AMPK activation attenuates high glucose-induced oxidative stress and apoptosis through regulation of TRPM2-mediated signaling pathway in endothelial cells

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Methods and Results Human umbilical vein vascular endothelial cells (HUVECs) were pretreated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (AMPK activator), Compound C (CC) (AMPK inhibitor), or N-(p-amylcinnamoyl) Anthranilic Acid (ACA) [transient receptor potential melastatin 2 (TRPM2) inhibitor]. Subsequently, the cells in the intervention groups were exposed to high glucose. Reactive oxygen species levels, endothelial apoptosis, calcium entry, and protein expression levels in the HUVECs were detected. The AMPK activator, AICAR, exhibited a significant reduction in high glucose-stimulated ROS production and apoptosis in HUVECs, whereas the AMPK inhibitor, CC, significantly enhanced these effects. Pretreatment with the TRPM2 inhibitor, ACA, significantly abrogated high glucose-induced calcium entry, high glucose-stimulated oxidative stress, and apoptosis, indicating the role of TRPM2-mediated intracellular calcium ([Ca 2+ ]i) influx in these cellular processes. Furthermore, the AMPK inhibitor, CC, significantly increased the levels of TRPM2-related proteins,including the primary calcium sensor calmodulin (CaM) and NADPH oxidase (NOX). Conversely, the AMPK activator exhibited opposite effects on these proteins. Conclusions AMPK activation could attenuate high glucose-stimulated oxidative stress and play a protective role in ECs partly through inhibiting the TRPM2-CaM-NOX pathway. AMP-activated protein kinase endothelial cells transient receptor potential melastatin 2 oxidative stress high glucose Figures Figure 1 Figure 2 Figure 3 Introduction Atherosclerosis, characterized by the build-up of lipids and persistent inflammation in large arteries, is a leading contributor to cardiovascular diseases, such as myocardial infarction and stroke. In patients with diabetes, atherosclerosis is highly prevalent, and it is considered that the development of diabetes-associated atherosclerosis is largely driven by endothelial dysfunction [ 1 ]. Oxidative stress, which commonly occurs in diabetics, plays a notable role in endothelial dysfunction [ 2 ]. Endothelial cell (EC) injury resulting from oxidative stress can impair endothelial integrity and barrier function, thereby accelerating the development and progression of atherogenesis. Elevated intracellular glucose level can increase the mitochondrial electron transport system, leading to excessive generation of reactive oxygen species (ROS) [ 3 ]. Previous research has indicated the beneficial effects of antioxidants in ameliorating endothelial dysfunction in the thoracic aorta of diabetic rats through mitigating oxidative stress [ 4 ]. Moreover, the elevated levels of ROS in the vasculature could be strongly associated with the development of atherosclerosis among diabetic patients. Although antioxidant therapy may still be a potential treatment or preventive strategy for diabetes-related atherosclerosis, it is essential to find a more selective approach for antioxidant medications [ 5 ]. However, the precise mechanisms of high glucose-induced oxidative stress and its related endothelial dysfunction have not yet been fully elucidated. Mitochondria are subcellular organelles with various critical cellular functions, including modulation of cytosolic calcium level, apoptosis, and ROS production [ 6 ]. Excessive Ca 2+ overload, especially in the mitochondria, can result in superoxide production and mitochondrial dysfunction, ultimately leading to apoptosis [ 7 ]. Chen et al. found that exposure to high glucose level led to an elevation in intracellular calcium [Ca 2+ ]i level in ECs and in turn caused an increase in ROS production [ 8 ]. Additionally, prior study demonstrated that pretreatment with the calcium channel inhibitor, ruthenium, reduced high glucose-induced ROS production, while the calcium channel activator, spermine, significantly enhanced high glucose-induced ROS levels [ 8 ]. Transient receptor potential melastatin 2 (TRPM2), a well-recognized cation channel permeable to Ca 2+ , is expressed in various cells, including EC, and it is activated under stimuli, such as excessive ROS production [ 9 ]. Recently, accumulating evidence has revealed that TRPM2 channel-mediated cell death plays a critical role in connecting various oxidative stress-inducing factors to diabetes [ 10 – 12 ]. Furthermore, it has been pointed out that ROS generated by polymorphonuclear neophiles could be sufficient to induce an increased [Ca 2+ ]i concentration through TRPM2 channels in ECs, leading to endothelial barrier dysfunction [ 13 ]. In murine aortic ECs, both current and Ca 2+ responses were effectively suppressed by a nonselective TRPM2 channel inhibitor, N-(p-amylcinnamoyl) anthranilic acid (ACA). These findings consistently indicated a critical function of TRPM2 channel in the mediation of ROS-induced Ca 2+ influx into ECs [ 14 ]. However, it remains elusive whether the TRPM2-mediated calcium entry could lead to EC apoptosis. Furthermore, the role of TRPM2-mediated calcium entry in high glucose-induced injury to ECs has been poorly assessed. In the present study, human umbilical vein vascular endothelial cells (HUVECs) were utilized to examine the influences of AMP-activated protein kinase (AMPK) on high glucose-induced oxidative stress and related endothelial impairment, and to investigate the function of TRPM2-mediated signaling pathway. The findings of this study could help identify potential therapeutic targets for developing new treatments or preventive strategies for patients with diabetes-related atherosclerosis and cardiovascular diseases. Materials and methods Cell culture and treatment HUVECs used in this study were obtained from the Chinese Academy of Sciences Cell Bank (Beijing, China), and the cells were cultured in a Dulbecco’s modified Eagle’s medium (DMEM; Hyklong, Boston, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, New York, NY, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin in standard conditions of 5% CO 2 and 37°C. Before the experiments, the cells were synchronized in cell culture media with reduced FBS (0.5%) for 12 h. Subsequently, the cells were exposed to the culture medium in the control group (CON group) or a high-glucose-containing cell culture medium in the high glucose group (HG group, 30 mmol/L glucose in the cell culture medium) for 48 h. To assess the potential roles of AMPK in the HG-mediated effects and the underlying mechanisms, HUVECs were pretreated for 1 h with 1 mM 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a well-known activator of AMPK, or 10 uM Compound C, a small molecule inhibitor of AMPK. Subsequently, the cells were treated with 30 mmol/L glucose in the HG + AICAR group and HG + CC group, respectively. For making comparison, HUVECs were also treated with 1 mM AICAR (AICAR group) or Compound C (CC group) alone, and these groups were included along with other experimental groups (HG + AICAR group, HG + CC group, CON group, and HG group). Cell viability assessment To assess the impact of HG on cell viability, HUVECs were exposed to 5 mM glucose in the control group or to a high-glucose-containing cell culture medium (30 mM glucose) in the high glucose (HG) group for 12, 24, and 48 h. Cell viability was determined using the cell counting kit-8 (CCK-8) kit, following the manufacturer's guidelines. In brief, a 96-well plate was loaded with 10 µl CCK-8 solution (5 mg/ml) in each well and incubated for 2 h at 37°C. The optical density at a wavelength of 490 nm was recorded, and cell viability was calculated with the values normalized to the normal control. To ensure reliability and accuracy, independent experiments were repeated five times. Examination of intracellular ROS levels in HUVECs To assess intracellular ROS levels in HUVECs, 1×10 4 cells per well were seeded into a 96-well plate. After the treatments were completed, cells were harvested and stained with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, Haimen, China) for 30 min, followed by flow cytometry (BD FACSCalibur™; BD Biosciences, Franklin Lakes, NJ, USA). The intracellular ROS levels in HUVECs were measured and normalized versus the control values. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for apoptosis TUNEL staining was used to detect DNA fragmentation of cells as a marker of apoptosis using a TUNEL fluorescence FITC kit (Roche, Indianapolis, IN, USA) on the basis of the manufacturer’s instructions. In brief, HUVECs grown on coverslips were treated with 4% paraformaldehyde for fixation and with 0.1% Triton X-100 for permeabilization. The cells were subsequently exposed to the TUNEL reaction mixture and maintained at 37°C for 1 h. Following the incubation period, the presence of apoptotic cells was assessed by examining TUNEL-positive cells using a fluorescence microscope (DMI3000 B; Leica, Berlin, Germany). Measurement of [Ca]i level To measure [Ca 2+ ]i level, HUVECs were loaded with the calcium indicator Fluo-3AM (5 µM) in HEPES-buffered saline solution. The fluorescence intensity of Fluo-3 bound to [Ca 2+ ]i was quantified using flow cytometry. To determine the maximum fluorescence intensity (Fmax), calcium ion carrier A23187 (1×10 − 5 mmol/L) and 1 mmol/L CaCl 2 were added to saturate the extracellular calcium. The minimum fluorescence intensity (Fmin) was measured by adding the fluorescence quencher MnCl 2 (2 mmol/L). Finally, [Ca 2+ ]i level was calculated using the following formula: [Ca 2+ ]i = Kd (F - Fmin)/(Fmax - F) (nmol/L). HUVECs were incubated with high glucose in the HG group, ACA (an inhibitor of TRPM2) in the ACA group, or a combination of HG and ACA in the HG + ACA group for 2 h, with untreated cells serving as the control in the control group. The [Ca 2+ ]i level was measured in the four groups. Western blotting (WB) To examine the protein expression levels of CaM, NOX, and TRPM2 in HUVECs, WB was conducted. In brief, HUVECs were collected through centrifugation at 700 ×g for 10 min at 4°C, followed by cell lysis to extract cellular proteins. After determining the protein concentration with Lowry’s method, equal amounts of proteins were then loaded on 10% polyacrylamide gel for separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes through electroblotting at 150 mA for 3 h. After blocking the PVDF membranes for 1 h in TBS containing 5% low-fat milk powder, they were incubated overnight with the primary antibody in TBS supplemented with either 5% BSA or 5% low-fat milk. The specific primary antibodies and dilutions were summarized as follows: anti-CaM (1:1000), anti-NOX (1:1000), anti-TRPM2 (1:1000; Abcam, Cambridge, UK), and anti-GAPDH (1:1000 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA). Thereafter, the secondary antibody linked with horseradish peroxidase (HRP) was added and incubated at room temperature for 1 h. Chemiluminescence detection was carried out with HRP Juice and charge-coupled device camera. Quantification of the densitometric signals was conducted using Quantity One Bioanalysis software (Bio-Rad Laboratories, Hercules, CA, USA). Quantitative polymerase chain reaction (qPCR) assay for determining TRPM2 gene expression level To determine the expression level of the TRPM2 gene, qPCR analysis was performed. Total RNA was extracted from the specific cell subgroups, and then transcribed into cDNA. The extracted cDNA was subjected to pretreatment with specific primers and fluorescent probes following the requirements of the qPCR kit. Subsequently, the reaction system was prepared and the PCR cycle was implemented according to the prescribed procedure. Finally, the fluorescence signal was measured and the number of threshold cycles (Ct value) during the qPCR was analyzed using software. The relative amount of TRPM2 was calculated on the basis of the increase in fluorescence signal. Statistical analysis Statistical analysis was carried out using SPSS 19.0 software (IBM, Armonk, NY, USA). Each experiment was conducted for at least three times. Data were presented as mean ± standard error of mean (SEM). An analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests was utilized for group comparisons. Abnormally distributed data were normalized using logarithms before being analyzed using either the Mann–Whitney test or Student’s t-test, depending on the specific conditions. P < 0.05 was considered statistically significant. Results AMPK attenuated high glucose-induced ROS generation and apoptosis in HUVECs Cell viability assay demonstrated that high glucose exposure (48 h) significantly diminished cell proliferation (HG vs. CON, P < 0.05, Fig. 1 A). The analysis of cellular ROS level (Fig. 1 B) and TUNEL staining for apoptosis (Fig. 1 C&D) revealed that high glucose treatment significantly increased ROS generation and promoted apoptosis in HUVECs (HG vs. CON, all P < 0.05). Notably, pretreatment with a small molecule inhibitor targeting AMPK, Compound C, in the HG + CC group significantly augmented cellular ROS levels (Fig. 1 B) and apoptosis (Fig. 1 C&D) (HG + CC vs. HG, all P < 0.05). Conversely, pretreatment with the AMPK activator, AICAR, in the HG + AICAR group significantly reduced cellular ROS levels (Fig. 1 B) and apoptosis in HUVECs (Fig. 1 C&D) (HG + AICAR vs. HG, all P < 0.05). These findings suggested that AMPK exerted a protective effect against the increased ROS generation and apoptosis induced by high glucose in HUVECs. TRPM2 mediated the increase in the Ca 2+ influx in ROS generation and apoptosis induced by high glucose in HUVECs Given that the TRPM2-mediated [Ca 2+ ]i influx into cells plays a crucial role in response to cellular stimuli, such as ROS generation and oxidative stress, the effects of high glucose on [Ca 2+ ]i level with or without an inhibitor of TRPM2, ACA, were assessed. The results of the CON, HG, ACA, and HG + ACA groups are presented in Fig. 2 . It was revealed that [Ca 2+ ]i level was significantly elevated in HUVECs (HG vs. CON, P < 0.05, Fig. 2 A). Notably, the combined treatment of HG and ACA significantly abrogated the high glucose-associated increase of [Ca 2+ ]i level (HG + ACA vs. HG, P 0.05, Fig. 2 A). Furthermore, cellular ROS levels and TUNEL staining for apoptosis were assessed in the CON, HG, ACA, and HG + ACA groups, and the findings are illustrated in Fig. 2 B, C, and D. High glucose treatment in the HG group significantly promoted apoptosis (HG vs. CON, P < 0.05), while this effect was significantly reversed in the HG + ACA group (Fig. 2 B &C), suggesting the potential role of TRPM2-mediated [Ca 2+ ]i in the high glucose-induced apoptosis in HUVECs. In terms of ROS, treatment with high glucose in the HG group significantly elevated intracellular ROS levels (HG vs. CON, P < 0.05), and inhibition of TRPM2 with its inhibitor ACA significantly attenuated the high glucose-promoted ROS production (HG + ACA vs. HG, P < 0.05) (Fig. 2 D). AMPK exerted its inhibitory effect on high glucose-stimulated [Ca 2+ ]i level via suppressing the TRPM2-related proteins It was attempted to assess whether AMPK could play its role in modulating [Ca 2+ ]i level upon high glucose stimulation by targeting TRPM2-associated signaling pathways. As illustrated in Fig. 3 A, exposure to high glucose significantly elevated [Ca 2+ ]i level in HUVECs (HG vs. CON, P < 0.05). However, activation of AMPK with its activator, AICAR, significantly reversed the high glucose-induced [Ca 2+ ]i level (HG + AICAR vs. HG, P < 0.05), while inhibition of AMPK with its inhibitor, Compound C, in the HG + CC group significantly increased the high glucose-induced [Ca 2+ ]i level ( P < 0.05). This finding indicated that AMPK could attenuate high glucose-induced ROS production and apoptosis via abrogation of [Ca 2+ ]i level in HUVECs. The expression levels of key molecules in TRPM2-associated signaling pathways, including CaM and NOX, were subsequently determined, and the results are presented in Fig. 3 B, C. The results of WB revealed that treatment with high glucose significantly elevated protein expression levels of CaM and NOX in HUVECs (HG vs. CON, P < 0.05) (Fig. 3 B, C). Moreover, pretreatment with the activator of AMPK, AICAR, significantly abrogated the high glucose-associated upregulation of CaM and NOX (HG + AICAR vs. HG, P < 0.05), and conversely pretreatment with the inhibitor of AMPK, CC, significantly augmented the high glucose-associated upregulation of CaM and NOX (HG + CC vs. HG, P < 0.05) (Fig. 3 B&C). AICAR or CC alone altered the CaM and NOX protein levels compared with the control group (Fig. 3 B, C). Moreover, the expression level of the TRPM2 protein was not significantly altered by the activator or inhibitor of AMPK (Fig. 3 D). However, as illustrated in Fig. 3 E, the TRPM2 gene expression level was significantly upregulated in response to high glucose stimulation, as well as in cells pretreated with the AMPK inhibitor. Collectively, these findings suggested that AMPK could exert its inhibitory effect on the high glucose-promoted oxidative stress that was potentially mediated via regulatingTRPM2-related proteins, especially CaM and NOX, in HUVECs (Fig. 3 ). Discussion This study demonstrated that exposure to high glucose stimulated ROS production in HUVECs through TRPM2-mediated elevation of [Ca 2+ ]i level and excessive apoptosis. Notably, pretreatment with an AMPK activator attenuated the high glucose-induced ROS production by restoring cellular calcium homeostasis, as well as regulating the expression levels of TRPM2-related proteins (i.e. CaM and NOX). The findings indicated that the elevation of [Ca 2+ ]i level through TRPM2 activation could be a crucial mechanism for the development of oxidative stress and subsequent endothelial injury in diabetes. Furthermore, AMPK may play a protective role against diabetes-related vascular complications by inhibiting ROS production and apoptosis induced by high glucose through regulating key molecules in the TRPM2-related signaling pathways, such as CaM and NOX, in ECs. Oxidative stress, known to cause endothelial dysfunction, is central to the development and progression of diabetes-associated complications [ 15 ]. Recent research suggested that hyperglycemia-induced ROS production could contribute to calcium entry and endothelial apoptosis [ 16 ]. The present study demonstrated that AMPK activation inhibited high glucose-stimulated ROS production, [Ca 2+ ]i level, and endothelial apoptosis, aligning with the antioxidative property of AMPK. The findings of this study suggested that the beneficial effects of AMPK on [Ca 2+ ]i level could be attributed to the antioxidative activity of AMPK. It is broadly accepted that hyperglycemia leads to metabolic disorders by triggering “aberrant” pathways that promote oxidative stress in human tissues [ 17 , 18 ]. Endothelial dysfunction is primarily associated with upregulation of cell-surface adhesion molecules in both ECs and blood immune cells, disrupting the redox serum balance, thereby resulting in ROS accumulation, oxidative stress, and hyperglycemia [ 19 ]. High glucose levels could induce ROS production, which may promote the release of cytochrome C. This, in turn, triggers caspase-dependent apoptosis, resulting in the loss of ECs and ultimately causing diabetic endothelial dysfunction [ 20 ]. Furthermore, hyperglycemia and hyperlipidemia increase the acetylation of dynamin-related protein 1, impairing mitochondrial dynamics and biogenesis, thereby exacerbating apoptosis [ 21 ]. These findings have provided direct evidence, linking diabetic endothelial dysfunction to apoptosis. In the present study, it was revealed that high glucose significantly decreased cell viability and increased apoptosis of ECs, which were effectively abolished by AMPK activation. Therefore, the restoration of injured ECs through regulating proliferation and apoptosis of the cells may hold significant importance. Notably, Ca 2+ and ROS are widely recognized as the major transduction signals that link the sarcoplasmic reticulum to mitochondrion. It has been shown that Ca 2+ overload could induce oxidative stress mainly through activation of the CaM-dependent signaling pathway, leading to an increase in ROS production [ 22 ]. CaM regulates EC function mainly through Ca 2+ /CaM-dependent protein kinase II (CaMKII). Activation of Ca 2+ /CaMKII triggers ROS production by activating NOX [ 23 ]. TRPM2, a cation channel permeable to Ca 2+ , is triggered by excessive ROS production [ 9 ]. The findings of the present study suggested that high glucose exposure caused [Ca 2+ ]i overload via the TRPM2-CaM-NOX signaling pathway, leading to the induction of ROS production and apoptosis in HUVECs. This is in line with outcomes of several previous studies, indicating that TRPM2-mediated Ca 2+ homeostasis plays a critical role in intracellular processes, such as gene expression, cell signaling, cell proliferation, and apoptosis [ 24 – 26 ]. Consequently, the modulation of TRPM2 and [Ca 2+ ]i might participate in high glucose-induced oxidative stress. When the TRPM2 inhibitor, ACA, was added along with Ca 2+ , a reduction was found in Ca 2+ level and ROS production, aligning with the result that oxidative stress induced impairment of endothelial barrier function through enhancing Ca 2+ influx into ECs, subsequently disrupting tight junctions between the cells. Overactivation of TRPM2-mediated Ca 2+ signaling results in the degradation of ZO-1 and internalization of VE-Cadherin, further enhancing the trans-endothelial migration of neutrophils in response to ROS-triggered different pathological stimuli [ 27 ]. Moreover, the present study indicated that pretreatment with an AMPK activator or inhibitor did not inhibit TRPM2 protein, while the high glucose-induced TRPM2 gene expression was significantly elevated after pretreatment with an AMPK inhibitor. This suggests that TRPM2-mediated Ca 2+ could be regulated by AMPK. In the present study, AMPK significantly suppressed the levels of CaM and NOX proteins, partially explaining the mechanism underlying oxidative stress. Moreover, this finding suggests that the activation of AMPK could be a promising strategy in mitigating high glucose-promoted oxidative stress and protecting ECs against oxidative stress. Conclusions This study demonstrated that AMPK activation could mitigate oxidative stress induced by high glucose partly via inhibiting the TRPM2-CaM-NOX pathway. The findings highlighted the potential protective effect of AMPK on attenuating diabetes-associated vascular complications. Therefore, AMPK and TRPM2-CaM-NOX signaling pathways are promising therapeutic targets for developing treatments or preventive strategies for diabetes-related atherosclerosis and cardiovascular diseases. Abbreviations ACA, N-(p-amylcinnamoyl) anthranilic acid; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK, AMP-activated protein kinase; Ca 2+ , calcium; [Ca 2+ ]i, intracellular calcium; CaM; calmodulin; CC, Compound C; EC, endothelial cell; HG, high glucose; HUVECs, human umbilical vein vascular endothelial cells; NOX, NADPH oxidase; ROS, reactive oxygen species; TRPM2, transient receptor potential melastatin 2. Statements & Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author contributions Ting Lu: Writing-original draft, Methodology. Yihua Zhang: Investigation. Zhe Wang: Writing-review & editing. Lu Zhao: Data curation. Dayan Zhou: Writing -review & editing. Qiang Xu: Supervision, Conceptualization. All the authors have contributed substantially to the study and approved the final manuscript. Ethics approval Not applicable Consent to participate Not applicable Consent to publish Not applicable Acknowledgements We thank Medjaden Inc. for scientific editing of this manuscript. This study was funded by the Scientific Research Project of Chongqing Nanan District Health Commission and Nanan District Science and Technology Bureau (Grant No. 2020-06). 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Arch Toxicol 95:2007-2018. https://doi.org/10.1007/s00204-021-03032-0 Sakurada R, Odagiri K, Hakamata A, Kamiya C, Wei J, Watanabe H (2019) Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells. Int J Mol Sci 20:1644. https://doi.org/10.3390/ijms20071644 Maliougina M, El Hiani Y (2023) TRPM2: bridging calcium and ROS signaling pathways-implications for human diseases. Front Physiol 14:1217828. https://doi.org/10.3389/fphys.2023.1217828 Xu J, Zhang W, Dong J, Cao L, Huang Z (2023) A New Potential Strategy for Treatment of Ischemic Stroke: Targeting TRPM2-NMDAR Association. Neurosci Bull 39:703-706. https://doi.org/10.1007/s12264-022-00971-1 Tektemur A, Ozaydin S, Etem Onalan E et al (2019) TRPM2 mediates distruption of autophagy machinery and correlates with the grade level in prostate cancer. J Cancer Res Clin Oncol 145:1297-1311. https://doi.org/10.1007/s00432-019-02898-z Ding R, Yin YL, Jiang LH (2021) Reactive Oxygen Species-Induced TRPM2-Mediated Ca(2+) Signalling in Endothelial Cells. Antioxidants (Basel) 10:718. https://doi.org/10.3390/antiox10050718 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5312586","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":371822172,"identity":"e7f616e1-077d-4644-81c5-24d9b9c1e8aa","order_by":0,"name":"Ting Lu","email":"","orcid":"","institution":"Chongqing Fifth People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Lu","suffix":""},{"id":371822173,"identity":"3bb8c9bc-1271-408a-b3a8-f795bd5df182","order_by":1,"name":"Yihua Zhang","email":"","orcid":"","institution":"Chongqing Fifth People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yihua","middleName":"","lastName":"Zhang","suffix":""},{"id":371822174,"identity":"d7d91a44-51b8-4ae7-8300-2a34ceb7c405","order_by":2,"name":"Dayan Zhou","email":"","orcid":"","institution":"Chongqing Fifth People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dayan","middleName":"","lastName":"Zhou","suffix":""},{"id":371822175,"identity":"be41868b-660d-46d9-9a00-37981b2f8322","order_by":3,"name":"Zhe Wang","email":"","orcid":"","institution":"Chongqing Fifth People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Wang","suffix":""},{"id":371822176,"identity":"c5463a9b-7f46-4065-b0a3-917a1c19d526","order_by":4,"name":"Lu Zhao","email":"","orcid":"","institution":"Chongqing Fifth People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Zhao","suffix":""},{"id":371822177,"identity":"e405b308-6196-48a7-99dd-9545a1d87f40","order_by":5,"name":"Qiang Xu","email":"data:image/png;base64,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","orcid":"","institution":"Chongqing Fifth People’s Hospital","correspondingAuthor":true,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-10-22 14:23:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5312586/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5312586/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68691373,"identity":"86016202-939d-49d1-8e71-54d2b4361211","added_by":"auto","created_at":"2024-11-11 06:00:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":190488,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of inhibition or activation of AMPK on high glucose-induced ROS production and apoptosis in HUVECs.\u003c/strong\u003e (a) Effects of high glucose on cell proliferation; (b) Effects of high glucose, AMPK inhibitor, and AMPK activator on ROS levels in HUVECs; (c) Representative images of TUNEL staining for effects of high glucose, AMPK inhibitor, and AMPK activator on apoptosis. Apoptotic cells were stained green, and nuclei were counterstained blue with DAPI (magnification, 9100); (d) Quantitative analysis of the TUNEL staining images in (c). Data were expressed as mean ± SEM. *P\u0026lt;0.05 compared with the control group, #P\u0026lt;0.05, ** P\u0026lt;0.05 compared with the HG group.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5312586/v1/1315a9a5c93bd146f44acbb2.png"},{"id":68691375,"identity":"42b50fba-6719-45dc-8e19-14d294fe468c","added_by":"auto","created_at":"2024-11-11 06:00:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":185334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of inhibition of TRPM2-mediated Ca\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e influx on high glucose-induced apoptosis and ROS production in HUMECs. \u003c/strong\u003eHUMECs were treated with HG, ACA (a blocker of TRPM2), HG+ACA, or grown in a culture medium alone as control (CON) for different time periods. [Ca\u003csup\u003e2+\u003c/sup\u003e]i level was measured. (a) Effects of high glucose on [Ca\u003csup\u003e2+\u003c/sup\u003e]I level with or without involvement of an inhibitor of TRPM2, ACA; (b) Representative images of TUNEL staining for effects of high glucose with or without involvement of ACA on apoptosis. Apoptotic cells were stained green, and nuclei were counterstained blue with DAPI (magnification, 9100); (c) Quantitative analysis of the TUNEL staining images in (b); (d) Effects of high glucose with or without involvement of ACA on ROS levels in HUVECs. ACA significantly decreased HG-stimulated apoptosis and ROS production (magnification, X100). Data were expressed as mean ± SEM. *P\u0026lt;0.05 vs. CON group, \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05 vs. HG group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5312586/v1/7250d29f5203b7b4422cea05.png"},{"id":68691374,"identity":"99949103-d855-46f0-ba9d-23e1b87f8016","added_by":"auto","created_at":"2024-11-11 06:00:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of activation or inhibition of AMPK on TRPM2 and its related proteins CaM and NOX upon high glucose stimulation in HUMECs\u003c/strong\u003e. (a) Effects of the AMPK inhibitor or activator on high glucose-induced [Ca\u003csup\u003e2+\u003c/sup\u003e]i level. Data were expressed as mean ± SEM; (b) and (c) Effects of the AMPK inhibitor or activator on high glucose-induced expression levels of CaM and NOX; (d) Effects of the AMPK inhibitor or activator on the protein expression level of TRPM2; (e) Effects of the AMPK inhibitor on high glucose-induced mRNA expression level of the TRPM2 gene. *P\u0026lt;0.05 vs. CON group, \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05 vs. HG group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5312586/v1/48196d45cd9374666ce25192.png"},{"id":85380985,"identity":"d08375e6-2e11-4a43-b1a0-ec027065eb37","added_by":"auto","created_at":"2025-06-25 09:09:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1315032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5312586/v1/1e2a6d3f-8d77-4bd3-b47e-aec86101e000.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"AMPK activation attenuates high glucose-induced oxidative stress and apoptosis through regulation of TRPM2-mediated signaling pathway in endothelial cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAtherosclerosis, characterized by the build-up of lipids and persistent inflammation in large arteries, is a leading contributor to cardiovascular diseases, such as myocardial infarction and stroke. In patients with diabetes, atherosclerosis is highly prevalent, and it is considered that the development of diabetes-associated atherosclerosis is largely driven by endothelial dysfunction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Oxidative stress, which commonly occurs in diabetics, plays a notable role in endothelial dysfunction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Endothelial cell (EC) injury resulting from oxidative stress can impair endothelial integrity and barrier function, thereby accelerating the development and progression of atherogenesis. Elevated intracellular glucose level can increase the mitochondrial electron transport system, leading to excessive generation of reactive oxygen species (ROS) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Previous research has indicated the beneficial effects of antioxidants in ameliorating endothelial dysfunction in the thoracic aorta of diabetic rats through mitigating oxidative stress [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, the elevated levels of ROS in the vasculature could be strongly associated with the development of atherosclerosis among diabetic patients. Although antioxidant therapy may still be a potential treatment or preventive strategy for diabetes-related atherosclerosis, it is essential to find a more selective approach for antioxidant medications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the precise mechanisms of high glucose-induced oxidative stress and its related endothelial dysfunction have not yet been fully elucidated.\u003c/p\u003e \u003cp\u003eMitochondria are subcellular organelles with various critical cellular functions, including modulation of cytosolic calcium level, apoptosis, and ROS production [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Excessive Ca\u003csup\u003e2+\u003c/sup\u003e overload, especially in the mitochondria, can result in superoxide production and mitochondrial dysfunction, ultimately leading to apoptosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Chen et al. found that exposure to high glucose level led to an elevation in intracellular calcium [Ca\u003csup\u003e2+\u003c/sup\u003e]i level in ECs and in turn caused an increase in ROS production [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Additionally, prior study demonstrated that pretreatment with the calcium channel inhibitor, ruthenium, reduced high glucose-induced ROS production, while the calcium channel activator, spermine, significantly enhanced high glucose-induced ROS levels [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Transient receptor potential melastatin 2 (TRPM2), a well-recognized cation channel permeable to Ca\u003csup\u003e2+\u003c/sup\u003e, is expressed in various cells, including EC, and it is activated under stimuli, such as excessive ROS production [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, accumulating evidence has revealed that TRPM2 channel-mediated cell death plays a critical role in connecting various oxidative stress-inducing factors to diabetes [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Furthermore, it has been pointed out that ROS generated by polymorphonuclear neophiles could be sufficient to induce an increased [Ca\u003csup\u003e2+\u003c/sup\u003e]i concentration through TRPM2 channels in ECs, leading to endothelial barrier dysfunction [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In murine aortic ECs, both current and Ca\u003csup\u003e2+\u003c/sup\u003e responses were effectively suppressed by a nonselective TRPM2 channel inhibitor, N-(p-amylcinnamoyl) anthranilic acid (ACA). These findings consistently indicated a critical function of TRPM2 channel in the mediation of ROS-induced Ca\u003csup\u003e2+\u003c/sup\u003e influx into ECs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, it remains elusive whether the TRPM2-mediated calcium entry could lead to EC apoptosis. Furthermore, the role of TRPM2-mediated calcium entry in high glucose-induced injury to ECs has been poorly assessed.\u003c/p\u003e \u003cp\u003eIn the present study, human umbilical vein vascular endothelial cells (HUVECs) were utilized to examine the influences of AMP-activated protein kinase (AMPK) on high glucose-induced oxidative stress and related endothelial impairment, and to investigate the function of TRPM2-mediated signaling pathway. The findings of this study could help identify potential therapeutic targets for developing new treatments or preventive strategies for patients with diabetes-related atherosclerosis and cardiovascular diseases.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eHUVECs used in this study were obtained from the Chinese Academy of Sciences Cell Bank (Beijing, China), and the cells were cultured in a Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Hyklong, Boston, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, New York, NY, USA), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin in standard conditions of 5% CO\u003csub\u003e2\u003c/sub\u003e and 37\u0026deg;C. Before the experiments, the cells were synchronized in cell culture media with reduced FBS (0.5%) for 12 h. Subsequently, the cells were exposed to the culture medium in the control group (CON group) or a high-glucose-containing cell culture medium in the high glucose group (HG group, 30 mmol/L glucose in the cell culture medium) for 48 h.\u003c/p\u003e \u003cp\u003eTo assess the potential roles of AMPK in the HG-mediated effects and the underlying mechanisms, HUVECs were pretreated for 1 h with 1 mM 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a well-known activator of AMPK, or 10 uM Compound C, a small molecule inhibitor of AMPK. Subsequently, the cells were treated with 30 mmol/L glucose in the HG\u0026thinsp;+\u0026thinsp;AICAR group and HG\u0026thinsp;+\u0026thinsp;CC group, respectively. For making comparison, HUVECs were also treated with 1 mM AICAR (AICAR group) or Compound C (CC group) alone, and these groups were included along with other experimental groups (HG\u0026thinsp;+\u0026thinsp;AICAR group, HG\u0026thinsp;+\u0026thinsp;CC group, CON group, and HG group).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell viability assessment\u003c/h3\u003e\n\u003cp\u003eTo assess the impact of HG on cell viability, HUVECs were exposed to 5 mM glucose in the control group or to a high-glucose-containing cell culture medium (30 mM glucose) in the high glucose (HG) group for 12, 24, and 48 h. Cell viability was determined using the cell counting kit-8 (CCK-8) kit, following the manufacturer's guidelines. In brief, a 96-well plate was loaded with 10 \u0026micro;l CCK-8 solution (5 mg/ml) in each well and incubated for 2 h at 37\u0026deg;C. The optical density at a wavelength of 490 nm was recorded, and cell viability was calculated with the values normalized to the normal control. To ensure reliability and accuracy, independent experiments were repeated five times.\u003c/p\u003e\n\u003ch3\u003eExamination of intracellular ROS levels in HUVECs\u003c/h3\u003e\n\u003cp\u003eTo assess intracellular ROS levels in HUVECs, 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per well were seeded into a 96-well plate. After the treatments were completed, cells were harvested and stained with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, Haimen, China) for 30 min, followed by flow cytometry (BD FACSCalibur\u0026trade;; BD Biosciences, Franklin Lakes, NJ, USA). The intracellular ROS levels in HUVECs were measured and normalized versus the control values.\u003c/p\u003e\n\u003ch3\u003eTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for apoptosis\u003c/h3\u003e\n\u003cp\u003eTUNEL staining was used to detect DNA fragmentation of cells as a marker of apoptosis using a TUNEL fluorescence FITC kit (Roche, Indianapolis, IN, USA) on the basis of the manufacturer\u0026rsquo;s instructions. In brief, HUVECs grown on coverslips were treated with 4% paraformaldehyde for fixation and with 0.1% Triton X-100 for permeabilization. The cells were subsequently exposed to the TUNEL reaction mixture and maintained at 37\u0026deg;C for 1 h. Following the incubation period, the presence of apoptotic cells was assessed by examining TUNEL-positive cells using a fluorescence microscope (DMI3000 B; Leica, Berlin, Germany).\u003c/p\u003e\n\u003ch3\u003eMeasurement of [Ca]i level\u003c/h3\u003e\n\u003cp\u003eTo measure [Ca\u003csup\u003e2+\u003c/sup\u003e]i level, HUVECs were loaded with the calcium indicator Fluo-3AM (5 \u0026micro;M) in HEPES-buffered saline solution. The fluorescence intensity of Fluo-3 bound to [Ca\u003csup\u003e2+\u003c/sup\u003e]i was quantified using flow cytometry. To determine the maximum fluorescence intensity (Fmax), calcium ion carrier A23187 (1\u0026times;10\u0026thinsp;\u0026minus;\u0026thinsp;5 mmol/L) and 1 mmol/L CaCl\u003csub\u003e2\u003c/sub\u003e were added to saturate the extracellular calcium. The minimum fluorescence intensity (Fmin) was measured by adding the fluorescence quencher MnCl\u003csub\u003e2\u003c/sub\u003e (2 mmol/L). Finally, [Ca\u003csup\u003e2+\u003c/sup\u003e]i level was calculated using the following formula: [Ca\u003csup\u003e2+\u003c/sup\u003e]i\u0026thinsp;=\u0026thinsp;Kd (F - Fmin)/(Fmax - F) (nmol/L).\u003c/p\u003e \u003cp\u003eHUVECs were incubated with high glucose in the HG group, ACA (an inhibitor of TRPM2) in the ACA group, or a combination of HG and ACA in the HG\u0026thinsp;+\u0026thinsp;ACA group for 2 h, with untreated cells serving as the control in the control group. The [Ca\u003csup\u003e2+\u003c/sup\u003e]i level was measured in the four groups.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting (WB)\u003c/h2\u003e \u003cp\u003eTo examine the protein expression levels of CaM, NOX, and TRPM2 in HUVECs, WB was conducted. In brief, HUVECs were collected through centrifugation at 700 \u0026times;g for 10 min at 4\u0026deg;C, followed by cell lysis to extract cellular proteins. After determining the protein concentration with Lowry\u0026rsquo;s method, equal amounts of proteins were then loaded on 10% polyacrylamide gel for separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes through electroblotting at 150 mA for 3 h. After blocking the PVDF membranes for 1 h in TBS containing 5% low-fat milk powder, they were incubated overnight with the primary antibody in TBS supplemented with either 5% BSA or 5% low-fat milk. The specific primary antibodies and dilutions were summarized as follows: anti-CaM (1:1000), anti-NOX (1:1000), anti-TRPM2 (1:1000; Abcam, Cambridge, UK), and anti-GAPDH (1:1000 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA). Thereafter, the secondary antibody linked with horseradish peroxidase (HRP) was added and incubated at room temperature for 1 h. Chemiluminescence detection was carried out with HRP Juice and charge-coupled device camera. Quantification of the densitometric signals was conducted using Quantity One Bioanalysis software (Bio-Rad Laboratories, Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eQuantitative polymerase chain reaction (qPCR) assay for determining TRPM2 gene expression level\u003c/h3\u003e\n\u003cp\u003eTo determine the expression level of the TRPM2 gene, qPCR analysis was performed. Total RNA was extracted from the specific cell subgroups, and then transcribed into cDNA. The extracted cDNA was subjected to pretreatment with specific primers and fluorescent probes following the requirements of the qPCR kit. Subsequently, the reaction system was prepared and the PCR cycle was implemented according to the prescribed procedure. Finally, the fluorescence signal was measured and the number of threshold cycles (Ct value) during the qPCR was analyzed using software. The relative amount of TRPM2 was calculated on the basis of the increase in fluorescence signal.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was carried out using SPSS 19.0 software (IBM, Armonk, NY, USA). Each experiment was conducted for at least three times. Data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (SEM). An analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s multiple comparison tests was utilized for group comparisons. Abnormally distributed data were normalized using logarithms before being analyzed using either the Mann\u0026ndash;Whitney test or Student\u0026rsquo;s t-test, depending on the specific conditions. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAMPK attenuated high glucose-induced ROS generation and apoptosis in HUVECs\u003c/h2\u003e \u003cp\u003eCell viability assay demonstrated that high glucose exposure (48 h) significantly diminished cell proliferation (HG vs. CON, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The analysis of cellular ROS level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and TUNEL staining for apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026amp;D) revealed that high glucose treatment significantly increased ROS generation and promoted apoptosis in HUVECs (HG vs. CON, all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, pretreatment with a small molecule inhibitor targeting AMPK, Compound C, in the HG\u0026thinsp;+\u0026thinsp;CC group significantly augmented cellular ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026amp;D) (HG\u0026thinsp;+\u0026thinsp;CC vs. HG, all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, pretreatment with the AMPK activator, AICAR, in the HG\u0026thinsp;+\u0026thinsp;AICAR group significantly reduced cellular ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and apoptosis in HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026amp;D) (HG\u0026thinsp;+\u0026thinsp;AICAR vs. HG, all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings suggested that AMPK exerted a protective effect against the increased ROS generation and apoptosis induced by high glucose in HUVECs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTRPM2 mediated the increase in the Ca\u003c/b\u003e \u003csup\u003e \u003cb\u003e2+\u003c/b\u003e \u003c/sup\u003e \u003cb\u003einflux in ROS generation and apoptosis induced by high glucose in HUVECs\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven that the TRPM2-mediated [Ca\u003csup\u003e2+\u003c/sup\u003e]i influx into cells plays a crucial role in response to cellular stimuli, such as ROS generation and oxidative stress, the effects of high glucose on [Ca\u003csup\u003e2+\u003c/sup\u003e]i level with or without an inhibitor of TRPM2, ACA, were assessed. The results of the CON, HG, ACA, and HG\u0026thinsp;+\u0026thinsp;ACA groups are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. It was revealed that [Ca\u003csup\u003e2+\u003c/sup\u003e]i level was significantly elevated in HUVECs (HG vs. CON, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Notably, the combined treatment of HG and ACA significantly abrogated the high glucose-associated increase of [Ca\u003csup\u003e2+\u003c/sup\u003e]i level (HG\u0026thinsp;+\u0026thinsp;ACA vs. HG, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas ACA alone had no significant effect on [Ca\u003csup\u003e2+\u003c/sup\u003e]i level in HUVECs (ACA vs. CON, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, cellular ROS levels and TUNEL staining for apoptosis were assessed in the CON, HG, ACA, and HG\u0026thinsp;+\u0026thinsp;ACA groups, and the findings are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C, and D. High glucose treatment in the HG group significantly promoted apoptosis (HG vs. CON, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while this effect was significantly reversed in the HG\u0026thinsp;+\u0026thinsp;ACA group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB \u0026amp;C), suggesting the potential role of TRPM2-mediated [Ca\u003csup\u003e2+\u003c/sup\u003e]i in the high glucose-induced apoptosis in HUVECs. In terms of ROS, treatment with high glucose in the HG group significantly elevated intracellular ROS levels (HG vs. CON, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and inhibition of TRPM2 with its inhibitor ACA significantly attenuated the high glucose-promoted ROS production (HG\u0026thinsp;+\u0026thinsp;ACA vs. HG, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAMPK exerted its inhibitory effect on high glucose-stimulated [Ca\u003csup\u003e2+\u003c/sup\u003e]i level via suppressing the TRPM2-related proteins\u003c/h2\u003e \u003cp\u003eIt was attempted to assess whether AMPK could play its role in modulating [Ca\u003csup\u003e2+\u003c/sup\u003e]i level upon high glucose stimulation by targeting TRPM2-associated signaling pathways. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, exposure to high glucose significantly elevated [Ca\u003csup\u003e2+\u003c/sup\u003e]i level in HUVECs (HG vs. CON, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, activation of AMPK with its activator, AICAR, significantly reversed the high glucose-induced [Ca\u003csup\u003e2+\u003c/sup\u003e]i level (HG\u0026thinsp;+\u0026thinsp;AICAR vs. HG, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while inhibition of AMPK with its inhibitor, Compound C, in the HG\u0026thinsp;+\u0026thinsp;CC group significantly increased the high glucose-induced [Ca\u003csup\u003e2+\u003c/sup\u003e]i level (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This finding indicated that AMPK could attenuate high glucose-induced ROS production and apoptosis via abrogation of [Ca\u003csup\u003e2+\u003c/sup\u003e]i level in HUVECs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression levels of key molecules in TRPM2-associated signaling pathways, including CaM and NOX, were subsequently determined, and the results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C. The results of WB revealed that treatment with high glucose significantly elevated protein expression levels of CaM and NOX in HUVECs (HG vs. CON, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Moreover, pretreatment with the activator of AMPK, AICAR, significantly abrogated the high glucose-associated upregulation of CaM and NOX (HG\u0026thinsp;+\u0026thinsp;AICAR vs. HG, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and conversely pretreatment with the inhibitor of AMPK, CC, significantly augmented the high glucose-associated upregulation of CaM and NOX (HG\u0026thinsp;+\u0026thinsp;CC vs. HG, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB\u0026amp;C). AICAR or CC alone altered the CaM and NOX protein levels compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Moreover, the expression level of the TRPM2 protein was not significantly altered by the activator or inhibitor of AMPK (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). However, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, the TRPM2 gene expression level was significantly upregulated in response to high glucose stimulation, as well as in cells pretreated with the AMPK inhibitor. Collectively, these findings suggested that AMPK could exert its inhibitory effect on the high glucose-promoted oxidative stress that was potentially mediated via regulatingTRPM2-related proteins, especially CaM and NOX, in HUVECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrated that exposure to high glucose stimulated ROS production in HUVECs through TRPM2-mediated elevation of [Ca\u003csup\u003e2+\u003c/sup\u003e]i level and excessive apoptosis. Notably, pretreatment with an AMPK activator attenuated the high glucose-induced ROS production by restoring cellular calcium homeostasis, as well as regulating the expression levels of TRPM2-related proteins (i.e. CaM and NOX). The findings indicated that the elevation of [Ca\u003csup\u003e2+\u003c/sup\u003e]i level through TRPM2 activation could be a crucial mechanism for the development of oxidative stress and subsequent endothelial injury in diabetes. Furthermore, AMPK may play a protective role against diabetes-related vascular complications by inhibiting ROS production and apoptosis induced by high glucose through regulating key molecules in the TRPM2-related signaling pathways, such as CaM and NOX, in ECs.\u003c/p\u003e \u003cp\u003eOxidative stress, known to cause endothelial dysfunction, is central to the development and progression of diabetes-associated complications [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recent research suggested that hyperglycemia-induced ROS production could contribute to calcium entry and endothelial apoptosis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The present study demonstrated that AMPK activation inhibited high glucose-stimulated ROS production, [Ca\u003csup\u003e2+\u003c/sup\u003e]i level, and endothelial apoptosis, aligning with the antioxidative property of AMPK. The findings of this study suggested that the beneficial effects of AMPK on [Ca\u003csup\u003e2+\u003c/sup\u003e]i level could be attributed to the antioxidative activity of AMPK.\u003c/p\u003e \u003cp\u003eIt is broadly accepted that hyperglycemia leads to metabolic disorders by triggering \u0026ldquo;aberrant\u0026rdquo; pathways that promote oxidative stress in human tissues [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Endothelial dysfunction is primarily associated with upregulation of cell-surface adhesion molecules in both ECs and blood immune cells, disrupting the redox serum balance, thereby resulting in ROS accumulation, oxidative stress, and hyperglycemia [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. High glucose levels could induce ROS production, which may promote the release of cytochrome C. This, in turn, triggers caspase-dependent apoptosis, resulting in the loss of ECs and ultimately causing diabetic endothelial dysfunction [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Furthermore, hyperglycemia and hyperlipidemia increase the acetylation of dynamin-related protein 1, impairing mitochondrial dynamics and biogenesis, thereby exacerbating apoptosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These findings have provided direct evidence, linking diabetic endothelial dysfunction to apoptosis. In the present study, it was revealed that high glucose significantly decreased cell viability and increased apoptosis of ECs, which were effectively abolished by AMPK activation. Therefore, the restoration of injured ECs through regulating proliferation and apoptosis of the cells may hold significant importance.\u003c/p\u003e \u003cp\u003eNotably, Ca\u003csup\u003e2+\u003c/sup\u003e and ROS are widely recognized as the major transduction signals that link the sarcoplasmic reticulum to mitochondrion. It has been shown that Ca\u003csup\u003e2+\u003c/sup\u003e overload could induce oxidative stress mainly through activation of the CaM-dependent signaling pathway, leading to an increase in ROS production [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. CaM regulates EC function mainly through Ca\u003csup\u003e2+\u003c/sup\u003e/CaM-dependent protein kinase II (CaMKII). Activation of Ca\u003csup\u003e2+\u003c/sup\u003e/CaMKII triggers ROS production by activating NOX [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. TRPM2, a cation channel permeable to Ca\u003csup\u003e2+\u003c/sup\u003e, is triggered by excessive ROS production [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The findings of the present study suggested that high glucose exposure caused [Ca\u003csup\u003e2+\u003c/sup\u003e]i overload via the TRPM2-CaM-NOX signaling pathway, leading to the induction of ROS production and apoptosis in HUVECs. This is in line with outcomes of several previous studies, indicating that TRPM2-mediated Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis plays a critical role in intracellular processes, such as gene expression, cell signaling, cell proliferation, and apoptosis [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consequently, the modulation of TRPM2 and [Ca\u003csup\u003e2+\u003c/sup\u003e]i might participate in high glucose-induced oxidative stress. When the TRPM2 inhibitor, ACA, was added along with Ca\u003csup\u003e2+\u003c/sup\u003e, a reduction was found in Ca\u003csup\u003e2+\u003c/sup\u003e level and ROS production, aligning with the result that oxidative stress induced impairment of endothelial barrier function through enhancing Ca\u003csup\u003e2+\u003c/sup\u003e influx into ECs, subsequently disrupting tight junctions between the cells. Overactivation of TRPM2-mediated Ca\u003csup\u003e2+\u003c/sup\u003e signaling results in the degradation of ZO-1 and internalization of VE-Cadherin, further enhancing the trans-endothelial migration of neutrophils in response to ROS-triggered different pathological stimuli [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, the present study indicated that pretreatment with an AMPK activator or inhibitor did not inhibit TRPM2 protein, while the high glucose-induced TRPM2 gene expression was significantly elevated after pretreatment with an AMPK inhibitor. This suggests that TRPM2-mediated Ca\u003csup\u003e2+\u003c/sup\u003e could be regulated by AMPK. In the present study, AMPK significantly suppressed the levels of CaM and NOX proteins, partially explaining the mechanism underlying oxidative stress. Moreover, this finding suggests that the activation of AMPK could be a promising strategy in mitigating high glucose-promoted oxidative stress and protecting ECs against oxidative stress.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrated that AMPK activation could mitigate oxidative stress induced by high glucose partly via inhibiting the TRPM2-CaM-NOX pathway. The findings highlighted the potential protective effect of AMPK on attenuating diabetes-associated vascular complications. Therefore, AMPK and TRPM2-CaM-NOX signaling pathways are promising therapeutic targets for developing treatments or preventive strategies for diabetes-related atherosclerosis and cardiovascular diseases.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACA, N-(p-amylcinnamoyl) anthranilic acid; AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK, AMP-activated protein kinase; Ca\u003csup\u003e2+\u003c/sup\u003e, calcium; [Ca\u003csup\u003e2+\u003c/sup\u003e]i, intracellular calcium; CaM; calmodulin; CC, Compound C; EC, endothelial cell; HG, high glucose; HUVECs, human umbilical vein vascular endothelial cells; NOX, NADPH oxidase; ROS, reactive oxygen species; TRPM2, transient receptor potential melastatin 2.\u0026nbsp;\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eTing Lu: Writing-original draft, Methodology. Yihua Zhang: Investigation. Zhe Wang: Writing-review \u0026amp; editing. Lu Zhao: Data curation. Dayan Zhou: Writing -review \u0026amp; editing. Qiang Xu: Supervision, Conceptualization. All the authors have contributed substantially to the study and approved the final manuscript.\u003c/p\u003e\n\u003cp skip=\"true\"\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Medjaden Inc. for\u0026nbsp;scientific editing\u0026nbsp;of this manuscript. This study was funded by the Scientific Research Project of Chongqing Nanan District Health Commission and Nanan District Science and Technology Bureau (Grant No. 2020-06).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePoznyak A, Grechko AV, Poggio P, Myasoedova VA, Alfieri V, Orekhov AN (2020) The Diabetes Mellitus-Atherosclerosis Connection: The Role of Lipid and Glucose Metabolism and Chronic Inflammation. Int J Mol Sci 21:1835. https://doi.org/10.3390/ijms21051835\u003c/li\u003e\n\u003cli\u003eAn Y, Xu BT, Wan SR et al (2023) The role of oxidative stress in diabetes mellitus-induced vascular endothelial dysfunction. 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Circ Res 126:456-470. https://doi.org/10.1161/circresaha.119.315252\u003c/li\u003e\n\u003cli\u003eVerma N, Pink M, Schmitz-Spanke S (2021) A new perspective on calmodulin-regulated calcium and ROS homeostasis upon carbon black nanoparticle exposure. Arch Toxicol 95:2007-2018. https://doi.org/10.1007/s00204-021-03032-0\u003c/li\u003e\n\u003cli\u003eSakurada R, Odagiri K, Hakamata A, Kamiya C, Wei J, Watanabe H (2019) Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells. Int J Mol Sci 20:1644. https://doi.org/10.3390/ijms20071644\u003c/li\u003e\n\u003cli\u003eMaliougina M, El Hiani Y (2023) TRPM2: bridging calcium and ROS signaling pathways-implications for human diseases. 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Antioxidants (Basel) 10:718. https://doi.org/10.3390/antiox10050718\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"AMP-activated protein kinase, endothelial cells, transient receptor potential melastatin 2, oxidative stress, high glucose","lastPublishedDoi":"10.21203/rs.3.rs-5312586/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5312586/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study aimed to determine the potential role of AMP-dependent protein kinase (AMPK) in high glucose-induced oxidative stress and to explore the underlying mechanisms in endothelial\u0026nbsp;cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman umbilical vein vascular endothelial cells (HUVECs) were pretreated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (AMPK activator), Compound C (CC) (AMPK inhibitor), or N-(p-amylcinnamoyl) Anthranilic Acid (ACA) [transient receptor potential melastatin 2 (TRPM2) inhibitor]. Subsequently, the cells in the intervention groups were exposed to high glucose. Reactive oxygen species levels, endothelial apoptosis, calcium entry, and protein expression levels in the HUVECs were detected. The AMPK activator, AICAR, exhibited a significant reduction in high glucose-stimulated ROS production and apoptosis in HUVECs, whereas the AMPK inhibitor, CC, significantly enhanced these effects. Pretreatment with the TRPM2 inhibitor, ACA, significantly abrogated high glucose-induced calcium entry, high glucose-stimulated oxidative stress, and apoptosis, indicating the role of TRPM2-mediated intracellular calcium ([Ca\u003csup\u003e2+\u003c/sup\u003e]i) influx in these cellular processes. Furthermore, the AMPK inhibitor, CC, significantly increased the levels of TRPM2-related proteins,including the primary calcium sensor calmodulin (CaM) and NADPH oxidase (NOX). Conversely, the AMPK activator exhibited opposite effects on these proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMPK activation could attenuate high glucose-stimulated oxidative stress and play a protective role in ECs partly through inhibiting the TRPM2-CaM-NOX pathway.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"AMPK activation attenuates high glucose-induced oxidative stress and apoptosis through regulation of TRPM2-mediated signaling pathway in endothelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 05:43:56","doi":"10.21203/rs.3.rs-5312586/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"145bf363-0b19-4484-a9f6-a1d2ad05bdf0","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-25T09:09:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-11 05:43:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5312586","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5312586","identity":"rs-5312586","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}