AD pathogenesis28. The study found that the level of mitophagy is increased in the early stages of AD to facilitate the removal of damaged mitochondria; however, mitophagy dysfunction during AD progression leads to the accumulation of damaged mitochondria, which further exacerbates the pathological changes of AD. Increased mitophagy levels inhibit AD-related tau hyperphosphorylation in human neurons and reverse memory impairment in transgenic tau nematodes and mice28. In cholestatic liver, PHB2 interacts with LC3 and sequestosome-1 to form a ternary complex, which plays an important role in bile acid-induced mitophagy29. In renal tubular epithelial cell injury, PHB2 regulates mitochondrial dysfunction and activates NOD-like receptor protein 3 pathway30. Our data showed that mitophagy was inhibited in HG- or AGE-treated HT-22 cells and DE mice, which was reversed by Phb2 OE.
Studies have shown that PHB complexes have neuroprotective effects. For instance, Phb2 KO can alter MMP, affecting electron transport chain complex formation and resulting in ROS accumulation in rat neurons31. OS can affect mitophagy by regulating the expression of fusion and fission proteins32. Parkin-mediated mitophagy is inhibited in type I DM mice and db/db mice, resulting in increased damaged mitochondria accumulation, decreased insulin secretion, and increased pancreatic β-cell death33. In this study, we found that Phb2 OE ameliorated the HG or AGE-induced mitochondrial dysfunction, as indicated by increased MMP, ATP levels, and respiratory function and decreased cellular and mitochondrial ROS generation. Additionally, we observed that DE mice showed decreased Phb2 expression and increased mitochondrial swelling, suggesting that PHB2 plays a vital role in the mitophagy of hippocampal neurons. Therefore, we further explored the mechanism of PHB2 regulation in DE.
PHB2 is an inner mitochondrial membrane mitophagy receptor. Nrf2 is an antioxidative transcription factor that can be activated by mitochondrial ROS and influences mitochondrial function34,35. Keap1, a Cul3-dependent ubiquitin ligase, is the anchor protein of Nrf2. Under normal conditions, Nrf2 localizes in the cytoplasm and binds with Keap1, where it is rapidly degraded by the ubiquitin–proteasome pathway. However, ROS can target the Nrf2–Keap1 complex, thereby dissociating Nrf2 from Keap1. The stable Nrf2 then translocates to the nucleus and binds to the antioxidant response elements to activate the transcription of its downstream target genes, such as heme oxygenase 1, which provide effective protection against OS in injured tissues. Previous studies have demonstrated that the Keap1–Nrf2 pathway is involved in OS damage in subarachnoid hemorrhage rats36. MitoQ can decline blood–brain barrier damage via the Nrf2–PHB2–optic atrophy 1 pathway48. Therefore, in this study, we used DMF (Nrf2 agonist) and ML-385 (Nrf2 inhibitor) to determine the role of upstream mechanisms of Nrf2 in DE. The results showed that the expression of PHB2 increased after DMF treatment and decreased after ML385 treatment in the HT-22 cells under OS. These results suggest that Nrf2 may be an upstream regulator of PHB2 in HT-22 cells.
We further explored the role and underlying mechanisms of PHB2 in the regulation of cognitive disorders in DE mice. MitoQ, a lipid-soluble mitochondria-targeting antioxidant, can effectively improve mitochondrial function and treat kidney injury37. Moreover, it has gained widespread attention owing to its ability to penetrate the blood–brain barrier and provide neuroprotective effects38. In this study, MitoQ increased PHB2 expression in the hippocampal region of DE mice, which further improved mitochondrial morphology. Moreover, MitoQ treatment or Phb2 OE increased the number of mitochondria autophagosomes in the DE mice. The MWM test was used to assess spatial and working memory, the NOR test was used to evaluate non-spatial short-term memory, and the OF test was used to estimate the anxiety behaviors of the mice. These behavioral tests showed that PHB2 upregulation improves cognitive functions and alleviates anxiety in DE mice.
In this study, we demonstrated that HG- or AGE-induced OS inhibits the mitophagy of hippocampal neurons by downregulating PHB2 expression, which contributes to the cognitive decline in DE. However, this study has several limitations that warrant consideration. First, the Keap1–Nrf2 pathway was found to be involved in the HG- or AGE-induced downregulation of PHB2 in HT-22 cells, which should be validated in DE mice. Second, this study did not investigate the downstream molecular mechanisms of PHB2-mediated regulation of mitophagy of hippocampal neurons in DE, necessitating further analysis.
Conclusion
In summary, our study demonstrated that HG- or AGE-induced OS inhibits hippocampal neuronal mitophagy via the Keap1–Nrf2–PHB2 pathway. Our results showed that HG and AGE-induced ROS generation, PHB2 downregulation, and mitophagy inhibition in HT-22 cells. Additionally, we found that Phb2 OE ameliorated the HG- or AGE-induced mitochondrial dysfunction and cognitive decline in DE mice. Altogether, these findings suggest that the Keap1–Nrf2–PHB2 pathway may serve as an effective therapeutic target for DE treatment.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Acknowledgements
This work was supported by the Hebei Provincial Natural Science Foundation (No. H2024206245 and H2024206280), the Key Projects of Hebei Administration of Traditional Chinese Medicine (Z2022015) and Foundation of XingHuo Project of the First Hospital of Hebei Medical University (XH202401).
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S.J.X contributed to the conception and design of the study. S.X. wrote the main manuscript text and prepared the data; L.J.; Z. G.; J. L.; Y.Q., D. Z.; P. T.; W. W.; N. Z. performed the experiments and analyzed the data; R. Z.; S.J.X. revised the paper. All authors have read and approved the final version of the manuscript to be published.
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Xu, S., Gao, Z., Jiang, L. et al. High glucose- or AGE-induced oxidative stress inhibits hippocampal neuronal mitophagy through the Keap1–Nrf2–PHB2 pathway in diabetic encephalopathy. Sci Rep 14, 24044 (2024). https://doi.org/10.1038/s41598-024-70584-3
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DOI: https://doi.org/10.1038/s41598-024-70584-3
