Oleuropein ameliorates isoproterenol-induced cardiac hypertrophy through SIRT1 activation via endoplasmic reticulum stress inhibition

Oleuropein ameliorates isoproterenol-induced cardiac hypertrophy through SIRT1 activation via endoplasmic reticulum stress inhibition | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 15 May 2025 V1 Latest version Share on Oleuropein ameliorates isoproterenol-induced cardiac hypertrophy through SIRT1 activation via endoplasmic reticulum stress inhibition Authors : Hao Wang , Yang Ran , Jialin Zhong , Shuting Xiang , Jun Xiao 0000-0003-1195-1031 , and Qiang She 0000-0001-5049-1264 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174729624.48352339/v1 211 views 126 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Cardiac hypertrophy is a major risk factor for cardiovascular disease worldwide, and Mediterranean diet has shown great benefits in reducing cardiovascular disease-related mortality and morbidity. Numerous studies have ascertained that the protective effect of olive oil on the heart is mainly attributed to its active component, oleuropein (OLE); however, the mechanism remains unknown. We hypothesized that OLE provides cardioprotective effects against cardiac hypertrophy through the alleviation of endoplasmic reticulum stress. In the present study, different dosage regimens (10, 30, and 60 mg/kg) of OLE were intragastrically administered to an isoproterenol (ISO) (7.5 mg/kg)-induced cardiac hypertrophy mouse model. OLE alleviates ISO-induced cardiac hypertrophy and apoptosis. In addition, the increased expression of endoplasmic reticulum (ER) stress-related genes, such as Glucose-Regulated Protein 78 (GRP78), activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein homologous protein (CHOP), and protein kinase RNA-like ER kinase (PERK), was blunted by OLE, with reduced Sirtuin-1 (SIRT1) levels. Furthermore, after pretreatment with EX527 (a SIRT1 inhibitor), the anti-hypertrophic, anti-apoptosis, and ER stress effects of OLE were diminished, implying a key role of SIRT1 in ER stress inhibition. Our study indicates that OLE ameliorates ISO-induced cardiac hypertrophy through SIRT1 activation via ER stress inhibition. Oleuropein ameliorates isoproterenol-induced cardiac hypertrophy through SIRT1 activation via endoplasmic reticulum stress inhibition Hao Wang, MD 1,2 , Yang Ran,MD 2 , Jialin Zhong,MD 2 , Shuting Xiang,MD 2 , Jun Xiao,PhD 2,* , and Qiang She,PhD 1,* 1 Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China 2 Department of Cardiovascular Medicine, Chongqing Emergency Medical Center，Chongqing University Central Hospital, Chongqing 400014, China *Co-corresponding authors: Jun Xiao: Department of Cardiovascular Medicine, Chongqing Emergency Medical Center，Chongqing University Central Hospital, Chongqing 400014, China. Email: [email protected] Qiang She: Department of Cardiology, The Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China. Email: [email protected] ABSTRACT Cardiac hypertrophy is a major risk factor for cardiovascular disease worldwide, and Mediterranean diet has shown great benefits in reducing cardiovascular disease-related mortality and morbidity. Numerous studies have ascertained that the protective effect of olive oil on the heart is mainly attributed to its active component, oleuropein (OLE); however, the mechanism remains unknown. We hypothesized that OLE provides cardioprotective effects against cardiac hypertrophy through the alleviation of endoplasmic reticulum stress. In the present study, different dosage regimens (10, 30, and 60 mg/kg) of OLE were intragastrically administered to an isoproterenol (ISO) (7.5 mg/kg)-induced cardiac hypertrophy mouse model. OLE alleviates ISO-induced cardiac hypertrophy and apoptosis. In addition, the increased expression of endoplasmic reticulum (ER) stress-related genes, such as Glucose-Regulated Protein 78 (GRP78), activating transcription factor 4 (ATF4), CCAAT/enhancer-binding protein homologous protein (CHOP), and protein kinase RNA-like ER kinase (PERK), was blunted by OLE, with reduced Sirtuin-1 (SIRT1) levels. Furthermore, after pretreatment with EX527 (a SIRT1 inhibitor), the anti-hypertrophic, anti-apoptosis, and ER stress effects of OLE were diminished, implying a key role of SIRT1 in ER stress inhibition. Our study indicates that OLE ameliorates ISO-induced cardiac hypertrophy through SIRT1 activation via ER stress inhibition. Keyword: Oleuropein, Cardiac hypertrophy, SIRT1, ER stress, Isoproterenol SIGNIFICANCE STATEMENT Oleuropein (OLE), a natural compound mainly in olive leaves and olive oil, has lots of benefits in human health and increases longevity. But the protection mechanism remains unknown. Here in our study, we identified the protection of OLE on cardiac hypertrophy both in vivo and in vitro. Furthermore, the protection was in close relation to SIRT1 expression. In addition, ER stress suppression induced by SIRT1 might be the key mechanism, therefore our study provided a novel therapy of OLE on cardiac hypertrophy. 1. INTRODUCTION Cardiac hypertrophy responds to physiological and pathological stresses, mainly presenting as individual cardiomyocyte enlargement and increased heart mass [1]. Cardiac hypertrophy sustains and preserves heart function according to Laplace’s law. Cardiomyocytes exhibited different types of cardiac phenotype and prognosis through various mechanisms as long as the condition persists. Physiological hypertrophy is fully reversible and maintains cardiac function over time without interstitial or replacement fibrosis, or cell death [2]. Pathological hypertrophy leads to adverse cardiovascular events such as heart failure, cardiac arrhythmia, and death [3]. Previous studies have interpreted the mechanisms involving myocardial inflammation, oxidative stress, intracellular Ca 2+ overload, apoptosis, subcellular alterations, and reduced capillary density that mediates the transition from physiological to pathological cardiac hypertrophy [4]. However, the exact mechanisms differentiating physiological and pathological hypertrophy remain unclear. The Mediterranean diet, rich in olive oil and nuts, has protective effects against diseases such as diabetes, asthma, and cancer. A large amount of medical evidence has shown that a Mediterranean diet can significantly reduce the mortality of cardiovascular diseases [5]. Olive oil is a major component of the Mediterranean diet and its role in the prevention of cardiovascular diseases has been confirmed. Its health promoting effects were attributed to its abundance in monounsaturated fatty acids, such as secoiridoids [6]. Among the secoiridoids, oleuropein (OLE), a natural compound found mainly in olive leaves and oils, exhibits cardioprotective, antioxidant, anti-inflammatory properties and increases longevity [7]. Previous studies implied that Hydroxytyrosol (HT) is the principal minor phenolic compound of olive oil [8]. Recently, plenty of reports in the literature demonstrate the multi-organ protection of oleuropein, which belongs to a group of coumarin derivative, secoiridoids [9]. It is an important component of the Mediterranean diet and is known for its potential health benefits, particularly against cardiovascular diseases [7,10]. Studies have verified its ability to lower blood pressure and cardioprotection [6, 11]. Additionally, OLE has been studied for its potential for anti-cancer, anti-inflammatory and antioxidant activities [12, 13]. OLE has also been studied for its potential to improve cognitive function and protect against neurodegenerative diseases by reducing inflammation and oxidation. A previous study showed that olive leaf extract attenuates cardiac hypertrophy and fibrosis in diabetic rats [14]. Moreover, Sirtuin1 (SIRT1) might be involved in the multi-protective mechanism of OLE [15]. Recent studies have implied that the benefits of oleuropein are related to its ability to inhibit endoplasmic reticulum (ER) [16]. ER stress refers to a state of malfunction and disruption of normal ER function, which can lead to cellular dysfunction and disease. Increasing evidence suggests a significant relationship between ER stress and cardiac hypertrophy. The heart, as a highly active organ with high energy demands, is susceptible to ER stress due to various cardiac stressors such as chronic hypertension, myocardial infarction, and biomechanical overload [17]. These stressors disrupt ER homeostasis and overwhelm the cellular capacity for proper protein folding and processing. ER stress activates a signalling pathway called the unfolded protein response (UPR), which initially aims to restore ER function and promote cell survival. However, persistent and unresolved ER stress can lead to UPR dysfunction and contribute to the development and progression of heart failure (HF). Maladaptive ER stress responses have been implicated in cardiomyocyte apoptosis, inflammation, hypertrophy, fibrosis, and impaired contractile function [18]. Therefore, we hypothesised that OLE would ameliorate cardiac hypertrophy by inhibiting ER stress. This study verified the inhibitory effects of OLE on isoproterenol(ISO)-induced cardiac hypertrophy and ER stress in mice, both in vivo and in vitro . Furthermore, ER stress related gene expression and apoptosis were examined to explore the underlying mechanisms. 2. Materials and Methods 2.1 Animals and treatment Eight-week-old male C57BL/6J mice were purchased from the Animal Centre of the Chongqing Medical University. The mice were housed in a specific pathogen free(SPF)-class environment where the room temperature was controlled at approximately 25 °C, and the humidity was around 50%. No restrictions were placed on the chow diet and water intake. Mice were exposed to equal light and dark periods throughout the day. In the ISO group, the mice received two weeks of isoproterenol (ISO, 7.5 mg/kg/d, MedChemExpress, Princeton, NJ, USA) administration via intraperitoneal injection to establish an ISO-induced cardiac hypertrophy animal model. In the OLE treatment group, the mice were pretreated with different doses of OLE (10, 30, or 60 mg/kg) (MedChemExpress, New Jersey, USA) by intragastric administration daily for 1 week, then the mice were treated with both OLE and ISO [19-21]. In the ISO+OLE+EX527 group, the mice were treated with OLE and EX527 (1 mg/kg i.p., MedChemExpress, New Jersey, USA) 1 week then co-administrated with ISO. The animal experiments complied with the requirements of the Animal Ethics Committee of Chongqing Medical University (approval no. IACUC-SAHCQMU-2023-0010). 2.2 Assessment of cardiac function Cardiac function was evaluated in mice using the VisualSonics Vevo 3100LT small-animal ultrasound system at the end of isoproterenol administration. Mice were anaesthetised with 2% isoflurane and fixed to the operating table. Representative ultrasound images were acquired in the M-mode. Ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter during diastole and left ventricular internal diameter during systole (LVIDd and LVIDs) were calculated using the Vevo Lab analysis software. 2.3 Immunocytochemical staining for WGA and cTnT The mice were sacrificed after ultrasound detection for wheat germ agglutinin (WGA) staining of heart tissues. The hearts were removed and fixed in a perfusion fixative (Biotechnology, Shanghai, China). Subsequently, the heart tissues were dehydrated, embedded in paraffin, and sectioned. Sections were de-paraffinized using Dewax Solution (Solaibao Biological Technology,Beijing, China). Sections were subjected to haematoxylin and eosin (H&E), WGA, and cardiac troponin T (cTnT) staining. The working concentration of WGA was 1:300, which was diluted using an antibody diluent solution (Biotechnology). The tissues were incubated with the WGA solution at room temperature for 60 min. After washing thrice with phosphate-buffered saline (PBS) (10 min each), the tissues were incubated with 4′,6-diamidino-2-phenylin-dole (DAPI) solution (Biotechnology) for 5 min. The cTnT staining of cardiomyocytes were performed by fixing the cells in 4% paraformaldehyde at room temperature for 10 min, washing with PBS solution, and incubation with cTnT antibody at 4 °C overnight. The following day, after washing with PBS solution, cardiomyocytes were incubated with a fluorescent green secondary antibody (Invitrogen, USA) at 37 ˚C for 30 min. Later, the cells were repeatedly washed with PBS and incubated with DAPI solution for 5 min. Images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan) and analysed using the Image J software v1.45. 2.4 The isolation of primary cardiomyocytes Primary cardiomyocytes were isolated using a previously reported density gradient centrifugation method [22]. Briefly, the ventricular tissues from the rats, seven days after birth, were cut into pieces, digested with 0.05% type II collagenase (Thermo Fisher Scientific, Uppsala, Sweden) in a 37 °C thermostatic water bath for 10 min, and further digested with 0.1% type II collagenase (Thermo Fisher) for 8 min. Repeated above steps until sufficient cell suspensions were obtained. Then, the collected cell suspensions were filtered through a 30 μm strainer, transferred to a new tube with an equal volume of ADS buffer, and centrifuged at 1,800 g for 5 min. The cells collected after centrifugation were resuspended in culture medium. Subsequently, the cell suspension was covered with the configured Percoll solutions of different densities (the low-density solution was located at the lowest layer), and then centrifuged at 1,800 g, for 40 min (acceleration was 1 without brake). The upper layer consists of fibroblasts and the lower layer consists of cardiomyocytes. Cardiomyocytes were seeded for further study after two more centrifugation steps in a preheated complete medium. Cell viability of primary cardiomyocytes was detected by cell counting kit-8(Beyotime, Shanghai, China). To induce the cardiomyocytes hypertrophy, the isolated primary cardiomyocytes were treated with ISO (10 µM, 24 h). In the OLE group, cardiomyocytes were treated with ISO and OLE. As in the EX527 group, cardiomyocytes were co-administrated with ISO, OLE and EX527 (30 μM, MedChemExpress, New Jersey, USA). 2.5 Real-time quantitative polymerase chain reaction (qPCR) RNA was extracted from the cells or tissues using a standard protocol. The quality and quantity of RNA were assessed using a spectrophotometer. RNA was reverse transcribed into cDNA using a commercial kit (Takara Bio, Shiga, Japan). Gene specific primers were designed using the NCBI Primer-BLAST tool, and the sequences of the genes were presented in the Supplemental Table. The reaction was run in a qPCR machine that measured the fluorescence in real time during amplification. The Ct value was used to calculate gene expression levels relative to the control gene or reference sample. 2.6 PCR Western blot Proteins were extracted from tissues or cells using RIPA protein extraction reagent (Beyotime). Protein concentration was measured using a bicinchoninic acid kit (Beyotime). Next, the protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto nitrocellulose or poly (vinylidene fluoride) (PVDF) membrane using a transfer apparatus. The membranes were blocked with blocking solution (Beyotime) at room temperature for 30 min and then incubated with a diluted primary antibody at 4 °C overnight. The next day, the membranes were washed to remove any unbound primary antibodies and incubated with secondary antibodies (SIRT1, Bax, Bcl-2, GRP78, CHOP, ATF4, P-PERK, PERK, and GAPDH) (Cell Signalling Technology, Danvers, MA, USA) at room temperature for 40 min. Finally, the protein expression in the different groups was detected using a chemiluminescence imaging system. 2.7 Data analysis The experimental data were collected in triplicates. All the numeric data were displayed as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA, followed by Tukey’s multiple comparison test. Comparisons between the two groups were performed using the Student’s t-test. Statistical significance was set at P < 0.05. significant. 3. Results 3.1 Oleuropein (OLE) improved isoproterenol (ISO)-induced cardiac dysfunction. An animal model of cardiac dysfunction was established by continuous intraperitoneal injection of isoproterenol (ISO) (7.5 mg/kg) for 14 days. To explore the role of oleuropein (OLE) in cardiac function, the mice were pretreated with intragastric administration of different doses of OLE (10, 30, and 60 mg/kg) one week before ISO injection. The cardiac function 9of mice in all groups was evaluated using echocardiography. As shown in Figure 1 A-E, ISO injection reduced the Ejection fraction (EF) and fractional shortening (FS) in mice and increased the left ventricular internal diameter during diastole and left ventricular internal diameter during systole (LVIDd and LVIDs) in a dose-dependent manner. OLE improved cardiac function at the optimum dose of 30 mg/kg, with increased EF and FS, and decreased LVIDs and LVIDd. 3.2 OLE improved cardiac function in ISO-treated mice by suppression of cardiac hypertrophy Cardiac hypertrophy is a common cause of cardiac dysfunction. We explored the effects of OLE on cardiac hypertrophy in ISO-treated mice. The administration of ISO in mice significantly induced cardiac hypertrophy as demonstrated by increased heart weight to tibia length (HW/TL) ratio (Figure 2A), increased expression of hypertrophic markers (atrial natriuretic peptide-ANP, brain natriuretic peptide-BNP, and β myosin heavy chain-MHC) (Figure 2B-D), and an enlarged cross-sectional area of the heart (Figure 2E-F). However, administration of OLE significantly reversed ISO-induced cardiac hypertrophy, accompanied by a reduced HW/TL ratio, expression of hypertrophic marker genes, and cardiomyocyte surface area (Figure 2A-F). Additionally, OLE administration did not affect the heart size of mice in the control group. 3.3 OLE attenuated ISO-induced cardiomyocyte hypertrophy by the inhibition of ER stress through SIRT1 Primary cardiomyocytes were treated with ISO (10 μM) for 48 h to establish an cardiac hypertrophy model in vitro . The effect of different concentrations of OLE on cell viability of primary cardiomyocytes was detected by Cell Counting Kit-8 assay (Supplemental Figure A). After treatment with ISO, the expression of hypertrophic genes in primary cardiomyocytes increased (Figure 3), and the cell surface of cardiomyocytes was stained with cardiac troponin T (cTnT) (Figure 3A-C). Sirtuin-1 (SIRT1) is involved in the protective effect of OLE [15]. SIRT1 is also involved in the development of cardiac hypertrophy, and its expression of SIRT1 was decreased in hypertrophied cardiomyocytes treated with ISO (Figure 3B-F). Subsequently, we explored the role of SIRT1 on the antihypertrophic effect of OLE in ISO-treated cardiomyocytes. OLE treatment increased the expression of SIRT1 (Figure 3A) and decreased the expression of hypertrophic marker genes (Figure 3D-F). Critically, inhibition of SIRT1 by EX527 (30 μM) significantly attenuated OLE-mediated anti-hypertrophic effects (P<0.05 vs. ISO+OLE group, Figure 3A-F). Endoplasmic reticulum (ER) is involved in proteostasis governed by the unfolded protein response (UPR), a signalling pathway that regulates the protein-folding capacity of the cell to sustain its secretory function [23]. Previous studies have suggested that SIRT1 regulates the UPR [24]. Moreover, UPR also activates apoptosis-related signalling pathways, promotes cardiomyocyte apoptosis, and participates in cardiac remodelling. Treatment with OLE reduced the increased mRNA expression levels of the proapoptotic gene (B-cell lymphoma 2-associated X, Bax ), the ER stress-related genes (activating transcription factor 4- ATF4 , Glucose-Regulated Protein 78- GPR78 , and CCAAT/enhancer-binding protein homologous protein - CHOP ) and increased the the anti-apoptotic gene (B-cell lymphoma 2, Bcl-2 ) expression (Figure 4A-E) in ISO-treated cardiomyocytes. Moreover, in the presence of SIRT1 inhibitor EX527, the effect of OLE against ER stress was abolished (Figure 4A-E). These results suggest that the antihypertrophic effect of OLE on cardiomyocytes is primarily dependent on the inhibition of ER stress through SIRT1. 3.4 The inhibition of SIRT1 abolished the anti-hypertrophic effect of OLE on ISO-induced cardiac hypertrophy in mice The antihypertrophic effect of OLE on cardiomyocytes was mediated by SIRT1 activation. We further studied the effect of SIRT1 on OLE treatment in ISO-treated mice. As depicted in Figure 5A-E, the intraperitoneal injection of EX527 almost abolished the rescue effect of OLE on cardiac function in ISO-treated mice. In addition, the HW/TL ratio (Figure 5F), expression of hypertrophic genes (ANP, BNP, and β-MHC) (Figure 5G-I), and cross-sectional area (Figure 5J-K), significantly increased in the OLE and ISO co-treated mice after the administration of EX527. Reduced SIRT1 expression in the heart tissues of the ISO group was recovered by OLE treatment, but this effect was almost diminished by EX527 treatment (Figure 6A, B). Additionally, we explored the role of SIRT1 in the anti-apoptotic effect of OLE and found that the administration of EX527 blocked the inhibition of Bax and activation of Bcl-2 by OLE (Figure 6A, C-D). Finally, the suppressive effect of OLE on ER stress in ISO-treated mice was also reduced by the administration of EX527, as demonstrated by the upregulation of ER stress-related proteins (Figure 6A, E-H) together with gene expression (Supplemental Figure B-G). These data demonstrated that the antihypertrophic effect of OLE in ISO-induced mice was achieved by the activation of SIRT1 to inhibit ER stress. 4. Discussion Pathological cardiac hypertrophy is a key process in the development of myocardial remodelling and HF, gaining increased attention in past years [18]. As heart failure was considered a syndrome with either reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF), treatment has developed into a paramount multidisciplinary approach [25]. Traditionally, nutrition management for HF has focused on sodium and fluid restrictions; however, in recent years, nutrition and dietary interventions have garnered research interest [26, 27]. It is increasingly evident that overarching dietary patterns, such as the Mediterranean dietary pattern, prevent the initial onset of HF and improve the outcomes in patients with HF [28]. OLE, a phenolic constituent of olives, is an essential component of the Mediterranean diet exerting multiple biological effects against oxidation, lipidaemia, etc [29]. Previous studies demonstrated that OLE alleviated cardiac hypertrophy induced by myocardial infarction, doxorubicin or diabetes [14, 30, 31]. In the present study, we identified the protective effect of OLE against ISO-induced cardiac hypertrophy in vivo and determined the optimal concentration. Consistent with cardiac hypertrophy in function, murine heart treated with ISO showed increased HW/TL ratio, mRNA levels of ANP, BNP and β-MHC. As indicated by morphology, OLE significantly reduced ISO-induced cardiomyocyte enlargement. These results were consistent with previous studies [29]. Further research implied that SIRT1 plays a pivotal role in the multi-protective effects of OLE. SIRT1, an important deacetylase expressed in numerous tissues and organs, which was found to be related the protections of OLE [32, 33]. SIRT1 mRNA levels and the SIRT1 inhibitor, EX527, was used to explore the underlying mechanisms. The results showed that the reduced SIRT1 mRNA level induced by ISO was significantly ameliorated by OLE in vitro , which was subsequently diminished by EX527, together with cell surface, ANP, BNP and β-MHC levels. Moreover, ISO-induced apoptosis was mitigated by OLE in cardiomyocytes, especially with a Bax and Bcl2 shift in the anti-apoptotic direction. Alleviation of cardiac hypertrophy by OLE was attenuated by EX527 in vivo . Furthermore, Elmazoglu et al. demonstrated the neuroprotective effects of oleuropein through the modulation of UPR and mitochondrial function [34]. Several studies have demonstrated that SIRT1 effectively inhibited ER stress [35-37]. Prolonged ER stress can activate apoptotic pathways and increase production of reactive oxygen species, causing mitochondrial dysfunction and triggering programmed cell death [38]. This loss of viable cardiomyocytes ultimately impairs cardiac function and contributes to the progression of cardiac hypertrophy and HF [39]. ER stress can also stimulate inflammatory responses in the heart, promoting the infiltration of immune cells and release of pro-inflammatory cytokines. Inflammatory processes further exacerbate ER stress, creating a vicious cycle that perpetuates cardiac dysfunction and remodelling [40]. While the precise mechanisms linking ER stress to heart failure are complex and multifaceted, targeting ER stress has emerged as a potential therapeutic strategy [38, 41]. Taken together, these results revealed that SIRT1 demonstrated the amelioration of septic associated lung-injury and apoptosis via inhibition of ER stress through the PERK/eIF2-α/ATF4/CHOP pathway [35, 42, 43]. Therefore, the ER stress-related genes GRP78, ATF4, CHOP, and PERK were examined. PERK signalling significantly increased in cardiac hypertrophy, subsequently attenuated by OLE.However, our study has some limitations which must be considered. It was reported that OLE can reduce bodyweight gain and abdominal adipose tissue level by repressing mitochondrial activity [44]. Further researches indicated its promising roles in metabolic syndrome through anti-oxidation, anti-inflammation, and improvements in autophagy [44]. So the role of mitochondrial activity needs further comprehensive investigation in cardiomyocyte hypertrophy. In contrast, the SIRT1 inhibitor EX527 almost abolished the effect of OLE on PERK signalling. 5. Conclusion Taken together, our results identified that OLE ameliorated ISO-induced cardiac hypertrophy by inhibiting ER stress and apoptosis. This protective effect is closely related to SIRT1 activation during ER stress inhibition. The present study suggestes that OLE inhibits ER stress response by reducing cardiac hypertrophy and cardiomyocyte apoptosis via the activation of SIRT1. Acknowledgment The authors have no acknowledgments to declare. Sources of Support This work was supported by the grant from Natural Science Foundation of Chongqing (No. CSTB2023NSCQ-MSX1041), the Joint project between the Science and Technology Bureau and the Health Commission of Chongqing (No. 2023MSXM019), 2023 key Disciplines On Public Health Construction in Chongqing, the Yong and Middle-aged Senior Medical Talents studio of Chongqing. Author Declarations The authors have no conflict of interest. REFERENCES 1. Nakamura M, Sadoshima J. Mechanisms of physiological and pathological cardiac hypertrophy. Nat Rev Cardiol. 2018;15(7):387-407. 2. Shimizu I, Minamino T. Physiological and pathological cardiac hypertrophy. J Mol Cell Cardiol. 2016;97:245-262. 3. Ritterhoff J, Tian R. 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Quercetin attenuates sepsis-induced acute lung injury via suppressing oxidative stress-mediated ER stress through activation of SIRT1/AMPK pathways. Cell Signal. 2022;96:110363. 37. Zhang J, Wang L, Gong D, Yang Y, Liu X, Chen Z. Inhibition of the SIRT1 signaling pathway exacerbates endoplasmic reticulum stress induced by renal ischemia/reperfusion injury in type 1 diabetic rats. Mol Med Rep. 2020;21(2):695-704. 38. Okada K, Minamino T, Tsukamoto Y, et al. Prolonged endoplasmic reticulum stress in hypertrophic and failing heart after aortic constriction: possible contribution of endoplasmic reticulum stress to cardiac myocyte apoptosis. Circulation. 2004;110(6):705-712. 39. Matsuura TR, Leone TC, Kelly DP. Fueling Cardiac Hypertrophy. Circ Res. 2020;126(2):197-199. 40. Zhang Y, Chen W, Wang Y. STING is an essential regulator of heart inflammation and fibrosis in mice with pathological cardiac hypertrophy via endoplasmic reticulum (ER) stress. Biomed Pharmacother. 2020;125:110022. 41. Liu MQ, Chen Z, Chen LX. Endoplasmic reticulum stress: a novel mechanism and therapeutic target for cardiovascular diseases. Acta Pharmacol Sin. 2016;37(4):425-443. 42. Wang F, Ma J, Wang J, et al. SIRT1 ameliorated septic associated-lung injury and macrophages apoptosis via inhibiting endoplasmic reticulum stress. Cell Signal. 2022;97:110398. 43. Yun YR, Lee JE. Kimchi attenuates endoplasmic reticulum stress-induced hepatic steatosis in HepG2 cells and C57BL/6N mice. Nutr Res. 2024;124:43-54. 44. Ahamad J, Toufeeq I, Khan MA, et al. Oleuropein: A natural antioxidant molecule in the treatment of metabolic syndrome. Phytother Res. 2019;33(12):3112-3128. Figure legends FIGURE 1 Oleuropein (OLE) ameliorates ISO-induced cardiac dysfunction in mice. A-D. The systolic function of heart was measured by EF, FS, LVIDs and LVIDd (n=5). E. Representative echocardiography images for each group. *P<0.05 versus Control group, #P<0.05 versus ISO group. OLE, oleuropein; ISO, isoproterenol; EF, left ventricular ejection fraction; FS, fraction shortening; LVIDs, left ventricular internal diameter during systole; LVIDd, left ventricular internal diameter during diastole. FIGURE 2 OLE alleviated ISO-induced cardiac hypertrophy in mouse. A. The HW/TL ratio of mice in each group (n=5). B-D. The mRNA expression levels of cardiac hypertrophy indicators (including ANP, BNP, and β-MHC) in heart tissues of each group (n=4). E. Quantification cardiomyocyte cross-sectional area in heart sections of each group (n=4). F. The WGA staining and H&E staining of heart sections in each group (n=4). All the scale bars in WGA staining represent 20 μm, and the scale bars in H&E staining represent 100 μm. *P<0.05 versus Control group, #P<0.05 versus ISO group. OLE, oleuropein; ISO, isoproterenol; HW/TL, heart weight to tibia length ratio; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β myosin heavy chain; WGA, wheat germ agglutinin; H&E, haematoxylin and eosin. FIGURE 3 OLE alleviated ISO-induced cardiomyocyte hypertrophy through the up-regulating SIRT1. A. The mRNA expression level of SIRT1 in cardiomyocytes of each group (n=4). B. The cTnT staining of cardiomyocytes of each group. C. The quantification of cardiomyocyte cell surface under cTnT staining (n=4). D-F The mRNA expression levels of hypertrophy markers (including ANP, BNP, and β-MHC) (n=4). *P<0.05 versus Control group, #P<0.05 versus ISO group, &P<0.05 versus ISO+OLE group. OLE, oleuropein; ISO, isoproterenol; SIRT1, Sirtuin-1; cTnT, cardiac troponin T; DAPI, 4′,6-diamidino-2-phenylin-dole; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β myosin heavy chain. FIGURE 4 OLE inhibited ISO-induced apoptosis and ER stress through the activation of SIRT1 in cardiomyocytes. A. The mRNA expression level of proapoptotic gene Bax (n=4). B. The mRNA expression level of anti-apoptotic gene Bcl-2 (n=4). C-E. The mRNA expression level of ER stress-related genes ( ATF4 , GRP78 , and CHOP ) (n=4). *P<0.05 versus Control group, #P<0.05 versus ISO group, &P<0.05 versus ISO+OLE group. OLE, oleuropein; ISO, isoproterenol; ER, endoplasmic reticulum; SIRT1, Sirtuin-1; Bcl2, B-cell lymphoma 2; Bax, B-cell lymphoma 2-associated X; ATF4, activating transcription factor 4; GRP 78, Glucose-Regulated Protein 78; CHOP, CCAAT/enhancer-binding protein homologous protein. FIGURE 5 The inhibition of SIRT1 blunted the anti-hypertrophy effect of OLE in ISO-induced mice. A-D. The evaluation of cardiac function by EF, FS, LVIDs and LVIDd (n=5). E. Representative echocardiography images for each group. F. The HW/TL ratio in each group (n=5). G-I. The expression of ANP, BNP, and β-MHC detected by RT-PCR (n=4). J. Quantification of cardiomyocyte cross sectional area evaluation in myocardial section (n=4). K. Upper panel, representative images for WGA statining; down panel, H&E staining. All the scale bars in WGA staining represent 20 μm, and the scale bars in H&E staining represent 100 μm. *P<0.05 versus Control group, #P<0.05 versus ISO group, &P<0.05 versus ISO+OLE group. SIRT1, Sirtuin-1; OLE, oleuropein; ISO, isoproterenol; EF, ejection fraction; FS, fractional shortening; LVIDd; left ventricular dimensions in diastole LVIDs, left ventricular dimensions in systole; HW/TL, heart weight to tibia length ratio; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β myosin heavy chain; RT-PCR, reverse transcription-polymerase chain reaction; WGA, wheat germ agglutinin; H&E, haematoxylin and eosin. FIGURE 6 The cardio-protection of OLE in ISO-induced mice was depended on the inhibition of ER stress through the activation of SIRT1. A. Protein expression of SIRT1, Bax, Bcl-2, GRP 78, CHOP, ATF4, P-PERK, and PERK detected by Western blot in hearts of each group (n=4). B-D Quantification of SIRT1, Bax and Bcl-2 expression in hearts of each group (n=4). E-H. Quantification of ER stress-related proteins (GRP78, CHOP, P-PERK, and ATF4) expression in each group (n=4). *P<0.05 versus Control group, #P<0.05 versus ISO group, &P<0.05 versus ISO+OLE group. OLE, oleuropein; ISO, isoproterenol; ER, endoplasmic reticulum; SIRT1, Sirtuin-1; Bax, B-cell lymphoma 2-associated X; Bcl2, B-cell lymphoma 2; GRP 78, Glucose-Regulated Protein 78; CHOP, CCAAT/enhancer-binding protein homologous protein; P-PERK, phosphate-protein kinase RNA-like ER kinase; ATF4, activating transcription factor 4. Supplemental Figure. A. The cell viability of primary cardiomyocytes after treatment with ISO or different concentrations of OLE. *P<0.05 versus Control group; #P<0.05 versus ISO group. B-G. The mRNA expression levels of SIRT1, Bax, Bcl-2, and ER stress-related genes (GRP78, CHOP, and ATF4) were detected by RT-PCR (n=4). *P<0.05 versus Control group, #P<0.05 versus ISO group, &P<0.05 versus ISO+OLE group. ISO, isoproterenol; OLE, oleuropein; SIRT1, Sirtuin-1; Bax, B-cell lymphoma 2-associated X; Bcl2, B-cell lymphoma 2; ER, endoplasmic reticulum; GRP 78, Glucose-Regulated Protein 78; CHOP, CCAAT/enhancer-binding protein homologous protein; ATF4, activating transcription factor 4; RT-PCR, reverse transcription-polymerase chain reaction. Supplementary Material File (fig2.pdf) Download 17.86 MB File (fig5.pdf) Download 14.06 MB Information & Authors Information Version history V1 Version 1 15 May 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cardiac hypertrophy er stress isoproterenol oleuropein sirt1 Authors Affiliations Hao Wang The Second Affiliated Hospital of Chongqing Medical University View all articles by this author Yang Ran Chongqing Emergency Medical Center View all articles by this author Jialin Zhong Chongqing Emergency Medical Center View all articles by this author Shuting Xiang Chongqing Emergency Medical Center View all articles by this author Jun Xiao 0000-0003-1195-1031 Chongqing Emergency Medical Center View all articles by this author Qiang She 0000-0001-5049-1264 [email protected] The Second Affiliated Hospital of Chongqing Medical University View all articles by this author Metrics & Citations Metrics Article Usage 211 views 126 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Hao Wang, Yang Ran, Jialin Zhong, et al. Oleuropein ameliorates isoproterenol-induced cardiac hypertrophy through SIRT1 activation via endoplasmic reticulum stress inhibition. 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