Curcumin mitigates high glucose-induced cardiac oxidative stress via Notch1 pathway activation

Curcumin mitigates high glucose-induced cardiac oxidative stress via Notch1 pathway activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Curcumin mitigates high glucose-induced cardiac oxidative stress via Notch1 pathway activation Xia Wu, ZhuangYin Qu, PeiYan Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6331619/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract This study aims to investigate the protective effects of curcumin (CUR) in high glucose (HG)-induced oxidative stress and apoptosis of primary cardiomyocytes by activating the Notch1 signaling pathway. CUR is a natural polyphenol isolated from turmeric rhizomes and plays an important role in the antioxidant, anti-apoptotic and anti-inflammatory effects of diabetes.Therefore, we established a in neonate rat myocardial cells induced by HG, followed by treatment with CUR and DAPT, respectively.We detected and assessed myocardial cells viability and antioxidant enzyme activity by CCK-8 reagent and antioxidant enzyme kit. Apoptosis was detected by flow cytometry. The production of reactive oxygen species was detected by fluorescence labeling, and the expression of related genes and proteins was detected by q-PCR and Western blot. HG-induced primary rat cardiomyocytes not only increased apoptosis and ROS production, but also decreased the activity of antioxidant enzymes and the expression of Notch1 and Hes1 proteins. After pre-treatment by CUR, surprisingly, we found that CUR can effectively increase the activity of rat cardiomyocytes induced by HG. The results showed that CUR could inhibit the apoptosis of rat cardiomyocytes, inhibit the production of intracellular ROS, and increase the activity of antioxidant enzymes. Further, we also found that CUR can upregulate the expression of Notch1 and Hes1 proteins and related genes, suggesting that the protective effect of CUR on HG-induced damage involves the Notch1/Hes1 signaling. These results suggest that CUR protects cardiomyocytes from HG-induced oxidative stress by activating Notch1 and its downstream target genes. Biological sciences/Cell biology Biological sciences/Molecular biology Curcumin Oxidative damage High glucose Diabetic cardiomyopathy Notch1 signaling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Diabetic cardiomyopathy (DCM) is a special kind of cardiomyopathy in the state of diabetes, that is, extensive focal myocardial necrosis on the basis of microangiopathy, leading to subclinical cardiac dysfunction. It is a special type of cardiomyopathy, it is not related to coronary artery disease, hypertension or other heart diseases. The incidence and prevalence of DCM have increased rapidly worldwide. Meanwhile, the incidence and mortality of cardiovascular diseases were associated with DCM in the past 50 years increased, too [1] . Hyperglycemia is a major feature of type I and type II diabetes, which can lead to excessive production of reactive oxygen species (ROS) , resulting in cardiomyocyte apoptosis, and then cause myocardial systolic dysfunction and promote the progression of DCM [2] . However, oxidative stress can be a double-edged sword, which can induce transient activation of antioxidant responses and prevent cytotoxicity [3] . Curcumin (CUR), known as two acetyl methane (1,7- double (4- hydroxyl -3- methoxy phenyl) -1,6- hepdiene -3, 5- two ketone), is an active ingredient in turmeric with non-toxic and non mutagenic properties.In addition, there is evidence that CUR has antiviral, anti-cancer,anti-inflammatory and strong antioxidant activities [4] . It is reported that CUR can protect liver and breast diseases, cancer, diabetes and heart disease and so on [5] . It has also been known that CUR protects cardiomyocytes against HG and ROS damage, and the antioxidant effect of CUR is to inhibit the production of ROS by inhibiting the opening of mitochondrial membrane potentials in cells [6] . Notch homolog1 translocation-associated (Drosophila) (Notch1) signaling pathway can regulate variety cellular process (such as development, differentiation, proliferation, apoptosis and regeneration), which plays an vital role in various organisms and cell type [7] . Notch1 signaling pathway is a cell communicate with each other ’ s platform, which plays an important regulatory role in the development of cardiac growth and differentiation in the embryonic stage and the progression of cardiomyopathy. Notch1 protein activated and released by the tumor necrosis factor -alpha (TNF-α) invertase (TACE) and g-secretase complex. It releases the Notch intracellular domain (N1ICD), and then combines the transcriptional factor CSL (CBF-1 in humans) [7,8] . After translocation of N1ICD into the nucleus, Notch promotes its target gene Hes. In the heart, Notch1 signaling not only regulates the development and differentiation of embryonic heart, but also stimulates the proliferation of immature cardiac myocytes [9,10] . Studies have shown that Notch1 signaling pathway is closely related to nuclear factor-erythroid-2-related factor 2 (Nrf2) signaling pathway and other signaling pathways, Notch ligand Dll1 and its target genes Hes1 and Hes3 can upregulate the expression of Nrf2 and its target genes, so as to reduce ROS formation and counteract oxidative stress [11,12] . However, it has remained elusive whether CUR ameliorates HG-mediated cardiomyocyte oxidative stress and apoptosis via activation of the Notch1 pathway. Therefore, the present study was designed to explore the potential protective mechanisms of CUR on primary rat cardiomyocytes using an in vitro model of HG-induced myocardial injury, and to study the role of Notch1 in the oxidative damage induced by HG. The present study may provide possible molecular mechanisms underlying the effect of CUR as a potent treatment for DCM. Materials and methods Cell culture and determination of CUR concentration and cell viability. The Sprague-Dawley rats were purchased from Nanjing Medical University’s Animal Centre. All animal experiments were performed under the procedures approved by the Animal Care and Use Committee of Nanjing first hospital. The previously described protocol was used to isolate neonatal rat cardiomyocytes from Sprague-Dawley rats (1 day old) [13] . The culture was incubated at 37℃, 5% CO 2 humidified incubator. The cells were interfered with the following experimental group for 12 h, 24 h, 36 h, 48 h, respectively. The experiment group: the normal glucose (NG) medium (5.5 mM) group, high glucose (HG, 33 mM) medium group, and HG + CUR (5, 10, 20, 40 μM, respectively) group (purity > 65%; Aladdin Biochemical Technology Company, Shanghai, China). Then 10 μL of Cell Counting Kit 8 (CCK-8, TransGen Biotech) solution was added to each well. Incubation in the CO 2 incubator was continued for 1.5 h. The absorbance at 450 nm was measured with a microplate reader (ThermoFisher Scientific). Use the same method to detect cell viability treated with X μM CUR and DAPT for 36 h. Detection of intracellular ROS . Myocardial cells were incubated in 6-well plates at 2x10 5 cells/well. The accumulation of ROS in the cells was detected by fluorescence probe 2, 7- two, chlorofluorescein two acetate (DCFH-DA). DCFH-DA can be transformed into DCFH2 by intracellular lactase and then oxidized to high fluorescence DCF by ROS. DCFH-DA is diluted with DMEM to 10 µM. H9C2 cardiacmyocytes were cultured in 60 mm cell dishes, removing cell culture medium and adding 1 mL diluted DCFH-DA. The cell incubator was incubated for 20 minutes at 37 ℃. The cells were washed with serum free medium for three times to remove DCFH-DA from the cells. Observation under fluorescence microscope. Annexin V/propidium iodide (PI) staining apoptosis assay. In a 10 cm culture dish, myocardial cells were grown at a density of 1x10 6 cells per dish. After allowing sufficient incubation, the resulting supernatant was subsequently removed. and the pellet was re-suspended in sterile phosphate-buffered saline (PBS). The suspension underwent two cycles of centrifugation at 1,after centrifugation at 1,000 rpm for 5 minutes at 4 ℃, the resulting precipitates were then re-dispersed in 200 µL of binding buffer, 10 µL of Annexin V-fluorescein isothiocyanate and 10 µL of propidium iodide (from Nanjing KeyGen Biotech Co.) were subsequently incorporated into the solution. Limitations. Nanjing, to which 300 µL of binding buffer was subsequently incorporated after gently mixing the solution in a dark room temperature setting for 15 minutes. and the rate of apoptosis was assessed using flow cytometry within 1 h. Biochemical analysis . The cellular supernatants or precipitates were gathered. The assessment of superoxide dismutase (SOD), malondialdehyde (MDA), and heme oxygenase-1 (HO-1) activities was carried out, alongside the measurement of nicotinamide adenine dinucleotide phosphate (NADPH) levels and glutathione peroxidase (GSH-Px) contents, utilizing the respective assay kits provided by Nanjing Jiancheng Bioengineering Institute, in accordance with the instructions given by the manufacturer. Quantitative reverse transcriptase polymerase chain reaction (q RT -PCR) . For this, total RNA was extracted and subsequently converted into cDNA through the use of a reverse transcription kit (TruScript First Strand cDNA Synthesis Kit) [2] . The StepOnePlus Real-Time PCR system was employed to carry out qRT-PCR, with β-actin mRNA serving as the internal control. The sequences of the primers utilized were: β-actin:forward, 5'-CATGTACGTTGCTATCCAGGC-3' and reverse,5'-CTCC TTAATGTCACGCACGAT-3'; Notch1:forward,5'-GAGGCGTGGCAGACTATGC-3' and reverse, 5'-CTTGTACTCCGTCAGCGTGA-3'; Hes1:forward,5'-TCAACACG ACACCGGATAAAC-3' and reverse, 5'-GCCGCGAGCTATCTTTCTTCA-3'. Western blot analysis. Myocardial cells were cultured in 6 cm culture dish treated with CUR and DAPT for 36h. Adding lysate into the culture dish.After lysis, the cells were scraped with one clean scraper on one side of the dish, and the cell debris and lysate were transferred to a 1.5 mL EP tube and centrifuged at 12000 g for 15 min at 4 ℃. Take the supernatant, 100 μL/tube aliquot, take a small amount of BCA protein quantification, the remaining -80 ℃ preservation. Take 1 tube protein plus 25 μL SDS-PAGE loading buffer, mixed, boiled for 10 min, and stored at -80 ℃ for Western blotting detection. Proteins were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis using primary antibodies for Notch1, N1ICD, Hes1 (Abcam,USA) and β-actin (Abcam, USA). The intensity of each band was analyzed using Image Lab 4.0.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Statistical analysis. For the analysis of data, SPSS 23.0 statistical software (SPSS) was employed to perform the statistical evaluations(SPSS, Inc., Chicago, IL, USA). The experimental results are reported as means ± standard deviation (SD). Group comparisons were conducted utilizing a one-way analysis of variance (ANOVA) [2] . For multiple comparisons where equal variances were not assumed, Tamhane's T2 method was employed. and the LSD method was used to test multiple comparisons of homogeneity of assumed variances A value of P < 0.05 was considered to indicate a statistically significant difference. A value of P < 0.01 was considered to indicate a statistically very significant difference. Results CUR increased the viability of myocardial cells with HG-induced damage . The CCK-8 assay was used to detect the effective concentration of CUR on myocardial cells. We can find that the cell viability of HG + CUR group was significantly dose-dependent with CUR ( P < 0.01), and the cell viability was highest in the HG + 20 μM CUR group ( P < 0.01) (Fig.1A). However, interestingly, we found that the cell viability of the HG + CUR group treated with a higher concentration of CUR (40 μM) was reduced, and the cell culture time is best at 36 h (Fig.1C). Therefore, in this study, we chose 20 μM concentration of CUR and cultivated for 36h. As shown in Fig. 1B, the cell viability in the HG group was significantly lower than that in the normal group ( P < 0.01). The cell viability of the HG + CUR group was significantly higher than that of the HG group. Furthermore the cell viability of the HG + DAPT group was significantly lower than that of the HG group. The results showed that DAPT significantly inhibited the protective effect of CUR ( P < 0.01) (Fig.1B). CUR can reduce the formation of ROS under condition s of HG damage . A high glucose injury model was established and DCFH-DA was used to detect intracellular ROS. The results showed that ROS formation in the HG-treated myocardial cells increased significantly ( P < 0.01), while the HG + CUR group decreased intracellular ROS levels ( P < 0.01). Compared with HG, the formation of ROS in the HG + DAPT group increased.It is worth noting that DAPT significantly increased ROS levels compared HG + CUR + DAPT with HG + CUR groups ( P < 0.01). HG + CUR + DAPT and HG group had no significant changes,indicating that DAPT increased ROS production (Fig. 2). CUR inhibited cardiomyocyte apoptosis under HG . The HG model was established and Annexin V/PI was used to detect apoptosis. The double staining flow cytometry analysis showed that there was no significant difference between the normal group and the mannitol group (Fig. 3A). The apoptosis rate of the HG + CUR group was lower than that of the HG group, indicating that activation of Notch1 signal can reduce cardiomyocyte apoptosis after HG ( P < 0.01). However, the HG + DAPT group significantly increase the rate of cardiomyocyte apoptosis than the HG group ( P < 0.01). It proved that activation of Notch1 signal can reduce myocardial apoptosis induced by high glucose. The apoptosis rate of the HG + CUR+ DAPT and the HG + DAPT group were higher than that of the HG + CUR group ( P < 0.01) (Fig. 3B). There was no significant difference in apoptosis between the control group and the mannitol group. CUR reduces HG-induced oxidative stress injury . The activities of SOD, MDA, HO-1, NADPH and GSH-Px were measured using the related kit. The activity of the HG + DAPT group was significantly lower than that of the HG group. However, the activity of the antioxidant enzymes in the HG + CUR group was significantly higher than that of the HG group. the activity of the antioxidant enzymes in the HG + CUR + DAPT group was lower than the HG + CUR group, but higher than the HG + DAPT groups. (except MDA) ( P < 0.01) (Fig. 4). CUR upregulated Notch1 and N1ICD expression in cardiomyocytes subjected to HG-induced damage . The result shows that the gene and protein of Notch1 and Hes1 levels are highly expressed in the HG + CUR group (Fig. 5A-D). However, the HG + DAPT groups had significantly lower protein levels compared to the HG group ( P < 0.01). Similarly, the expression of Hes1 protein in the HG + CUR group was similar to that of N1ICD. The HG + CUR protein expression level was higher than that of the HG, and the HG + CUR + DAPT ( P < 0.01). There is no difference between Control group and Mannitol group (Fig. 5E). DAPT and CUR affected the translocation of N1ICD from cytoplam to the nucles, CUR counteracted the blocking effect of DAPT on Notch1 signaling (Fig. 5F-H). Discussion This investigation revealed that exposure to high glucose levels diminished cell viability, intensified oxidative stress, and triggered apoptosis in the cardiomyocytes obtained from rats. Conversely, treatment with CUR led to an enhancement in cell viability in a manner that was both dependent on dosage and duration, while also alleviating oxidative stress within the cells; this effect correlated with a decrease in the production of reactive oxygen species and a reduction in apoptosis, alongside an increase in the activity of antioxidant enzymes. In addition, CUR treatment enhanced the levels of Notch1 and hes1 expression under high glucose conditions. The application of the Notch1 antagonist DAPT significantly diminished the CUR-induced elevation of Notch1 and Hes1, thereby hindering the protective effects of CUR on cardiomyocytes. This indicates that CUR may provide cardioprotective effects partly through the Notch1/Hes1 signaling pathway. The natural compound CUR, which is derived from the root of the everlasting herb curcuma longa, is a polyphenolic substance [4] . often referred to as curcumin, it has been extensively utilized in traditional Chinese medicine for the management of cardiac complications associated with diabetes. neuropathy, Kidney disease, Retina, dysfunction of β cells in the islets and vascular disorders. It significantly contributes to cardiovascular treatment by modulating antioxidant activity, effects that protect against cell death and inflammation [5,6 , 13] . Last yearthe beneficial impacts of CUR on heart health are well acknowledged, its role in mitigating cardiotoxicity caused by doxorubicin has been demonstrated for several years [14] . Research involving animals has indicated that CUR can also aid in lessening cardiac hypertrophy induced by lipopolysaccharides and in relieving myocardial fibrosis resulting from spontaneous hypertension, it also plays a role in averting heart failure [15] . Furthermore, CUR has been shown to facilitate cardiac regeneration and enhance heart function in a rat model of myocardial infarction [16] . In this study, The results indicated a notable dose-dependent enhancement in the viability of cardiomyocytes with CUR treatment. To determine if the protective effect of CUR against HG-induced apoptosis in cardiomyocytes was linked to the reduction of oxidative stress, the levels of intracellular ROS were assessed using the DCFH-DA probe, revealing that CUR enhanced the function of antioxidant enzymes such as SOD. HO-1， NADPH and GSH Px, excluding MDA. At a CUR concentration of 20 µM, the viability of the cells reached its peak. suggesting that CUR has the ability to safeguard cardiomyocytes against injury from high glucose levels. In the case of the HG + CUR group, administering a more elevated dose (40 μM) resulted in increased effects. Toxic cells suggesting that excessive doses of CUR could lead to detrimental effects (Fig. 1). CUR participates in myocardial protection through a variety of pathways, such as SIRT1, NF-κB, JNK, and so on [17] . The Notch1 signaling pathway involves a wide range of physiological processes, including mitosis, cell survival, metastasis and transcription in the state of stress [18] . In addition,the Notch1/Hes1 signaling pathway has been shown to protect myocardial cells and reduce myocardial injury [19] . To investigate the Notch1 pathway and whether it has a pivotal role in the effects of CUR on HG-induced cardiomyocyte apoptosis, the highly active γ-secretase inhibitor DAPT was used to block the Notch1 pathway. Our results show that when exposed to HG injury, the expression of Notch1 protein is highest when 20 µM CUR is administered. Meanwhile, we used DAPT (α-secretase inhibitor, which inhibits Notch1 signaling pathways) to eliminate these effects. In fact, these effects were eliminated when DAPT was used, indicating that the cardioprotective effects of CUR are related to the Notch1 signaling pathway. Therefore, we conferred that Notch1 may be involved in the protective effect of CUR on HG-induced cardiomyocyte injury. CUR exerts a protective effect on cardiomyocytes against injuries induced by high glucose by blocking the Notch1 signaling pathway, which results in enhanced cell viability, a lower rate of apoptosis, and diminished production of reactive oxygen species. In a similar way, the activation of the Notch1 signaling pathway resulted in enhanced cardiomyocyte survival, a reduction in apoptosis among the cardiomyocytes, and a decrease in damage caused by ROS. In SK-N-MC cells, the signaling pathway of Notch1 can be stimulated by ROS, which results in cell death via apoptosis. Nonetheless, the compound EUK134 demonstrated its ability to inhibit cell death in SK-N-MC cells triggered by H 2 O 2 /menadione through the modulation of the Notch1 signaling pathway [20] . Elevated glucose levels can trigger significant levels of reactive oxygen species (ROS), which contribute to oxidative stress damage [21] . The findings of this investigation illustrated that treatment with CUR resulted in a decrease in MDA levels and an enhancement in the activities of HO-1, SOD, GSH-Px, and NADPH. This suggests that CUR has the potential to mitigate the harm caused by HG in oxidative stress (Fig. 4). Research demonstrates that treatment with CUR can lower ROS levels that are provoked by HG. Research indicates that CUR may also inhibit the generation of ROS in N2A cells while alleviating oxidative stress effects in endothelial cells derived from human umbilical veins [22] . This research does have certain constraints. It has been proposed that the generation of reactive oxygen species and the resulting oxidative stress could potentially play a role in cardioprotective mechanisms associated with CUR; However, the precise pathways involved remain to be elucidated. Initially, only western blot assays were employed in the examination of the signaling pathways to assess the expression levels of N1ICD and Hes1 proteins. And we have no performed experiments to determine the mRNA level of N1ICD. Besides, there is a lack of in vivo experiments to justify the entire study. In summary, we demonstrated that activation of the Notch1 signaling pathway protects cardiomyocytes by enhancing cell viability, reducing ROS formation, and inhibiting the production of apoptosis. In addition, most of the cardioprotective effects were abolished by DAPT. Although the activity of SOD, HO-1, NADPH and GSH-Px antioxidant enzymes impaired DAPT damage to cardiomyocytes. The findings show that pretreating with CUR can enhance the levels of Notch1 and Hes1 proteins, which implies that CUR has a protective role against damage induced by high glucose (HG). Abbreviations ANOVA a one-way analysis of variance CCK Cell Counting Kit CUR curcumin DCM diabetic cardiomyopathy DMEM dulbecco's modified eagle medium GSH-Px glutathione peroxidase HG high glucose HO-1 heme oxygenase-1 H hour MDA malondialdehyde NADPH nicotinamide adenine dinucleotide phosphate NG normal glucose N1ICD notch 1 intracellular domain Notch1 Notch homolog 1, translocation-associated (Drosophila) NRF2 nuclear factor-erythroid-2-related factor 2 PBS phosphate-buffered saline PI propidium iodide ROS reactive oxygen species SD standard deviation SOD superoxide dismutase TNF-α tumor necrosis factor -alpha Declarations Acknowledgements Not applicable. Funding All research costs were supplied by Nanjing Medical University Science and Technology Development Fund (No. NMUB20220079). Data Availability All datasets are included as Supplementary Files S1 . A statement for use of experimental animals The study is reported in accordance with ARRIVE guidelines. Author Contributions X.W. researched, analyzed the data and reviewed the article. X.W. and Z.Y. Q. wrote and edited the article. X.W. analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft, performed statistical analysis. P.Y. L. contributed initial discussion of the project and reviewed the article. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval and consent to participate This study was approved (NMUC20240358) by Laboratory Animal Ethics Committee of Nanjing First Hospital. Patient consent for publication Not applicable. Authors' information Dr PeiYan Liu ORCID iD: 0009‑0003‑7492‑2227. References Dillmann WH. Diabetic cardiomyopathy. Circ Res 2019;124(8), 1160-1162. Wu X, Zhou XL, Lai SQ, Liu JC, Qi JW. 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Supplementary Files S1rawimages20250326221751.pdf FilesS1.doc Cite Share Download PDF Status: Published Journal Publication published 02 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 13 May, 2025 Reviews received at journal 12 May, 2025 Reviewers agreed at journal 12 May, 2025 Reviews received at journal 08 May, 2025 Reviewers agreed at journal 06 May, 2025 Reviewers invited by journal 05 May, 2025 Editor assigned by journal 05 May, 2025 Editor invited by journal 29 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 28 Mar, 2025 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. 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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-6331619","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":452949214,"identity":"004330b4-de6f-49df-af8b-ee95c76849c7","order_by":0,"name":"Xia Wu","email":"","orcid":"","institution":"Nanjing first hospital","correspondingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Wu","suffix":""},{"id":452949215,"identity":"a1b746b1-f304-4c4f-8be4-520b32debb08","order_by":1,"name":"ZhuangYin Qu","email":"","orcid":"","institution":"Nanjing first hospital","correspondingAuthor":false,"prefix":"","firstName":"ZhuangYin","middleName":"","lastName":"Qu","suffix":""},{"id":452949216,"identity":"4f2fc655-71ab-4906-b87c-0fe7f82352a5","order_by":2,"name":"PeiYan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYFACxgYgYcEDZn8wsJEjVosEWAvjjII0Y2KtkgCTzDwfDicSVGvO3twm8XOHhIzB8bPHpG0MmBMY2A8f3YBPi2XPwTbJ3jMSPAZn8tKkcwzY8hh40tJu4NNicCOxTYK3DajlBo8ZUAtPMdBfZvi13H/YJvkXpsXCQCKxgaCWG4xt0nBbGAwMCGux7ElstpYFapE8k5ds2WOQYMxGyC/m7Mcf3nzbZmPPd/zswRs//vyX42c/fAy/wxgYWCCRwgBJAAxs+JRDtTB/QNEyCkbBKBgFowAdAABTg0N59mEzGgAAAABJRU5ErkJggg==","orcid":"","institution":"Nanjing first hospital","correspondingAuthor":true,"prefix":"","firstName":"PeiYan","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-03-29 03:38:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6331619/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6331619/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-09105-9","type":"published","date":"2025-07-02T15:58:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82560718,"identity":"5cff52ff-224d-4729-a846-c769d1e67cb9","added_by":"auto","created_at":"2025-05-13 01:36:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55866,"visible":true,"origin":"","legend":"\u003cp\u003eCUR increased viability of myocardial cells subjected to HG injury. (A) Rat myocardial cells were incubated with CUR (concentration gradient 5, 10, 20, 40 μM, respectively). (B) The HG grourp decreased the viability of myocardial cells, whereas HG + CUR treatment increased the cell viability, while treatment of the Notch1 inhibitor DAPT abolished the effects of CUR on cell viability. (C) Cell viability was determined using the TransDetect\u003csup\u003eTM\u003c/sup\u003e Cell Counting Kit (CCK)-8 assay at varying concentrations of CUR at different timepoints (12, 24, 36, and 48 h).**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ΔΔ\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, and ##\u003cem\u003eP \u003c/em\u003e\u0026lt;0.01 versus 20 μM CUR group at 12, 24, 36, 48 h, respectively (A). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 HG group vs. HG +CUR group, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 HG group vs. HG + DAPT group, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 HG + CUR group vs. HG + CUR + DAPT group (B). Data are expressed as the mean ± SD, n = 3.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6331619/v1/e647304033c622139cb40a3b.png"},{"id":82560722,"identity":"0ffe7eeb-e3d0-43ec-9678-24e5d2d23074","added_by":"auto","created_at":"2025-05-13 01:36:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":325766,"visible":true,"origin":"","legend":"\u003cp\u003eCUR pretreatment decreased the reactive oxygen species (ROS) generation. (A) ROS generation was measured using DCF fluorescence analysis. Representative images of the intracellular ROS production are shown (100´). (B) The DCF\u003csup\u003e+\u003c/sup\u003e numbers significantly increased in the HG group and HG + DAPT group (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. HG + CUR group) and decreased in HG + CUR + DAPT groups (**\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 vs.HG + CUR group).Data are expressed as the mean ± SD, n = 3.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6331619/v1/4561d2c13ebda9cdce694108.png"},{"id":82560721,"identity":"61bb425d-5a41-4369-a107-2614c7d557a9","added_by":"auto","created_at":"2025-05-13 01:36:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292078,"visible":true,"origin":"","legend":"\u003cp\u003eCUR inhibited the apoptosis of cardiomyocytes, and can regulate the activity of antioxidant enzymes. (A) Representative dot plots of flow cytometry (the x-axis and y-axis represent Annexin V and PI staining, respectively). (B) Evaluation of apoptotic cell populations.The apoptosis rate of HG + CUR group was significantly lower than that of HG group and HG + DAPT group, there were obvious statistical significance (**\u003cem\u003eP \u003c/em\u003e\u0026lt;\u003cem\u003e \u003c/em\u003e0.01). Data are expressed as the mean ± SD, n = 3.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6331619/v1/e420afb6949d94e32ed090c8.png"},{"id":82560720,"identity":"9774c4b4-bc2f-4778-8941-3c4275e4e98a","added_by":"auto","created_at":"2025-05-13 01:36:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45508,"visible":true,"origin":"","legend":"\u003cp\u003eThe contents of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), heme oxygenase-1 (HO-1), nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione peroxidase (GSH-Px) induced by high glucose (HG) injury. The activities of SOD, HO-1, NADPH, and GSH-Px were significantly increased in the HG + CUR group (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. HG group) and were decreased in both the HG + CUR + DAPT and HG + DAPT groups (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. HG + CUR group). However, the contents of MDA showed the opposite trends. Data are expressed as the mean ± SD, n = 3.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6331619/v1/91ccabf4d7221396afd5c5f4.png"},{"id":82560724,"identity":"6523906d-5bfd-4e1a-b6a5-367d1d0b3095","added_by":"auto","created_at":"2025-05-13 01:36:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":109422,"visible":true,"origin":"","legend":"\u003cp\u003eCUR activated Notch1 signaling pathways. (A,B) Rat myocardial cells were incubated in HG-treated with or without CUR for 36 h, respectively. qRT-PCR was applied to detect the mRNA expression of Notch1 and Hes1 genes. (C) Notch1, N1ICD and Hes1 protein expression were evaluated by western blotting; b-actin was used as an internal control. (D) DAPT and CUR affected the translocation of N1ICD from cytoplam to the nucles.CUR promoted N1ICD translocation into the nucleus. (E-G) The expression levels of Notch1, N1ICD and Hes1 proteins significantly increased in the HG + CUR group (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. HG group) and decreased in both the HG + CUR + DAPT (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. HG + CUR group). (H) The expression levels of N1ICD proteins in nuclear significantly increased in both the HG + CUR group (**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. HG group, and **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. HG + CUR + DAPT). Data are expressed as the mean ± SD, n = 3.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6331619/v1/10ad062ccc0851fccecbc19a.png"},{"id":86179194,"identity":"a4512f90-cfc6-4f02-bd13-255b68158b03","added_by":"auto","created_at":"2025-07-07 16:17:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1203135,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6331619/v1/99931865-7871-46b5-b17d-f395b1d96fbd.pdf"},{"id":82562314,"identity":"fc378b3f-7fba-41e0-aa4f-6a4b0f004e97","added_by":"auto","created_at":"2025-05-13 01:44:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":244853,"visible":true,"origin":"","legend":"","description":"","filename":"S1rawimages20250326221751.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6331619/v1/fe626a560ea0a6000ff004d7.pdf"},{"id":82560719,"identity":"4ce5fe9e-d8e1-4896-9531-eee595bbe456","added_by":"auto","created_at":"2025-05-13 01:36:32","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":203264,"visible":true,"origin":"","legend":"","description":"","filename":"FilesS1.doc","url":"https://assets-eu.researchsquare.com/files/rs-6331619/v1/e071835f51f76c29991ee083.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Curcumin mitigates high glucose-induced cardiac oxidative stress via Notch1 pathway activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDiabetic cardiomyopathy (DCM) is a special kind of cardiomyopathy in the state of diabetes, that is, extensive focal myocardial necrosis on the basis of microangiopathy, leading to subclinical cardiac dysfunction. It is a special type of cardiomyopathy, it is not related to coronary artery disease, hypertension or other heart diseases. The incidence and prevalence of DCM have increased rapidly worldwide. Meanwhile, the incidence and mortality of cardiovascular diseases were associated with DCM in the past 50 years increased, too\u003csup\u003e[1]\u003c/sup\u003e.\u0026nbsp;Hyperglycemia is a major feature of type I and type II diabetes,\u0026nbsp;which can lead to excessive production\u0026nbsp;of\u0026nbsp;reactive oxygen species (ROS) ,\u0026nbsp;resulting in\u0026nbsp;cardiomyocyte apoptosis,\u0026nbsp;and then cause myocardial systolic dysfunction and promote the progression of DCM\u003csup\u003e[2]\u003c/sup\u003e.\u0026nbsp;However,\u0026nbsp;oxidative stress can be a double-edged sword,\u0026nbsp;which can induce transient activation of antioxidant responses and prevent cytotoxicity\u003csup\u003e[3]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCurcumin (CUR), known as two acetyl methane (1,7- double (4- hydroxyl -3- methoxy phenyl) -1,6- hepdiene -3, 5- two ketone), is an active ingredient in turmeric with non-toxic and non mutagenic properties.In addition, there is evidence that CUR has antiviral, anti-cancer,anti-inflammatory and strong antioxidant activities\u003csup\u003e[4]\u003c/sup\u003e.\u0026nbsp;It is reported that CUR can protect liver and breast diseases, cancer, diabetes and heart disease and so on\u003csup\u003e[5]\u003c/sup\u003e.\u0026nbsp;It\u0026nbsp;has also been known that CUR protects cardiomyocytes against HG and ROS damage, and\u0026nbsp;the antioxidant effect of CUR is to inhibit the production of ROS by inhibiting the opening of mitochondrial membrane potentials in cells\u003csup\u003e[6]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNotch homolog1 translocation-associated (Drosophila) (Notch1) signaling pathway can regulate variety cellular process (such as development, differentiation, proliferation, apoptosis and regeneration), which plays an vital role in various organisms and cell type\u003csup\u003e[7]\u003c/sup\u003e. Notch1 signaling pathway is a cell communicate with each other\u003csup\u003e\u0026rsquo;\u003c/sup\u003es platform, which plays an important regulatory role in the development of cardiac growth and differentiation in the embryonic stage and the progression of cardiomyopathy. Notch1 protein activated and released by the tumor necrosis factor -alpha (TNF-\u0026alpha;) invertase (TACE) and\u0026nbsp;g-secretase complex. It releases the Notch intracellular domain (N1ICD), and then combines the transcriptional factor CSL (CBF-1 in humans)\u003csup\u003e[7,8]\u003c/sup\u003e. After translocation of N1ICD into the nucleus, Notch promotes its target gene Hes. In the heart, Notch1 signaling not only regulates the development and differentiation of embryonic heart, but also stimulates the proliferation of immature cardiac myocytes\u003csup\u003e[9,10]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eStudies have shown that Notch1 signaling pathway is closely related to nuclear factor-erythroid-2-related factor 2 (Nrf2)\u0026nbsp;signaling pathway and other signaling pathways,\u0026nbsp;Notch ligand Dll1 and its target genes Hes1 and Hes3 can upregulate the expression of Nrf2 and its target genes,\u0026nbsp;so as to reduce ROS formation and counteract oxidative stress\u003csup\u003e[11,12]\u003c/sup\u003e. However, it has remained elusive whether CUR ameliorates HG-mediated cardiomyocyte oxidative stress and apoptosis via activation of the Notch1 pathway. Therefore, the present study was designed to explore the potential protective mechanisms of CUR on primary rat cardiomyocytes using an in vitro model of HG-induced myocardial injury, and to study the role of Notch1 in the oxidative damage induced by HG. The present study may provide possible molecular mechanisms underlying the effect of CUR as a potent treatment for DCM.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eCell culture and determination of\u0026nbsp;\u003c/em\u003e\u003cem\u003eCUR\u003c/em\u003e\u003cem\u003e\u0026nbsp;concentration and cell viability.\u0026nbsp;\u003c/em\u003eThe Sprague-Dawley rats were purchased from Nanjing Medical University\u0026rsquo;s Animal Centre. All animal experiments were performed under the procedures approved by the Animal Care and Use Committee of Nanjing first hospital. The previously described protocol was used to isolate neonatal rat cardiomyocytes from Sprague-Dawley rats (1 day old)\u003csup\u003e[13]\u003c/sup\u003e. The culture was incubated at 37℃,\u0026nbsp;5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator. The cells were interfered with the following experimental group for 12 h, 24 h, 36 h, 48 h, respectively. The experiment group: the normal glucose (NG) medium (5.5 mM) group, high glucose (HG, 33 mM) medium group, and HG + CUR (5, 10, 20, 40 \u0026mu;M, respectively) group (purity \u0026gt; 65%; Aladdin Biochemical Technology Company, Shanghai, China). Then 10 \u0026mu;L of Cell Counting Kit 8 (CCK-8, TransGen Biotech) solution was added to each well. Incubation in the CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eincubator was continued for 1.5 h. The absorbance at 450 nm was measured with a microplate reader (ThermoFisher Scientific). Use the same method to detect cell viability treated with X \u0026mu;M CUR and DAPT for 36 h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDetection of intracellular ROS\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eMyocardial cells were incubated in 6-well plates at 2x10\u003csup\u003e5\u003c/sup\u003e cells/well. The accumulation of ROS in the cells was detected by fluorescence probe 2, 7- two, chlorofluorescein two acetate (DCFH-DA). DCFH-DA can be transformed into DCFH2 by intracellular lactase and then oxidized to high fluorescence DCF by ROS. DCFH-DA is diluted with DMEM to 10 \u0026micro;M. H9C2 cardiacmyocytes were cultured in 60 mm cell dishes, removing cell culture medium and adding 1 mL diluted DCFH-DA. The cell incubator was incubated for 20 minutes at 37 ℃. The cells were washed with serum free medium for three times to remove DCFH-DA from the cells. Observation under fluorescence microscope.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnnexin V/propidium iodide (PI) staining apoptosis assay.\u0026nbsp;\u003c/em\u003e In a 10 cm culture dish, myocardial cells were grown at a density of 1x10\u003csup\u003e6\u003c/sup\u003e cells per dish. After allowing sufficient incubation, the resulting supernatant was subsequently removed. and the pellet was re-suspended in sterile phosphate-buffered saline (PBS). The suspension underwent two cycles of centrifugation at 1,after centrifugation at 1,000 rpm for 5 minutes at 4 ℃, the resulting precipitates were then re-dispersed in 200 \u0026micro;L of binding buffer, 10 \u0026micro;L of Annexin V-fluorescein isothiocyanate and 10 \u0026micro;L of propidium iodide (from Nanjing KeyGen Biotech Co.) were subsequently incorporated into the solution. Limitations. Nanjing, to which 300 \u0026micro;L of binding buffer was subsequently incorporated after gently mixing the solution in a dark room temperature setting for 15 minutes. and the rate of apoptosis was assessed using flow cytometry within 1 h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBiochemical analysis\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe cellular supernatants or precipitates were gathered. The assessment of superoxide dismutase (SOD), malondialdehyde (MDA), and heme oxygenase-1 (HO-1) activities was carried out, alongside the measurement of nicotinamide adenine dinucleotide phosphate (NADPH) levels and glutathione peroxidase (GSH-Px) contents, utilizing the respective assay kits provided by Nanjing Jiancheng Bioengineering Institute, in accordance with the instructions given by the manufacturer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQuantitative reverse transcriptase polymerase chain reaction\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003e(q\u003c/em\u003e\u003cem\u003eRT\u003c/em\u003e\u003cem\u003e-PCR)\u003c/em\u003e\u003cem\u003e.\u003c/em\u003eFor this, total RNA was extracted and subsequently converted into cDNA through the use of a reverse transcription kit (TruScript First Strand cDNA Synthesis Kit)\u003csup\u003e[2]\u003c/sup\u003e. The StepOnePlus Real-Time PCR system was employed to carry out qRT-PCR, with \u0026beta;-actin mRNA serving as the internal control. The sequences of the primers utilized were: \u0026beta;-actin:forward, 5\u0026apos;-CATGTACGTTGCTATCCAGGC-3\u0026apos; and reverse,5\u0026apos;-CTCC TTAATGTCACGCACGAT-3\u0026apos;; Notch1:forward,5\u0026apos;-GAGGCGTGGCAGACTATGC-3\u0026apos; and reverse, 5\u0026apos;-CTTGTACTCCGTCAGCGTGA-3\u0026apos;; Hes1:forward,5\u0026apos;-TCAACACG ACACCGGATAAAC-3\u0026apos; and reverse, 5\u0026apos;-GCCGCGAGCTATCTTTCTTCA-3\u0026apos;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWestern blot analysis.\u0026nbsp;\u003c/em\u003eMyocardial cells were cultured in 6 cm culture dish treated with CUR and DAPT for 36h. Adding lysate into the culture dish.After lysis, the cells were scraped with one clean scraper on one side of the dish, and the cell debris and lysate were transferred to a 1.5 mL EP tube and centrifuged at 12000 g for 15 min at 4 ℃. Take the supernatant, 100 \u0026mu;L/tube aliquot, take a small amount of BCA protein quantification, the remaining -80 ℃ preservation. Take 1 tube protein plus 25 \u0026mu;L SDS-PAGE loading buffer, mixed, boiled for 10 min, and stored at -80 ℃ for Western blotting detection. Proteins were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis using primary antibodies for Notch1, N1ICD, Hes1 (Abcam,USA) and \u0026beta;-actin (Abcam, USA). The intensity of each band was analyzed using Image Lab 4.0.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis.\u0026nbsp;\u003c/em\u003eFor the analysis of data, SPSS 23.0 statistical software (SPSS) was employed to perform the statistical evaluations(SPSS, Inc., Chicago, IL, USA). The experimental results are reported as means \u0026plusmn; standard deviation (SD). Group comparisons were conducted utilizing a one-way analysis of variance (ANOVA)\u003csup\u003e[2]\u003c/sup\u003e. For multiple comparisons where equal variances were not assumed, Tamhane\u0026apos;s T2 method was employed. and the LSD method was used to test multiple comparisons of homogeneity of assumed variances A value of \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered to indicate a statistically significant difference. A value of \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 was considered to indicate a statistically very significant difference.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eCUR increased the viability of\u0026nbsp;\u003c/em\u003e\u003cem\u003emyocardial cells\u003c/em\u003e\u003cem\u003e\u0026nbsp;with HG-induced damage\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe\u003cem\u003e\u0026nbsp;\u003c/em\u003eCCK-8 assay was used to detect the effective concentration of CUR on myocardial cells. We can find that the cell viability of HG + CUR group was significantly dose-dependent with CUR (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026lt;\u0026nbsp;0.01),\u0026nbsp;and the cell viability was highest\u0026nbsp;in the\u0026nbsp;HG\u0026nbsp;+\u0026nbsp;20\u0026nbsp;\u0026mu;M\u0026nbsp;CUR group (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026lt;\u0026nbsp;0.01)\u0026nbsp;(Fig.1A).\u0026nbsp;However,\u0026nbsp;interestingly, we found that\u0026nbsp;the cell viability of the HG\u0026nbsp;+\u0026nbsp;CUR group treated with a higher concentration of CUR (40\u0026nbsp;\u0026mu;M) was reduced,\u0026nbsp;and the cell culture time is best at 36\u0026nbsp;h\u0026nbsp;(Fig.1C). Therefore, in this study, we chose 20\u0026nbsp;\u0026mu;M concentration of CUR and cultivated for 36h.\u0026nbsp;As shown in Fig.\u0026nbsp;1B,\u0026nbsp;the cell viability\u0026nbsp;in the HG group was significantly lower than that in the normal group\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). The cell viability of the HG + CUR group was significantly higher than that of the HG group. Furthermore the cell viability of the HG + DAPT group was significantly lower than that of the HG group. The results showed that DAPT significantly inhibited the protective effect of CUR (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig.1B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCUR can reduce the formation of ROS under condition\u003c/em\u003e\u003cem\u003es\u003c/em\u003e\u003cem\u003e\u0026nbsp;of\u0026nbsp;\u003c/em\u003e\u003cem\u003eHG\u003c/em\u003e\u003cem\u003e\u0026nbsp;damage\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eA high glucose injury model was established and DCFH-DA was used to detect intracellular ROS. The results showed that ROS formation in the HG-treated myocardial cells increased significantly (\u003cem\u003eP\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u0026lt;\u0026nbsp;0.01),\u0026nbsp;while\u0026nbsp;the\u0026nbsp;HG\u0026nbsp;+\u0026nbsp;CUR group decreased intracellular ROS levels (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Compared with HG, the formation of ROS in the HG + DAPT group increased.It is worth noting that DAPT significantly increased ROS levels compared HG + CUR + DAPT with HG + CUR groups (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). HG + CUR + DAPT and HG group had no significant changes,indicating that DAPT increased ROS production (Fig. 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCUR inhibited cardiomyocyte apoptosis under HG\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe HG model was established and Annexin V/PI was used to detect apoptosis. The double staining flow cytometry analysis showed that there was no significant difference between the normal group and the mannitol group (Fig. 3A). The apoptosis rate of the HG + CUR group was lower than that of the HG group, indicating that activation of Notch1 signal can reduce cardiomyocyte apoptosis after HG (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). However, the HG + DAPT group significantly increase the rate of cardiomyocyte apoptosis than the HG group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). It proved that activation of Notch1 signal can reduce myocardial apoptosis induced by high glucose. The apoptosis rate of the HG + CUR+ DAPT and the HG + DAPT group were higher than that of the HG + CUR group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig. 3B). There was no significant difference in apoptosis between the control group and the mannitol group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCUR reduces HG-induced oxidative stress injury\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe activities of SOD, MDA, HO-1, NADPH and GSH-Px were measured using the related kit. The activity of the HG + DAPT group was significantly lower than that of the HG group. However, the activity of the antioxidant enzymes in the HG + CUR group was significantly higher than that of the HG group. the activity of the antioxidant enzymes in the HG + CUR + DAPT group was lower than the HG + CUR group, but higher than the HG + DAPT groups. (except MDA) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) (Fig. 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCUR upregulated\u0026nbsp;\u003c/em\u003e\u003cem\u003eNotch1 and\u0026nbsp;\u003c/em\u003e\u003cem\u003eN1ICD expression in cardiomyocytes subjected to HG-induced damage\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThe result shows that the gene and protein of Notch1 and Hes1\u003cem\u003e\u0026nbsp;\u003c/em\u003elevels are highly expressed in the HG + CUR group (Fig. 5A-D). However, the HG + DAPT groups had significantly lower protein levels compared to the HG group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Similarly, the expression of Hes1 protein in the HG + CUR group was similar to that of N1ICD. The HG + CUR protein expression level was higher than that of the HG, and the HG + CUR + DAPT (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). There is no difference between Control group and Mannitol group (Fig. 5E). DAPT and CUR affected the translocation of N1ICD from cytoplam to the nucles, CUR counteracted the blocking effect of DAPT on Notch1 signaling (Fig. 5F-H).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis investigation revealed that exposure to high glucose levels diminished cell viability, intensified oxidative stress, and triggered apoptosis in the cardiomyocytes obtained from rats. Conversely, treatment with CUR led to an enhancement in cell viability in a manner that was both dependent on dosage and duration, while also alleviating oxidative stress within the cells; this effect correlated with a decrease in the production of reactive oxygen species and a reduction in apoptosis, alongside an increase in the activity of antioxidant enzymes. In addition, CUR treatment enhanced the levels of Notch1 and hes1 expression under high glucose conditions. The application of the Notch1 antagonist DAPT significantly diminished the CUR-induced elevation of Notch1 and Hes1, thereby hindering the protective effects of CUR on cardiomyocytes. This indicates that CUR may provide cardioprotective effects partly through the Notch1/Hes1 signaling pathway.\u003c/p\u003e\n\u003cp\u003eThe natural compound CUR, which is derived from the root of the everlasting herb curcuma longa, is a polyphenolic substance\u003csup\u003e[4]\u003c/sup\u003e. often referred to as curcumin, it has been extensively utilized in traditional Chinese medicine for the management of cardiac complications associated with diabetes. neuropathy, Kidney disease, Retina, dysfunction of \u0026beta; cells in the islets and vascular disorders. It significantly contributes to cardiovascular treatment by modulating antioxidant activity, effects that protect against cell death and inflammation\u003csup\u003e[5,6\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e13]\u003c/sup\u003e. Last yearthe beneficial impacts of CUR on heart health are well acknowledged, its role in mitigating cardiotoxicity caused by doxorubicin has been demonstrated for several years\u003csup\u003e[14]\u003c/sup\u003e. Research involving animals has indicated that CUR can also aid in lessening cardiac hypertrophy induced by lipopolysaccharides and in relieving myocardial fibrosis resulting from spontaneous hypertension, it also plays a role in averting heart failure\u003csup\u003e[15]\u003c/sup\u003e. Furthermore, CUR has been shown to facilitate cardiac regeneration and enhance heart function in a rat model of myocardial infarction\u003csup\u003e[16]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In this study, The results indicated a notable dose-dependent enhancement in the viability of cardiomyocytes with CUR treatment. To determine if the protective effect of CUR against HG-induced apoptosis in cardiomyocytes was linked to the reduction of oxidative stress, the levels of intracellular ROS were assessed using the DCFH-DA probe, revealing that CUR enhanced the function of antioxidant enzymes such as SOD. HO-1，\u0026nbsp;NADPH and GSH Px, excluding MDA. At a CUR concentration of 20 \u0026micro;M, the viability of the cells reached its peak. suggesting that CUR has the ability to safeguard cardiomyocytes against injury from high glucose levels. In the case of the HG + CUR group, administering a more elevated dose (40 \u0026mu;M) resulted in increased effects. Toxic cells suggesting that excessive doses of CUR could lead to detrimental effects (Fig. 1).\u003c/p\u003e\n\u003cp\u003eCUR participates in myocardial protection through a variety of pathways, such as SIRT1,\u0026nbsp;NF-\u0026kappa;B, JNK,\u0026nbsp;and so on\u003csup\u003e[17]\u003c/sup\u003e.\u0026nbsp;The Notch1 signaling pathway involves a wide range of physiological processes,\u0026nbsp;including mitosis,\u0026nbsp;cell survival,\u0026nbsp;metastasis and transcription in the state of stress\u003csup\u003e[18]\u003c/sup\u003e.\u0026nbsp;In addition,the Notch1/Hes1\u0026nbsp;signaling pathway has been shown to protect myocardial cells and reduce myocardial injury\u003csup\u003e[19]\u003c/sup\u003e.\u0026nbsp;To investigate the Notch1 pathway and whether it has a pivotal role in the effects of\u0026nbsp;CUR\u0026nbsp;on HG-induced cardiomyocyte apoptosis, the highly active \u0026gamma;-secretase inhibitor DAPT was used to block the Notch1 pathway. Our results show that when exposed to\u0026nbsp;HG\u0026nbsp;injury,\u0026nbsp;the expression of Notch1 protein is highest when 20\u0026nbsp;\u0026micro;M CUR is administered.\u0026nbsp;Meanwhile, we used DAPT (\u0026alpha;-secretase inhibitor, which inhibits Notch1 signaling pathways) to eliminate these effects.\u0026nbsp;In fact,\u0026nbsp;these effects were eliminated when DAPT was used,\u0026nbsp;indicating that the cardioprotective effects of CUR are related to the Notch1 signaling pathway.\u0026nbsp;Therefore,\u0026nbsp;we conferred that\u0026nbsp;Notch1 may be involved in the protective effect of CUR on HG-induced cardiomyocyte injury.\u003c/p\u003e\n\u003cp\u003eCUR exerts a protective effect on cardiomyocytes against injuries induced by high glucose by blocking the Notch1 signaling pathway, which results in enhanced cell viability, a lower rate of apoptosis, and diminished production of reactive oxygen species. In a similar way, the activation of the Notch1 signaling pathway resulted in enhanced cardiomyocyte survival, a reduction in apoptosis among the cardiomyocytes, and a decrease in damage caused by ROS. In SK-N-MC cells, the signaling pathway of Notch1 can be stimulated by ROS, which results in cell death via apoptosis. Nonetheless, the compound EUK134 demonstrated its ability to inhibit cell death in SK-N-MC cells triggered by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/menadione through the modulation of the Notch1 signaling pathway\u003csup\u003e[20]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eElevated glucose levels can trigger significant levels of reactive oxygen species (ROS), which contribute to oxidative stress damage\u003csup\u003e[21]\u003c/sup\u003e. The findings of this investigation illustrated that treatment with CUR resulted in a decrease in MDA levels and an enhancement in the activities of HO-1, SOD, GSH-Px, and NADPH. This suggests that CUR has the potential to mitigate the harm caused by HG in oxidative stress (Fig. 4). Research demonstrates that treatment with CUR can lower ROS levels that are provoked by HG. Research indicates that CUR may also inhibit the generation of ROS in N2A cells while alleviating oxidative stress effects in endothelial cells derived from human umbilical veins\u003csup\u003e[22]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis research does have certain constraints. It has been proposed that the generation of reactive oxygen species and the resulting oxidative stress could potentially play a role in cardioprotective mechanisms associated with CUR; However, the precise pathways involved remain to be elucidated. Initially, only western blot assays were employed in the examination of the signaling pathways to assess the expression levels of N1ICD and Hes1 proteins. And we\u0026nbsp;have no performed experiments to determine the mRNA level of N1ICD. Besides, there is a lack of in vivo experiments to justify the entire study.\u003c/p\u003e\n\u003cp\u003eIn summary, we demonstrated that activation of the Notch1 signaling pathway protects cardiomyocytes by enhancing cell viability, reducing ROS formation, and inhibiting the production of apoptosis. In addition, most of the cardioprotective effects were abolished by DAPT. Although the activity of SOD, HO-1, NADPH and GSH-Px antioxidant enzymes impaired DAPT damage to cardiomyocytes. The findings show that pretreating with CUR can enhance the levels of Notch1 and Hes1 proteins, which implies that CUR has a protective role against damage induced by high glucose (HG).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANOVA a one-way analysis of variance\u003c/p\u003e\n\u003cp\u003eCCK Cell Counting Kit \u003c/p\u003e\n\u003cp\u003eCUR curcumin\u003c/p\u003e\n\u003cp\u003eDCM diabetic cardiomyopathy\u003c/p\u003e\n\u003cp\u003eDMEM dulbecco\u0026apos;s modified eagle medium\u003c/p\u003e\n\u003cp\u003eGSH-Px glutathione peroxidase\u003c/p\u003e\n\u003cp\u003eHG high glucose\u003c/p\u003e\n\u003cp\u003eHO-1 heme oxygenase-1 \u003c/p\u003e\n\u003cp\u003eH hour \u003c/p\u003e\n\u003cp\u003eMDA malondialdehyde\u003c/p\u003e\n\u003cp\u003eNADPH nicotinamide adenine dinucleotide phosphate\u003c/p\u003e\n\u003cp\u003eNG normal glucose\u003c/p\u003e\n\u003cp\u003eN1ICD notch 1 intracellular domain\u003c/p\u003e\n\u003cp\u003eNotch1 Notch homolog 1, translocation-associated (Drosophila)\u003c/p\u003e\n\u003cp\u003eNRF2 nuclear factor-erythroid-2-related factor 2\u003c/p\u003e\n\u003cp\u003ePBS phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003ePI propidium iodide\u003c/p\u003e\n\u003cp\u003eROS reactive oxygen species\u003c/p\u003e\n\u003cp\u003eSD standard deviation\u003c/p\u003e\n\u003cp\u003eSOD superoxide dismutase\u003c/p\u003e\n\u003cp\u003eTNF-\u0026alpha; tumor necrosis factor -alpha\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll research costs were supplied by Nanjing Medical University Science and Technology Development Fund (No. NMUB20220079). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets are included as Supplementary Files \u003cem\u003eS1\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA statement for \u003cstrong\u003euse of experimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study is reported in accordance with ARRIVE guidelines. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eX.W. researched, analyzed the data and reviewed the article. X.W. and Z.Y. Q. wrote and edited the article. X.W. analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft, performed statistical analysis. P.Y. L. contributed initial discussion of the project and reviewed the article. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved (NMUC20240358) by Laboratory Animal Ethics Committee of Nanjing First Hospital.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDr PeiYan Liu ORCID iD: 0009‑0003‑7492‑2227.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDillmann WH. 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Curcumin Attenuates Colistin-Induced Neurotoxicity in N2a Cells via Anti-inflammatory Activity, Suppression of Oxidative Stress, and Apoptosis.\u003cem\u003e Mol Neurobiol\u003c/em\u003e 2018;55(1):421-434.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Curcumin, Oxidative damage, High glucose, Diabetic cardiomyopathy, Notch1 signaling","lastPublishedDoi":"10.21203/rs.3.rs-6331619/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6331619/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aims to investigate the protective effects of curcumin (CUR) in high glucose (HG)-induced oxidative stress and apoptosis of primary cardiomyocytes by activating the Notch1 signaling pathway. CUR is a natural polyphenol isolated from turmeric rhizomes and plays an important role in the antioxidant, anti-apoptotic and anti-inflammatory effects of diabetes.Therefore, we established a in neonate rat myocardial cells induced by HG, followed by treatment with CUR and DAPT, respectively.We detected and assessed myocardial cells viability and antioxidant enzyme activity by CCK-8 reagent and antioxidant enzyme kit. Apoptosis was detected by flow cytometry. The production of reactive oxygen species was detected by fluorescence labeling, and the expression of related genes and proteins was detected by q-PCR and Western blot. HG-induced primary rat cardiomyocytes not only increased apoptosis and ROS production, but also decreased the activity of antioxidant enzymes and the expression of Notch1 and Hes1 proteins. After pre-treatment by CUR, surprisingly, we found that CUR can effectively increase the activity of rat cardiomyocytes induced by HG. The results showed that CUR could inhibit the apoptosis of rat cardiomyocytes, inhibit the production of intracellular ROS, and increase the activity of antioxidant enzymes. Further, we also found that CUR can upregulate the expression of Notch1 and Hes1 proteins and related genes, suggesting that the protective effect of CUR on HG-induced damage involves the Notch1/Hes1 signaling. These results suggest that CUR protects cardiomyocytes from HG-induced oxidative stress by activating Notch1 and its downstream target genes.\u003c/p\u003e","manuscriptTitle":"Curcumin mitigates high glucose-induced cardiac oxidative stress via Notch1 pathway activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 01:36:27","doi":"10.21203/rs.3.rs-6331619/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-13T06:06:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-12T21:01:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240530964380550431865111633307579369717","date":"2025-05-12T20:49:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-09T00:54:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"78916333551250298774160477029037904942","date":"2025-05-07T01:59:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-06T03:15:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-06T03:13:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-29T05:33:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T12:34:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-29T03:22:26+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90c4e9cb-8689-4491-bb97-d41a13d88aa0","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48169760,"name":"Biological sciences/Cell biology"},{"id":48169761,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-07-07T16:06:54+00:00","versionOfRecord":{"articleIdentity":"rs-6331619","link":"https://doi.org/10.1038/s41598-025-09105-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-02 15:58:23","publishedOnDateReadable":"July 2nd, 2025"},"versionCreatedAt":"2025-05-13 01:36:27","video":"","vorDoi":"10.1038/s41598-025-09105-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-09105-9","workflowStages":[]},"version":"v1","identity":"rs-6331619","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6331619","identity":"rs-6331619","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}