Geraniin attenuates isoproterenol-induced cardiac hypertrophy by inhibiting inflammation, oxidative stress and cellular apoptosis

Jiaqi Ding; Shenjie Zhang; Qi Li; Boyu Xia; Jingjing Wu; Xu Lu; Chao Huang; Xiaomei Yuan; Qingsheng You

doi:10.4196/kjpp.24.200

Journal List > Korean J Physiol Pharmacol > v.29(3) > 1516090426

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Ding, Zhang, Li, Xia, Wu, Lu, Huang, Yuan, and You: Geraniin attenuates isoproterenol-induced cardiac hypertrophy by inhibiting inflammation, oxidative stress and cellular apoptosis

Original Article

Korean J. Physiol. Pharmacol. 2025;29(3):307-319.

Published online: 22 November 2024

DOI: https://doi.org/10.4196/kjpp.24.200

Geraniin attenuates isoproterenol-induced cardiac hypertrophy by inhibiting inflammation, oxidative stress and cellular apoptosis

Jiaqi Ding^1,^#, Shenjie Zhang^1,^#, Qi Li^1,^#, Boyu Xia^1,^#, Jingjing Wu², Xu Lu³, Chao Huang³, Xiaomei Yuan^4,^*, Qingsheng You^1,^*

¹Department of Cardiothoracic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China

²Department of Cardiology, Suzhou Kowloon Hospital of Shanghai Jiaotong University School of Medicine, Suzhou 215027, Jiangsu, China

³Department of Pharmacology, School of Pharmacy, Nantong University, Nantong 226001, Jiangsu, China

⁴Department of Cardiology, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu 610072, Sichuan, China

^*Correspondence Qingsheng You, E-mail: yqsyy@ntu.edu.cn, Xiaomei Yuan, E-mail: yuanxiaomei@med.uestc

Contributed by:

^# These authors contributed equally to this work.

Contributed by:

Author contributions: J.D. and Q.Y. designed the experiments. J.D., Q.L., B.X., S.Z., J.W., X.L., C.H., and X.Y. performed the experiments. J.D. analyzed the data. J.D. and Q.Y. wrote the manuscript. All authors read and approved the final manuscript.

Received 18 June 2024 Revised 8 October 2024 Accepted 13 October 2024

(open-access):

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Geraniin, a polyphenol derived from the fruit peel of Nephelium lappaceum L., has been shown to possess anti-inflammatory and antioxidant properties in the cardiovascular system. The present study explored whether geraniin could protect against an isoproterenol (ISO)-induced cardiac hypertrophy model. Mice in the ISO group received an intraperitoneal injection of ISO (5 mg/kg) once daily for 9 days, and the administration group were injected with ISO after 5 days of treatment with geraniin or spironolactone. Potential therapeutic effects and related mechanisms analysed by anatomical coefficients, histopathology, blood biochemical indices, reverse transcription-PCR and immunoblotting. Geraniin decreased the cardiac pathologic remodeling and myocardial fibrosis induced by ISO, as evidenced by the modifications to anatomical coefficients, as well as the reduction in collagen I/III á1mRNA and protein expression and cross-sectional area in hypertrophic cardiac tissue. In addition, geraniin treatment reduced ISO-induced increase in the mRNA and protein expression levels of interleukin (IL)-6, IL-1β and tumor necrosis factor-α, whereas ISO-induced IL-10 showed the opposite behaviour in hypertrophic cardiac tissue. Further analysis showed that geraniin partially reversed the ISO-induced increase in malondialdehyde and nitric oxide, and the ISO-induced decrease in glutathione, superoxide dismutase and glutathione. Furthermore, it suppressed the ISO-induced cellular apoptosis of hypertrophic cardiac tissue, as evidenced by the decrease in B-cell lymphoma-2 (Bcl-2)-associated X/caspase-3/caspase-9 expression, increase in Bcl-2 expression, and decrease in TdT-mediated dUTP nick-end labeling-positive cells. These findings suggest that geraniin can attenuate ISO-induced cardiac hypertrophy by inhibiting inflammation, oxidative stress and cellular apoptosis.

Keywords: Cardiomegaly, Geraniin, Inflammation, Oxidative stress

INTRODUCTION

Cardiac hypertrophy is a prevalent cardiovascular disease characterized by enlarged cardiomyocytes, which has the potential to cause heart failure. It is classified into two types: Physiological and pathological. Physiological hypertrophy often occurs in a normally developing heart or in well-trained athletes. This type presents with normal cardiac structure or organization, and normal or increased systolic function [1]. On the other hand, pathological hypertrophy is common in patients with hypertension, myocardial infarction, cardiomyopathy or structural heart disease, and is usually associated with systolic dysfunction, interstitial fibrosis and re-expression of fetal cardiac genes [2]. Although certain treatments have been developed, the socioeconomic burden of hypertrophic complications remains a challenge globally [3]. Therefore, it is imperative to develop new drugs that can adequately treat cardiac hypertrophy.

Physiological hypertrophy is usually accompanied by increased cell growth and protein synthesis, whereas pathological hypertrophy is often associated with dysregulation of responses such as cellular apoptosis, fibrosis, mitochondrial dysfunction, metabolic reprogramming, inadequate blood vessel production and changes in sarcomere structure [4]. Therefore, abnormalities in the signaling pathways that trigger these responses are potential mechanisms contributing to the pathogenesis of cardiac hypertrophy. Based on this hypothesis, the signaling pathway mediated by β-adrenergic receptors has been postulated to be an important mechanism leading to cardiac hypertrophy [5]. Over-activation of β-adrenergic receptors and subsequent production of pro-inflammatory cytokines and oxido-nitrosative stress, as well as abnormal increase in the expression of hypertrophic genes induced by isoproterenol (ISO), a potent β-adrenergic receptor agonist, has been linked to cardiac hypertrophy [5]. For these reasons, β-adrenoceptor blockers, angiotensin converting enzyme inhibitors and angiotensin receptor blockers are often used to treat cardiac hypertrophy. However, the therapeutic efficacy of these drugs is suboptimal [6].

Geraniin is a polyphenol isolated from medicinal plants, which possesses antioxidant, anti-inflammatory and anti-thrombotic effects. Several studies have demonstrated its pharmacological effects. For example, geraniin treatment was found to protect bone marrow-derived mesenchymal stem cells from hydrogen peroxide-induced oxidative stress damage via the phosphatidylinositol-3-hydroxykinase-protein kinase B signaling pathway [7]. Elsewhere, it was found to increase the survival of mice infected with human enterovirus 71 [8], and to attenuate lipopolysaccharide-induced neuroinflammation and cognitive impairment [9]. Although the antitumor effect of geraniin has been described in previous studies [10,11], the cardiovascular effects and molecular mechanisms of geraniin are not fully understood. Therefore, the current study used an ISO-induced cardiac hypertrophy mouse model to investigate the pharmacological effects and molecular mechanisms of geraniin on cardiac hypertrophy with the aim of providing a theoretical and experimental basis for the adoption of geraniin as a new therapeutic agent for cardiac hypertrophy.

METHODS

Materials

Geraniin (#HY-N0472), ISO (#HY-N1952) and spironolactone (SPI) (#HY-B0561) were purchased from MedChem Express. Antibodies against B-cell lymphoma-2 (Bcl-2)-associated X (Bax) (#50599-2-lg), Bcl-2 (#12789-1-AP), caspase-3 (#19677-1-AP), caspase-9 (#10380-1-AP), interleukin (IL)-1β (#16806-1-AP), IL-6 (#21865-1-AP), tumor necrosis factor-α (TNF-α) (#17590-1-AP) and IL-10 (#60269-1-lg) were purchased from Proteintech. Anti-atrial natriuretic polypeptide (ANP) antibody (#ab225844) is the product of Abcam. Antibodies against brain natriuretic peptide (BNP) (#sc-18817), collagen I (#sc-8784) and collagen III (#sc-8781) were purchased from Santa Cruz Biotechonology. Geraniin, ISO and SPI were dissolved in 10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300 (PEG300), 5% Tween-80 and 45% saline. A solution containing only DMSO with PEG300, Tween-80 and saline was used as a vehicle.

Animals

Adult male C57BL/6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were housed under standard conditions (22˚C, 55% relative humidity, 12-h light/darkness) for 5 days and fed ad libitum. All animal experiments were approved by the Animal Ethics Committee of Nantong University (Permit Number: 2110836) and conformed to the internationally accepted guidelines for the use of animals in toxicology adopted by the Society of Toxicology in 1999. When the purpose of the experiment has been achieved or the physical condition of the experimental mice has changed drastically, we chose to treat them humanely to terminate the experiment in order to alleviate the pain of the mice. The mice were observed to be dead or not by observing the rise and fall of their thorax. All animals were humanely euthanized at Nantong University Animal Centre by inhalation of an overdose of CO₂ (60% vol/min).

Experimental setup and drug treatment

After 1 week of adaptation, 80 mice were randomly divided into control (n = 20), ISO (n = 20), geraniin (20 mg/kg, n = 20) [12] and positive control (200 mg/kg SPI administered intragastrically, n = 20) groups [13]. Mice in the ISO group received an intraperitoneal injection of ISO (5 mg/kg) once daily for 9 days to establish a model of cardiac hypertrophy for a controlled in vivo study [14]. Mice in the administration group were injected with ISO after 5 days of treatment with geraniin or SPI. Fig. 1 represents the detailed experimental design. Obtain tissues for storage in a –80°C refrigerator. All experimental assay data were completed within 1 month of tissue collection.

Western blot

To collect heart tissue samples, mice were anesthetized with an intraperitoneal injection of 1% barbiturates (800 mg/kg) and then fixed on an operating table. Next, the ribs were cut open to expose the heart, and the right auricle was cut open to allow bleeding. A saline solution was then injected until the outflow was free of blood, and the heart was subsequently excised and homogenized with an ultrasonic cryomill (Shanghai Jingxin; FENGYUE Industrial Company). Proteins were extracted from the homogenized heart tissue, and the protein concentration was determined by the BCA method (Beyotime Institute of Biotechnology). Protein samples were boiled at 95˚C for 10 min, separated by 10% SDS-PAGE at 80 V for 1.5 h, and transferred onto nitrocellulose membranes at 260 mA for 1.5 h. The membranes were blocked with milk for 2 h and treated overnight at 4˚C with antibodies against Bax (1:500), Bcl-2 (1:500), caspase-3 (1:500) and caspase-9 (1:500). The following day, the membranes were washed three times with TBS-Tween 20 and incubated with IRDye680-labelled antibodies (1:3,000) (P0023, Beyotime Institute of Biotechnology) for 2 h at room temperature. The protein blots were visualized using the Odyssey CLX Western Blot Detection System (LI-COR Biosciences) and analyzed using Image J software (Version 1.8.0, National Institute of Health).

Histopathological examinations

Heart tissue was harvested and fixed with 10% formalin buffer, embedded in paraffin and cut into 5-μm-thick sections, before being stained with a hematoxylin and eosin (H&E) and wheat germ agglutinin (WGA) working solution. Photographs were obtained with a light microscope at ×400 magnification to assess the extent of myocardial fibrosis. Image J (Version 1.8.0, National Institute of Health) was used for automatic analysis of the cross-sectional area (CSA).

Measurement of reactive oxygen species (ROS)

Unfixed frozen sections of 10–20 μm in thickness were prepared for examination. A total of 200 μl washing solution (E004-1-1, Nanjing Jiancheng Bioengineering Institute) was added to the section and incubated for 3–5 min. Next, 100–200 μl staining probe (E004-1-1, Nanjing Jiancheng Bioengineering Institute) working solution was added drop-wise. The sections were then incubated at 37˚C for 20–60 min under light. Finally, the sections were sealed with coverslips or glycerol, and fluorescence emission was detected using a confocal laser.

TdT-mediated dUTP nick-end labeling (TUNEL) assay

Cellular apoptosis kit (G002-1-2) was purchased from Nanjing Jiancheng Bioengineering Institute. Tissue sections were collected and fixed in a 4% paraformaldehyde solution for 60 min. The paraformaldehyde solution was discarded, and the sections were washed twice with phosphate-buffered saline (PBS), mixing gently for 10 min each time. Next, the sections were incubated for 5 min at room temperature with 0.5% Triton X-100 to allow permeabilization and washed twice with PBS for 10 min each time. Subsequently, 25 μl thawed TdT enzyme and 225 μl fluorescent labeling solution were added to a 1.5-ml Eppendorf tube, and mixed thoroughly to produce the TUNEL assay detection solution. Next, 50 μl of this solution was evenly added to each tissue section, and incubated at 37˚C for 60 min in the dark. Subsequently, the sections were washed three times with PBS for 10 min each time. Finally, the tissue sections were blocked with 30% glycerol and observed under a fluorescence microscope (Leica Microsystems GmbH).

Measurement of malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) concentrations

The concentrations of MDA, GSH, NO, SOD and T-AOC were measured in heart tissue homogenates using corresponding commercial kits (cat. nos. A003-1-2, A005-1-2, A012-1-2, A001-4-1 and A015-1-2, respectively) obtained from Nanjing Jiancheng Bioengineering Institute according to the manufacturer’s instructions. Cardiac tissue homogenates were ground using an ultrasonic cryomill (Shanghai Jingxin; FENGYUE Industrial Company). The results were normalized to total protein concentration, as determined using the BCA method (Beyotime Institute of Biotechnology), and expressed as nmol MDA, nmol GSH, U SOD, U NO and U T-AOC mg protein.

Reverse transcription (RT)-PCR

Total RNA was extracted from mice using RNeasy Mini Kit from Qiagen, Inc. RNA was reverse transcribed into cDNA using an RT system (Promega Corporation). The reaction mixture consisted of 1X Faststart SYBR Green Master Mix (Roche Molecular Diagnostics), 2 μl diluted cDNA, 2 mM MgCl₂ and 0.5 μM primers. The primers used were as follows: ANP, 5’-CACAGATCTGATGGATTTCAAGA-3’ (F) and 5’-CCTCATCTTCTACCGGCATC-3’ (R); BNP, 5’-GAAGGTGCTGTCCCAGATGA-3’ (F) and 5’-CCAGCAGCTGCATCTTGAAT-3’ (R) [15]; IL-6, 5’-TTCCATCCAGTTGCCTTCTT-3’ (F) and 5’-CAGAATTGCCATTGCACAAC-3’ (R); IL-10, 5’-GGCAGAGAACCATGGCCCAGAA-3’ (F) and 5’-AATCGATGACAGCGCCTCAGCC-3’ (R); IL-1â, 5’-TGGAAAAGCGGTTTGTCTTC-3’ (F) and 5’-TACCAGTTGGGGAACTCTGC-3’ (R); TNF-á, 5’-CTGTGAAGGGAATGGGTGTT-3’ (F) and 5’-GGTCACTGTCCCAGCATCTT-3’ (R) [16]; collagen I á1, 5’GCTCCTCTTAGGGGCCACT-3’ F and 5’-CCACGTCTCACCATTGGGG-3’ (R); collagen III á1, 5’-CTGTAACATGGAAACTGGGGAAA-3’ (F) and 5’-CCATAGCTGAACTGAAAACCACC-3’ (R) [15]; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5’-GCAAGTTCAACGGCACAG-3’ (F) and 5’-CGCCAGTAGACTCCACGAC-3’ (R) [16].

Statistical analysis

All data were analyzed using Graphpad Prism 8 (GraphPad; Dotmatics) and are expressed as the mean ± standard error of the mean (n = 8). Groups were compared with one-way ANOVA followed by Bonferroni post-hoc test. p < 0.05 was considered to indicate a statistically significant difference.

RESULTS

Geraniin protects against ISO-induced myocardial hypertrophy and improves cardiac anatomy-related parameters

Initially, alterations in cardiac volume across the control, ISO, geraniin administration and positive control groups were assessed (Fig. 1). The results revealed an increase in cardiac volume in the ISO group compared with the control group. However, both geraniin and SPI (Fig. 2A) administration effectively mitigated the ISO-induced rise in cardiac volume (Fig. 2B). Similar to the positive drug SPI, geraniin administration attenuated the ISO-induced increase in heart weight (HW)/body weight (BW) ratio (HW/BW, F_3,28 = 45.61, p < 0.001; Fig. 2C), as well as the ratio of left ventricular weight (LVW) to BW (LVW/BW, F_3,28 = 12.16, p < 0.001; Fig. 2D), heart weight-to-tibial length (TL) ratio (HW/TL, F_3,28 = 10.23, p < 0.001; Fig. 2E) and the LVW/TL ratio (F_3,28 = 7.30, p < 0.001; Fig. 2F). In addition, both geraniin and SPI down-regulated the ISO-induced increase in mRNA expression levels of two indicators of myocardial hypertrophy: ANP (F_3,28 = 131.30, p < 0.001; Fig. 2G) and BNP (F_3,28 = 836.10, p < 0.001; Fig. 2H). Changes in the expression of ANP and BNP in the myocardial tissue of mice treated with ISO and/or geraniin were also explored. The results showed that geraniin treatment, similar to SPI, inhibited the ISO-induced increase in the expression levels of ANP (F_3,28 = 27.20, p < 0.001; Fig. 2I, J) and BNP (F_3,28 = 30.87, p < 0.001; Fig. 2I, K). Altogether, these results suggest that geraniin may protect against ISO-induced myocardial hypertrophy.

Geraniin attenuates myocardial fibrosis in ISO-induced cardiac hypertrophy

To investigate the impact of geraniin on ISO-induced myocardial fibrosis, alterations in the expression levels of collagen I á1 and collagen III á1 were analyzed. Similar to SPI, geraniin alleviated the ISO-induced increase in the mRNA expression levels of collagen I á1 (F_3,28 = 99.55, p < 0.001; Fig. 3A) and collagen III á1 (F_3,28 = 73.46, p < 0.001; Fig. 3B) in hypertrophic cardiac tissue of mice. Analysis of WGA staining showed that the cardiomyocytes in the ISO group were irregularly arranged and morphologically diverse, but this change was reversed by geraniin and SPI treatment (Fig. 3C). Morphometric analysis revealed that geraniin and SPI treatment inhibited the ISO-induced increase in the CSA of cardiomyocytes (F_3,28 = 267.70, p < 0.001; Fig. 3D) in hypertrophic cardiac tissue of mice.

Next, changes in the expression of collagen I and collagen III in the myocardial tissue of mice treated with ISO and/or geraniin were examined. It was observed that the ISO-induced increase in the expression of collagen I (F_3,28 = 36.34, p < 0.001; Fig. 3E, F) and collagen III (F_3,28 = 35.33, p < 0.001; Fig. 3E, G), similar to SPI, was attenuated by geraniin treatment. Similarly, H&E staining indicated that both geraniin and SPI treatment suppressed myocardial fibrosis induced by ISO (Fig. 3H). Altogether, these results suggest that geraniin attenuates myocardial fibrosis in ISO-induced cardiac hypertrophy.

Geraniin attenuates inflammatory response in ISO-induced cardiac hypertrophy

Since inflammation has been linked to the development of cardiac hypertrophy, the mRNA expression of inflammation-associated factors such as IL-1β, IL-6, TNF-α and IL-10 in the hypertrophic heart tissue of mice treated with geraniin, SPI and/or ISO was examined. It was observed that geraniin treatment suppressed the ISO-induced increase in mRNA expression levels of IL-1β (F_3,28 = 38.59, p < 0.001; Fig. 4A), IL-6 (F_3,28 = 28.93, p < 0.001; Fig. 4B) and TNF-α (F_3,28 = 70.51, p < 0.001; Fig. 4C) in hypertrophic cardiac tissue, and the ISO-induced decrease in mRNA expression of IL-10 (F_3,28 = 33.57, p < 0.001; Fig. 4D). These results were comparable to those of SPI.

Furthermore, changes in the protein expression levels of IL-1β, IL-6, TNF-α, and IL-10 in myocardial tissue of mice treated with ISO and/or geraniin were explored. It was noted that the ISO-induced increase in IL-1β (F_3,28 = 20.00, p < 0.001; Fig. 4E, F), IL-6 (F_3,28 = 36.94, p < 0.001; Fig. 4E, G) and TNF-α (F_3,28 = 31.18, p < 0.001; Fig. 4E, H) expression levels, as well as the ISO-induced decrease in IL-10 expression levels (F_3,28 = 14.38, p < 0.001; Fig. 4E, I) were down-regulated by geraniin. These results support the hypothesis that geraniin reduces inflammatory response in myocardial hypertrophy.

Geraniin attenuates myocardial oxidative stress in ISO-induced cardiac hypertrophy

Changes in MDA, NO, T-AOC, SOD and GSH levels in the hypertrophic cardiac tissue of mice treated with geraniin, SPI and/or ISO were investigated. The results showed that geraniin suppressed the ISO-induced increase in MDA (F_3,28 = 19.50, p < 0.001; Fig. 5A) and NO (F_3,28 = 111.0, p < 0.001; Fig. 5B), as well as the ISO-induced decrease in T-AOC (F_3,28 = 150.40, p < 0.001; Fig. 5C), SOD (F_3,28 = 14.43, p < 0.001; Fig. 5D) and GSH (F_3,28 = 79.12, p < 0.001; Fig. 5E). These effects were similar to those of SPI. The present study also explored the changes in ROS in the myocardial tissue of mice treated with ISO and/or geraniin, and found that, similar to SPI, geraniin treatment suppressed elevated levels of ROS (F_3,28 = 24.89, p < 0.001; Fig. 5F, G). These results suggest that geraniin can re-balance the oxidative stress response in hypertrophic myocardial tissue.

Geraniin attenuates ISO-induced apoptosis in cardiomyocytes during cardiac hypertrophy

Finally, alterations in the expression of proteins associated with cellular apoptosis in the cardiac tissue of mice treated with geraniin, SPI and/or ISO were investigated. It was found that geraniin treatment, similar to SPI, inhibited the ISO-induced increase in the expression of Bax (F_3,16 = 259.60, p < 0.001; Fig. 6A, B), caspase-3 (F_3,16 = 65.33, p < 0.001; Fig. 6A, C) and caspase-9 (F_3,16 = 101.10, p < 0.001; Fig. 6A, D) in hypertrophic cardiac tissue, as well as the ISO-induced decrease in the expression of Bcl-2 (F_3,16 = 15.99, p < 0.001; Fig. 6A, E). In addition, geraniin treatment suppressed the ISO-induced increase in the number of TUNEL-positive cells in hypertrophic cardiac tissue (F_3,16 = 44.35, p < 0.001; Fig. 6F, G), which was similar to the effect of SPI. These results suggest that geraniin can inhibit cardiac apoptosis in hypertrophic myocardial tissue.

DISCUSSION

Cardiomyocyte hypertrophy, often associated with hypertension, cardiomyopathy, vascular disease and congenital heart disease, is a biological process by which the heart adapts to increased workload. However, persistent exposure to hypertrophy-inducing stimuli may cause systolic dysfunction and cardiac remodeling, increasing the risk of heart failure [17]. Although numerous drugs have been introduced to treat cardiac hypertrophy, the prognosis of the majority of patients remains poor. Therefore, further in-depth studies are needed to explore the pathogenesis and treatment strategies of myocardial hypertrophy.

Geraniin is a hydrolyzable polyphenol that has been shown to possess significant antioxidant effects. In a rat model of obesity induced by a high-fat diet, geraniin treatment was observed to enhance antioxidant capacity, offering protection to the organs of obese rats [18]. In an animal model of cerebral ischemia-reperfusion, geraniin inhibited oxidative stress and neuronal apoptosis via the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway [12]. In targeted therapeutic studies for gouty arthritis, geraniin inhibited the interaction between ACS and NLRP3 through its antioxidant effects, thereby suppressing inflammasome activation, sepsis, and IL-1β release associated with the disease [19]. In spontaneously hypertensive rats, intravenous administration of geraniin effectively controlled the progression of hypertension [20]. Geraniin has also been shown to exert anti-atherogenic effects via the intestinal trimethylamine N-oxide-dependent pathway [19]. Liu et al. [20] found that geraniin treatment suppressed the lipopolysaccharide-induced increase in the protein and mRNA levels of the M1 cytokines TNF-α and IL-6, thereby attenuating the pathogenesis of atherosclerosis. Considering the observed effects of geraniin on oxidative stress, cellular apoptosis and inflammation, it was hypothesized that geraniin may serve as a preventive agent against myocardial hypertrophy. As expected, the present study found that geraniin ameliorated ISO-induced myocardial hypertrophy in mice. These findings suggest that geraniin holds promise as a potential candidate for addressing myocardial hypertrophy.

ISO, a potent synthetic non-selective β-adrenoceptor agonist that activates β-adrenergic receptors, is commonly used to induce cardiac hypertrophy and fibrosis in rodents [21]. Using an ISO-induced model, the current study examined the cardioprotective effect of geraniin by evaluating changes in HW/BW, HW/TL, LVW/BW and LVW/TL ratios, and found that geraniin treatment suppressed the ISO-induced increase in HW/BW, HW/TL, LVW/BW and LVW/TL ratios. Cardiac fibroblasts, which serve as an important communication bridge between other cell types in the heart, account for ~15% of non-myocytes in adult mouse heart. Therefore, these cells may play an important role in the regulation of cardiomyocyte growth and death as well as cardiac fibrosis, by secreting collagen fibers [22]. Fibroblast-specific overexpression has been shown to induce cardiac fibrosis and contractile dysfunction, which is the main developmental process associated with pathological cardiac hypertrophy [23]. In the present study, significant fibrosis was detected in ISO-induced cardiac tissue, as evidenced by the marked increase in the expression of genes and proteins involved in hypertrophy and abnormal fibroblast proliferation. However, geraniin-treated mice showed opposite effects. WGA showed that disorganized cells, irregular morphology, enlarged cardiomyocytes and proliferation of fibrous bodies in the hypertrophic cardiac tissue of ISO-treated mice were improved following geraniin treatment, which was confirmed by H&E staining. Moreover, the ISO-induced increase in indicators of myocardial fibrosis such as collagen I á1 and collagen III á1 was suppressed by geraniin treatment. Based on these results, it can be postulated that geraniin may be a potential candidate for developing anti-fibrosis agents, although the mechanisms of its ant-fibrosis effects should be explored in further experiments.

It has been demonstrated that IL-6 is a strong predictor of prognosis in patients with heart failure [24]. Hirota et al. [25] investigated the role of IL-6 in myocardial hypertrophy by overexpressing IL-6 and IL-6 receptors in mice. The authors found that the mice developed ventricular hypertrophy and thickening of the ventricular wall at 5 months of age. In a study by Cha et al. [26], IL-6 was found to be up-regulated in the heart tissue of mice treated with ISO. Moreover, simvastatin suppressed the ISO-induced myocardial hypertrophy in rats by down-regulating IL-6 expression in hypertrophic heart tissue [27]. A previous study found that NF-κB expression was up-regulated in ISO-induced rat hearts, which was accompanied by an increase in IL-6 expression [28]. Overexpression of TNF-α could induce cardiac hypertrophy and decrease contractility [29], and ablation of TNF-α or inhibition of TNF-α production attenuated myocardial hypertrophy, inflammatory response, cellular apoptosis and fibrosis [30,31]. IL-10 is an anti-inflammatory cytokine known to inhibit macrophage infiltration into the heart, and has a direct effect on cardiomyocytes [32]. IL-10 knockout exacerbated ISO-induced cardiac hypertrophy, whereas supplementation with IL-10 attenuated or even reversed the onset of cardiac remodeling by inhibiting NF-κB. Treatment with IL-10 attenuated the damage caused by cardiac hypertrophy [33]. Clinical studies have shown that IL-10 levels are elevated in the serum of patients with cardiac hypertrophy, highlighting the role of IL-10 in clinical therapy [34]. A previous study reported that IL-1β induced a hypertrophic response in cardiomyocytes [35], while inhibition of IL-1β delayed the development of cardiac hypertrophy and myocardial fibrosis [36,37]. The present study found that geraniin treatment suppressed the ISO-induced increase in IL-6, TNF-α and IL-1β expression, as well as the ISO-induced decrease in IL-10 expression, which suggests that geraniin may inhibit myocardial hypertrophy by attenuating ISO-induced inflammation. The association between the inhibitory effect of geraniin on the ISO-induced increase in IL-6, TNF-α and IL-1β levels or the ISO-induced decrease in IL-10 levels in hypertrophic heart tissue should be further clarified.

The inflammatory response has been linked to oxido-nitrosative stress [38]. Production of ROS is a normal physiological process in the context of redox homeostasis, but uncontrolled production of ROS can potentially cause lipid peroxidation and DNA mutations, resulting in irreversible cell damage and oxidative stress [39]. Numerous studies have demonstrated that the accumulation of ROS stimulates cardiac hypertrophy and remodeling by increasing oxidative stress [40]. Therefore, antioxidant treatment may be a useful strategy for treating cardiac remodeling and dysfunction [41], although this hypothesis needs to be clarified given the inconsistencies in the current literature [42]. Therefore, further research into the mechanisms underlying the development of oxidative stress and adverse cardiac responses to oxidative stress is necessary to generate ideas for developing more effective anti-oxidative stress drugs. During the development of myocardial hypertrophy, the myocardium secretes vascular growth factors that accelerate angiogenesis to provide sufficient blood supply for the hypertrophic heart [43]. Endothelium-derived NO has been implicated in the development of cardiac hypertrophy [44]. In addition, high secretion of pro-inflammatory factors in the heart under hypertrophic conditions can result in abnormal production of NO, which promotes ROS production and oxido-nitrosative stress [45]. Therefore, inhibiting NO may be an effective strategy to treat myocardial hypertrophy. This hypothesis was supported in the present study. For instance, it was found that geraniin treatment suppressed the ISO-induced increase in indicators of oxidative and nitrosative stress, such as MDA and NO, in mouse cardiac tissue, as well as the ISO-induced decrease in indicators of anti-oxidation, such as GSH, SOD and T-AOC, in mouse cardiac tissue. Future studies should investigate the exact mechanisms by which geraniin regulates the pathological process of oxidative and oxido-nitrosative stress in hypertrophic heart tissue.

In addition to oxidative and nitrosative stress, cardiomyocyte apoptosis is a major factor in the transition from compensatory to decompensatory heart failure. The progression of apoptosis in cardiomyocytes is well established, and is driven by factors such as sustained hypertension, chronic pressure overload from angiotensin II, calcium overload, and the influence of cytokines and neurohormones [46,47]. Therefore, inhibition of cellular apoptosis may be a promising approach for the treatment of myocardial hypertrophy [48]. Numerous studies have reported that oxido-nitrosative stress can trigger various apoptotic processes [49]. Elevated NO produced by cardiomyocytes during oxidative stress can generate large quantities of ROS, which in turn activates cystatin proteases and cellular apoptosis by degrading key target proteins in the nucleus, cytoplasm and mitochondria [50]. In the present study, the results of TUNEL staining assay showed that ISO-treated mouse hearts exhibited increased cellular apoptosis, which was suppressed by geraniin treatment. Bcl-2, an anti-apoptotic protein, can reduce the production of ROS, thereby attenuating cellular apoptosis. Bax, a pro-apoptotic protein, promotes cardiac apoptosis by inhibiting Bcl-2 expression [51]. The present results showed that the ISO-induced increase in levels of pro-apoptotic factors such as Bax, caspase-3 and caspase-9, and the decrease in Bcl-2 in hypertrophic cardiac tissue were reversed by geraniin treatment. This suggests that geraniin confers protection against cardiac hypertrophy through a mechanism involving inhibition of apoptosis.

SPI effectively alleviates fibrosis associated with cardiac hypertrophy. However, as a potent diuretic, it induces hyperkalemia, rendering it unsuitable for patients with renal insufficiency. Moreover, many patients with cardiac hypertrophy experience intolerable gastrointestinal and endocrine side effects. Therefore, the development of a safe and well-tolerated therapeutic agent for patients with both cardiac hypertrophy and renal insufficiency is critically important [13]. The current results showed that geraniin attenuated the inflammatory response, oxidative stress and cellular apoptosis caused by ISO. This lays a foundation for further research into the specific mechanisms of the antioxidant and anti-fibrotic properties of geraniin. Sudden death is the most dangerous outcome in patients with pathological myocardial hypertrophy. Therefore, the current study on geraniin provides important ideas regarding the treatment of myocardial hypertrophy.

In our study, both geraniin and SPI demonstrated significant therapeutic effects in the treatment of myocardial hypertrophy. Geraniin was more effective than SPI in slowing the progression of myocardial fibrosis and reducing oxidative stress, while SPI was more effective than geraniin in decreasing apoptosis. These findings offer valuable insights for developing targeted therapies for cardiac hypertrophy in different patient populations.

A limitation of the present study is that experiments were only performed using an in vivo model of ISO-induced myocardial hypertrophy. Therefore, it is not clear whether geraniin can protect cardiomyocytes at the cellular level. Another limitation is that a gradient dose of geraniin was not administered. Although our previous gradient dosing studies in mice with heart disease showed that 20 mg/kg provided the best protection and did not cause any side effects, different doses and dosing schedules need to be evaluated in future studies to determine the optimal effective dose. In addition, the lack of Masson staining and immunohistochemical analysis showing the distribution of collagen I/III are also limitations of the current study.

In conclusion, the present study has shown that geraniin exerts important cardioprotective effects on mice with ISO-induced myocardial hypertrophy. Mechanistically, the effects of geraniin are mediated by inhibition of inflammation, oxido-nitrosative stress and cellular apoptosis (Fig. 7). Further research is needed to explore the molecular mechanisms by which geraniin alleviates the symptoms of myocardial hypertrophy.

ACKNOWLEDGEMENTS

None.

Notes

FUNDING

The present study was supported by the Natural Science Foundation of China (grant nos. 81974216 and 81974015), the Science and Technology Project of Nantong City (grant no. MS12020016), (grant no. Z2021005), and the High Technology Research Project of the Suzhou Science & Technology Division (grant no. SKY2021003).

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

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Fig. 1

A flowchart of the experimental design.

ISO, isoproterenol.

Fig. 2

Effect of geraniin on the ISO-induced cardiac hypertrophy indices.

(A) The chemical structure of geraniin. (B) Representative images showing that geraniin treatment, similar to SPI (a positive control), suppressed ISO-induced increase in cardiac volume in mice. (C–F) Quantitative analysis of the effects of geraniin and SPI treatment on the ratio of heart weight (HW) and left ventricular weight (LVW) to body weight (BW) (C, D) and the ratio of HW and LVW to tibial length (TL) (E, F) (n = 8, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO) in ISO-treated mice. (G, H) Quantitative analysis of the effects of geraniin and SPI on mRNA expression of ANP (G) and BNP (H) in mice treated with ISO (n = 8, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). (I–K) Representative images and quantitative analysis indicating the effects of geraniin and SPI on the protein expression of ANP (I, J) and BNP (I, K) in ISO-induced mouse hypertrophic cardiac tissue (n = 5, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). Data are shown as mean ± SEM. ISO, isoproterenol; SPI, spironolactone; ANP, atrial natriuretic polypeptide; BNP, brain natriuretic peptide; Con, control.

Fig. 3

Effect of geraniin on the indices of ISO-induced myocardial fibrosis.

(A, B) Quantitative analysis of the effects of geraniin and SPI on the mRNA expression of collagen I á1 and collagen III á1 in hypertrophic cardiac tissue of mice (n = 8, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). (C, D) Representative images (C) and quantitative analysis (D) of the impact of geraniin and SPI on cardiomyocyte fibrosis area (CSA) as detected by the wheat germ agglutinin (WGA) staining of hypertrophic cardiac tissue (n = 8, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). Scale bar: 20 μm. (E–G) Representative images and quantitative analysis of the effects of geraniin and SPI on collagen I (E, F) and collagen III (E, G) expression (n = 5, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). (H) Representative HE staining images of the left ventricle in mice treated with ISO and geraniin or SPI. Scale bar: 20 μm. Data are shown as mean ± SEM. ISO, isoproterenol; SPI, spironolactone; HE, hematoxylin and eosin; Con, control.

Fig. 4

Effect of geraniin on ISO-induced cardiac inflammation.

(A–D) Quantitative analysis of the effects of geraniin and SPI son the mRNA expression levels of IL-1β (A), IL-6 (B), and TNF-α (C) and mRNA expression levels of IL-10 (D) in hypertrophic heart tissues of mice (n = 8, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). (E–I) Representative images and quantitative analysis of the impact of geraniin and SPI on the protein expression of IL-1β (E, F), IL-6 (E, G), TNF-α (E, H), and IL-10 (E, I) (n = 5, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). Data are shown as mean ± SEM. ISO, isoproterenol; SPI, spironolactone; IL, interleukin; TNF-α, tumor necrosis factor-α; Con, control.

Fig. 5

Effect of geraniin on ISO-induced oxidative stress in mouse hypertrophic cardiac tissue.

(A, B) Quantitative analysis of the effects of geraniin and SPI on MDA (A) and NO (B) (n = 8, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). (C–E) Quantitative analysis of the impact of geraniin and SPI on T-AOC (C), SOD (D), and GSH (E) in hypertrophic heart tissue of mice (n = 8, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). (F, G) Representative images and quantitative analysis of the results of ROS assays indicating the effects of geraniin on ISO-induced increase in ROS production in hypertrophic heart tissue of mice (n = 5, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). Scale bar: 20 μm. Data are shown as mean ± SEM. ISO, isoproterenol; SPI, spironolactone; MDA, malondialdehyde; NO, nitric oxide; T-AOC, total antioxidant capacity; SOD, superoxide dismutase; GSH, glutathione; ROS, reactive oxygen species; Con, control.

Fig. 6

Effect of geraniin on ISO-induced cellular apoptosis in mouse hypertrophic cardiac tissue.

(A–E) Representative images and quantitative analysis for the effect of geraniin and SPI on Bax (A, B), caspase-3 (A, C), and caspase-9 (A, D) and on Bcl-2 (A, E) in the hypertrophic heart tissue of mice (n = 5, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). (F, G) Representative images and quantitative analysis of the results of TUNEL staining of hypertrophic heart tissue in the indicated groups (n = 5, *p < 0.05 vs. control, ^#p < 0.05 vs. ISO). Scale bar: 20 μm. Data are shown as mean ± SEM. ISO, isoproterenol; SPI, spironolactone; TUNEL, TdT-mediated dUTP nick-end labeling; Con, control.

Fig. 7

A schematic showing the protective effects of geraniin against ISO-induced cardiac hypertrophy by suppressing inflammation, oxidative stress, and cellular apoptosis.

ISO, isoproterenol.

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