Protective effect of apelin-13 in lens epithelial cells via inhibiting oxidative stress-induced apoptosis

Abstract

Background

It is widely accepted that glaucoma-induced oxidative stress expedites cataracts’ process. Therefore, we examined the effects of apelin-13 against oxidative stress-induced damage in human lens epithelial cells (HLECs) and investigated the potential pathogenic mechanism of acute primary angle-closure glaucoma.

Methods

This experiment included five groups: control, H₂O₂, apelin-13 + H₂O₂, ML221 + H₂O₂, and apelin-13 + ML221 + H₂O₂. ML221 was employed in rescue experiments as an APJ antagonist. HLECs were pretreated with or without apelin-13 and subsequently exposed to H₂O₂. HLECs’ viability was assessed by CCK8. Cell apoptosis was determined using Annexin V-FITC/PI staining. The mitochondrial membrane potential was assessed by fluorescent probe JC-1. Intracellular G6PD activity, NADPH/NADP+, and GSH/GSSG ratios were detected to assess the cells’ oxidative damage.

Result

Apelin-13 reversed the H₂O₂-induced decrease in cell viability. The increased expression of G6PD and GLTU1, the G6PD, GSH/GSSG and NADPH/NADP + levels showed that apelin-13 can mitigate the H₂O₂-induced inhibition of the pentose phosphate pathway and dysregulation of cell redox status in the apelin-13 + H₂O₂ group compared with the H₂O₂ group. In H₂O₂-treated HLECs, apelin-13 can mitigate cell apoptosis, promote Bcl-2 expression, and suppress the Bax and Caspase-3 expression. In addition, H₂O₂ substantially reduced the mitochondrial membrane potential in HLECs, which was reversed by apelin-13. Notably, the inhibition of APJ intensified oxidative damage in H₂O₂-induced HLECs, demonstrating that the effects of apelin-13 were hindered by ML221.

Conclutions

Apelin-13 reduced oxidative damage and apoptosis in HLECs through APJ. These results demonstrate that apelin-13 can be employed as a potential drug for glaucoma with cataracts to delay the progression of cataracts.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12886-024-03746-6.

Introduction

Globally, cataract is the primary cause of blindness among individuals aged 50 years and older, and as the population ages, the global burden of irreversible vision loss due to glaucoma continues to increase [1, 2]. Cataracts and glaucoma often exhibit a mutually causal relationship [2]. Some lens modifications can contribute to the development of primary angle-closure glaucoma (PACG) [3, 4], while glaucoma can also contribute to cataract progression [5, 6], Glaucoma is the primary independent risk factor that exacerbates cataract formation although its pathogenesis remains unclear [7]. High intraocular pressure in glaucoma can result in oxidative stress [8]. Glaucoma patients are more susceptible to oxidative stress than cataract patients [9]. Oxidative damage of the lens caused by the cellular reactive oxygen species (ROS) is a primary mechanism leading to cataracts [10].

In glaucoma, oxidative stress is a potential mechanism for promoting cataracts, which arises from the unbalanced generation and clearance of cellular ROS. In lens epithelial cells (LECs), excessive ROS can induce deoxyribonucleic acid (DNA) damage and lipid peroxidation, resulting in cell apoptosis and cataract formation [10]. Hydrogen peroxide (H₂O₂), a nonfree radical member of the reactive oxygen family, accumulates in significant amounts in the lens and serves as a major intracellular ROS. The ROS generated by H₂O₂ causes protein degradation and oxidative damage to cells, similar to the damage in the lens in cataract disease [11].

Generally, cells possess numerous antioxidants, including reduced glutathione (GSH), which regulate the production and clearance of ROS. The GSH/oxidized glutathione disulfide (GSSG) redox system maintains a reduced cell environment, ensuring lens proteins remain in their reduced state. To facilitate the regeneration of glutathione, the glutathione reductase requires nicotinamide adenine dinucleotide phosphate (NADPH) obtained from the pentose phosphate pathway (PPP) and glucose 6-phosphate [12]. An imbalance in redox homeostasis in LECs leads to oxidative stress. The antioxidant defense system plays a crucial role in oxidative stress and can effectively reduce ROS production and facilitate the elimination of excessive ROS from cells. Consequently, deficiencies in NADPH production and glutathione regeneration are considered primary factors contributing to cellular oxidative damage and apoptosis [13].

Apelin, a prohormone comprising 77 amino acids derived from bovine stomach extract, is an endogenous ligand of the G protein-coupled receptor, angiotensin receptor-like 1 (APJ) [14]. The most effective APJ activator expressed in cell lines is apelin-13 [14, 15]. Apelin/APJ are extensively expressed in diverse human tissues and cells, demonstrating various essential biological functions, including antioxidation, maintaining homeostasis, and anti-apoptosis in vivo [16, 17]. Numerous studies have demonstrated the protective effects of apelin on oxidative stress [17]. It can effectively reduce the intracellular ROS production induced by H₂O₂ and inhibit cell apoptosis [18, 19]. Furthermore, apelin can regulate redox homeostasis and mitigate oxidative damage in cells by enhancing endogenous oxygen-free radical scavenging agents [20]. The administration of apelin is anticipated to mitigate oxidative stress-induced damage and prevent the onset of associated diseases. Notably, in zebrafish, homologous genes for human AGTRL1 (angiotensin-like-receptor 1) have been observed in the developing lens [21]. However, further investigations are required to confirm whether apelin can inhibit the apoptosis of LECs triggered by oxidative stress and to elucidate its possible mechanism.

To provide a novel theoretical basis for the prevention and treatment of glaucoma complicated with cataracts in clinical practice, this study investigated the protective effects of apelin-13 against H₂O₂-induced oxidative stress and the associated molecular mechanisms in lens capsule tissue of patients with glaucoma complicated with cataracts and in human lens epithelial cells (HLECs).

Materials and methods

Collection and arrangement of anterior lens capsule tissues

In this study, we selected a total of 20 patients with cataract. The criteria of anterior lens capsule tissues’ collection were to select the patients with acute primary angle-closure glaucoma combined with different kinds of cataracts (nuclear, cortical, or posterior subcapsular cataracts). Additionally, the anterior lens capsule tissues of control selected the age-matched patients with cataract. In this study, these patients who was selected were admitted to the Department of Ophthalmology, Army Medical Center, Chongqing, China. And before collecting the samples, we obtained the informed consent and the written informed consent of all patients in this study. The inclusion criteria of cataract (or combined with acute primary angle-closure glaucoma) included: no history of systemic diseases, such as diabetes, hypertension, hyperlipidemia, chronic inflammatory diseases, autoimmune disease and cancer, and hepatic diseases or kidney. All procedures involving human material in this study were reviewed and approved by the Ethics Committee of the Army Medical Center, Chongqing, China (No.82070962), and performed in accordance with the Declaration of Helsinki. The continuous annular capsulorhexis procedure was used to abtain the anterior lens capsule tissues during the cataract surgery. The anterior lens capsule tissues were quickly washed with Phosphate Buffered Saline (PBS) and then immediately stored at − 80℃ to further molecular biological experiments.

HLEC-B3 cells culture and drug treatment

The human lens epithelial B3 (HLE-B3) cell line (CRL-11421; ATCC, Manassas, Virginia, USA) was used in this experiment. HLEC-B3 cells were cultured in a carbon dioxide incubator using Dulbecco’s modified Eagle’s medium (DMEM, BI, Bioind, Israel), which was containing the 10% fetal bovine serum (BSA, Gibco, Carlsbad, CA, USA) at 37℃ and 5% CO₂. Additionally, 0.25% trypsin/Ethylene Diamine Tetraacetic Acid (BI, Bioind, Israel) was used to detach the cells. When the cell density reached more than 80%, cell passage treatment was performed. Meanwhile, we used the HLE-B3 cells that was used between 3 passage and 10 passage to detect the related cell experiments. And cells were collected by centrifuge at 175 g for 5 min during cell culture experiment. HLEC-B3 cells were preincubated with different concentrations of apelin-13 (MCE, New Jersey, USA) and/or ML221 (Anaspec Inc, CA, USA) for 24 h, then treated with H₂O₂ (KEHBIO, China). In a cell culture incubator, all treatments were conducted at 37℃. (Pyr1)-apelin-13 was dissolved with ddH2O to 1mM as storage liquid to dilute to the corresponding concentration in the experiment. And ML221 was dissolved with dimethylsulfoxide (Sigma, St. Louis, MO) and ddH₂O to 50 mM as storage liquid, and it’s first diluted to 10mM, then dilute to 10 μm to use in the cell experiment.

Cell viability assay of HLEC-B3

The cell counting kit-8(CCK8 kit) (Dojindo, Tokyo, Japan) was used to detect HLE-B3 cells’ viability in this experiment. The cells were arranged in 96-well cell plates with 5000 cells per well with 100 µL DMEM medium contained 10% FBS (BI, Bioind, Israel). After the cell density reached 50–60%, different concentrations of H₂O₂ was used to incubate for 24 h to model cellular oxidative damage, either alone or after pretreatment with varying concentrations of apelin-13 and/or ML221 for 24 h. And the concentration of H₂O₂ is set to 0, 50, 100, 200, and 400 µM, the concentration of apelin-13 is set to 10^− 8 M, 10^− 7 M, 10^− 6 M, the concentration of ML221 was set to 10 µM. After the cell treatment was completed, the cell viability detection procedures were performed according to the CCK8 kit instructions. Cell viability values are calculated using this formula: cell viability (%) = (As − Ab)/(Ac − Ab), where As represents the average value of optical density (OD) of per cells of the experimental treatment group with H₂O_2, and/or apelin-13, Ab represents the average OD value of per cells of blank group, and Ac represents the average OD value of per cells of control group.

Cell apoptosis assay

HLE-B3 cells were seeded in 6-well cell plates at the cell density of 1 × 10⁵ cells/well. H₂O₂ (200 µM) was used to treated with the cells for 24 h, after the pretreatment of apelin-13, and/or ML221 for 24 h. Then all cells were collected separately for testing the cell apoptosis. The experimental procedure was carried out according to the instructions of apoptosis kit (Bioscience, San Jose, CA, USA). The apoptotic percentage of the prepared samples was detected by flow cytometry.

Mitochondrial membrane permeability (MMP) assay

LEC suspension was seeded on cell climbing films and cultured in 24 pore culture plates with a CO₂ concentration of 5% at 37℃. After grouping, the LECs were washed with PBS. MMP assay kit (MCE, New Jersey, USA) was used in this experiment. To prepare the working solution of JC-1, JC-1 (200X) was treated with ultra-pure water in accordance with dilution ratio. The solution was thoroughly dissolved by vigorous vortexing to ensure proper mixing with JC-1. Subsequently, the staining buffer (5X) was added in working solution of JC-1 according to the dilution ratio to form the staining working solution. Then the prepared dyeing solution was added to the pretreated cell culture plate, and treated with 37° constant temperature in a water bath for 20 min. Then, HLE-B3 cells LECs were washed with PBS, and cells were placed in culture medium to be measured. Finally, laser confocal microscope was used to observe fluorescent samples of cells.

Assays for G6PD activity, NADPH/NADP + ratio, and GSH/GSSG ratio

The cells were inoculated in the six-well cell culture plate with a cell density of 100,000, and were treated when the cells grew to about 50%. The experiment comprised five groups: control, H₂O₂, apelin-13 + H₂O₂, ML221 + H₂O₂, and apelin-13 + ML221 + H₂O₂ group. ML221 was used to pretreat the cells at the concentration of 10µM for 1 h. Then apelin-13 was used to process cells at the concentration of 0.1 µM for 24 h. Afterward, HLE-B3 cells were washed with PBS and then cultured with H₂O₂ at the concentration of 200 µM for 24 h. Then, Cells were collected for the following experiments. The glucose-6-phosphate dehydrogenase (G6PDH) assay kit (Sangon biotech, China) was used to test the activity of G6PD in this experiment, NADPH (nicotinamide adenine dinucleotide phosphate)/NADP + was used the EnzyChrom NADPH/NADP + assay kit (Beyotime, China) to detect, and GSH/GSSG ratios were tested using GSH/GSSG kit (Beyotime, China). All experiments were performed according to the kit instructions. Specifically, the NADP+/NADPH and G6PDH extracts were defrosted at 37℃ in a water bath and placed on ice for later use. Approximately 1 × 10⁶ HLE-B3 cells were required for the experiment. Then NADP+/NADPH working solution and G6PDH extract working solution were added to the collected HLEB-3 cells. HLE-B3 cells were collected and used the protein removal reagent M solution to precipitate and mix. Then HLE-B3 cells were centrifuged to collect the supernatant of samples to determine total and reduced glutathione levels. The amount of reduced glutathione was computed as the difference between the total amount of glutathione and the amount of glutathione. A series of standard products were prepared, and colorimetric determination was performed. The activity ratios of GSH/GSSG, NADPH/NADP+, and G6PD of the cell samples were computed based on the standard curve.

Western blotting (WB)

After HLE-B3 cells were preincubate with drugs, the cell protein was extracted and detected by WB. Cells subjected to different treatments for the indicated duration were employed for WB. Briefly, cells were used RIPA buffer with protease inhibitors and phosphatase inhibitor to extract cell protein, and cell lysis buffer was collected by centrifuge at 15,000 g for 15 min. The protein was diluted by loading buffer and bathed in water at 100 °C for 10 min to test by WB. The protein samples were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, ACE Biotechnology, China) and then the protein was transferred to polyvinylidene fluoride (PVDF) membrane (Roche, Basel, Switzerland). Then PVDF membranes with protein were incubated with 5% milk in Tris-buffered saline Tween-20 (TBST, solarbio, China) on a shaker at room temperature for 1.5 h. Membranes with protein were incubated with the primary antibodies overnight at 4℃ on a shaker. last, membranes with protein were incubated with the secondary antibody of the corresponding species at room temperature for 1.5 h after cleaned with TBST. PVDF Membranes were treated with enhanced chemiluminescence reagent (bioground, China) and detected by WB exposure instrument.

Statistical analysis

The statistical data presented in this paper consisted of at least three independently replicated experiments, and all expressed as the mean ± standard deviation (SD). Analysis of variance was employed to compare the means of multiple groups of samples, and pairwise comparisons between the experimental groups in this papper were conducted using the LSD-t-test. P < 0.05 represented a statistically significant difference.

Results

Protein expression of the capsular lens in acute primary angle-closure glaucoma and cataract patients

The protein expression of APJ, Glucose transporter 1(GLUT1), G6PD, and B-cell lymphoma-2 (Bcl-2) in the glaucoma capsule was lower compared to the simple cataract capsule, and the P value of each group was 0.0286, 0.01, 0.0107, 0.0252 respectively, as illustrated in Fig. 1. Whereas the protein expression of BCL2-Associated X (Bax) and Caspase-3 was higher compared to the simple cataract capsule, and the P value of each group was 0.0379, 0.0399, as illustrated in Fig. 1.

Fig. 1.

The relative APJ, PPP, and apoptosis-related protein expression in the lens capsule of acute primary angle-closure glaucoma patients with cataract and cataract alone. (a) The relative APJ, PPP, and apoptosis-related protein expression were measured using western blotting analyses. The full-length blots/gels are presented in Supplementary Figs. 1–7. (b) The relative protein expression levels of APJ, PPP, and apoptosis-related proteins. Representative images of three replicated experiments were presented. The significance marked at the top of the columns refers to comparison with the control group. Data are shown as mean ± SD (n = 3)

Effects of H₂O₂ and apelin‑13 on HLE-B3 cell viability

Apelin‑13 did not demonstrate any cytotoxic effects on HLE-B3 cells, as illustrated in Fig. 2. However, H₂O₂ impaired cell viability in a dose-dependent manner (Fig. 2. a). Compared with the control group (cells not treated with H₂O₂), treatment with H₂O₂ (at a concentration of 200 µΜ) for 24 h was selected for subsequent experiments as the effect on cell viability was about 59.32 ± 2.139% (Fig. 2. b), and the cell viability of this group was significantly lower compared to the blank control group (P = 0.0009). Pretreatment of HLE-B3 cells with apelin‑13 demonstrated a dose-dependent protective effect against H₂O₂ damage (Fig. 2. c). The cell viability of pretreatment via apelin-13 group was significantly increased compared to the H₂O₂ group, and P value of each group was 0.0283, 0.0024, 0.00013 respectively, as illustrated in Fig. 2(c). The concentration of 0.1 µM was selected for downstream approaches, consistent with a referenced experiment [22].

Fig. 2.

The effects of apelin-13 on the cell viability of H₂O₂-treated HLE-B3 cells. (a) HLE-B3 cells were incubated with various concentrations of apelin-13 (0.01 µM, 0.1 µM, 1 µM) for 24 h. (b) HLE-B3 cells were incubated with various concentrations of H₂O₂ (50 µM, 100 µM, 200 µM, 400 µM) for 24 h. (c) HLE-B3 cells were preincubated with apelin-13 (0.01 µM, 0.1 µM, 1 µM) for 24 h before being treated with H₂O₂ (200 µM) for 24 h. Cell viability was evaluated using CCK8 assay. Representative images of three replicated experiments were presented. The significance marked at the top of the columns refers to comparison with the control group. Data are shown as mean ± SD (n = 3)

Effect of Apelin‑13 on H₂O₂‑induced HLE-B3 cell viability via APJ

HLE-B3 cells were pretreated with ML221 and/or apelin-13 for 24 h to determine the impact of apelin‑13 on H₂O₂-induced HLE-B3 cell viability through APJ and then treated with H₂O₂ (200 µM) for 24 h. Cell viability decreased significantly after H₂O₂ treatment, while the addition of apelin-13 pretreatment could increase the cell viability compared with H₂O₂ group (P = 0.0473) as illustrated in Fig. 3. In addition, adding ML221 pretreatment improved the cell viability decline (P = 0.0269). The viability of cells pretreated with ML221 for 1 h in advance decreased (P = 0.0053) compared with the apelin-13 + H₂O₂ group. These results indicate that apelin-13/APJ is involved in regulating the activity of H₂O₂‑induced HLE-B3 cell viability.

Fig. 3.

Apelin-13 prevented H₂O₂-induced cellular viability in HLE-B3 cells. Viability of HLE-B3 cells as assessed using CCK8 assay. HLE-B3 cells were treated with ML221 (10 µmol) for 1 h, preincubated with apelin-13 (0.1 µM) for 24 h, and subsequently treated with H₂O₂ (200 µM) for 24 h in this experiment. Representative images of three replicated experiments were presented. The significance marked at the top of the columns refers to comparison with the control group. Data are shown as mean ± SD (n = 3)

Apelin‑13 inhibition of HLE-B3 cell apoptosis Induced by H₂O₂ via APJ

To assess the impact of apelin-13 on the apoptosis of H₂O₂-induced HLE-B3 cells, we employed the Annexin V-FITC/PI kit. H₂O₂ promoted the apoptosis of HLE-B3 cells (P = 0.0003), while the addition of apelin-13 attenuated the apoptosis induced by H₂O₂ (P = 0.0044), as demonstrated in Fig. 4. Subsequently, we investigated whether apelin-13 could inhibit H₂O₂-induced HLE-B3 cell death through APJ. Flow cytometry demonstrated that the combined application of ML221 blocked this protective effect. The APJ antagonist ML221 considerably incresed the number of apoptotic cells compared with H₂O₂ (P = 0.0005), and reduced the protective effect of apelin-13 on HLE-B3 cells (P = 0.0004). These results indicate that apelin-13/APJ is involved in regulating HLE-B3 cell apoptosis due to H₂O₂.

Fig. 4.

Apelin-13 prevented H₂O₂-induced cellular apoptosis in HLE-B3 cells. Based on the experimental statistics, apelin-13 substantially decreased the apoptosis rate of HLE-B3 cells through APJ. Representative images of three replicated experiments were presented. The significance marked at the top of the columns refers to comparison with the control group. Data are shown as mean ± SD (n = 3)

Effect of apelin‑13 on the mitochondrial apoptotic pathway in H₂O₂-induced HLE-B3 cells via APJ

As illustrated in Fig. 5a, changes in mitochondrial membrane permeability (MMP) were detected using fluorescent probe JC-1. H₂O₂ substantially reduced MMP in HLE-B3 cells (P = 0.0345), whereas the MMP of HLE-B3 cells in the apelin-13 + H₂O₂ group was higher than that in the H₂O₂ treatment alone group (P = 0.0017). Notably, the MMP of HLE-B3 cells in the ML221 + apelin-13 + H₂O₂ group was lower than that in the apelin-13 + H₂O₂ group (P = 0.0009), and the MMP of HLE-B3 cells in the ML221 + apelin-13 + H₂O₂ group was higher than that in the ML221 + H₂O₂ group (P < 0.0001). The Bcl-2 protein family members play crucial roles in regulating the mitochondrial apoptotic pathway. Apoptosis can be affected by both pro- and anti-apoptotic Bcl-2 family members. Western blotting analysis indicated that H₂O₂ effectively induced the upregulation of pro-apoptotic proteins (such as Bax and Caspase-3) and inhibited the expression of the anti-apoptotic protein (Bcl-2) in HLE-B3, as illustrated in Fig. 5b and c. However, pretreatment with apelin-13 reversed this effect. In addition, ML221 blocked the protective effect of Apelin-13. These findings indicate that apelin-13 suppressed H₂O₂-induced HLE-B3 cell death through APJ.

Fig. 5.

Effect of apelin‑13 on mitochondrial apoptotic pathway in H₂O₂-induced HLE-B3 cells via APJ. (a, d) The fluorescence microscopy findings (×100) on mitochondrial membrane potential (MMP) in each group obtained using the fluorescence probe JC-1. (b, c) The expression of mitochondrial apoptotic pathway-related proteins (Bax, Caspase-3, and Bcl-2) detected by western blotting. Representative images of three replicated experiments were presented. The significance marked at the top of the columns refers to comparison with the control group. Data are shown as mean ± SD (n = 3). The full-length blots/gels are presented in Supplementary Figs. 8–11

Apelin-13 regulation of intracellular PPP under H₂O₂ conditions

Detoxification of H₂O₂ in a lens is facilitated by the glutathione redox cycle, which relies on the reducing power generated by the PPP. A crucial enzyme in PPP is G6PD. The activity of G6PD and PPP proteins was assessed in HLE-B3 cells to investigate the impact of apelin-13 on PPP under H₂O₂ conditions. As illustrated in Fig. 6a, b, and d, H₂O₂ reduced G6PD protein expression (P = 0.0192), and the specific activity of G6PD was considerably lower than that in the control group (P = 0.0065). However, apelin-13 pretreatment increased protein expression and specific activity of G6PD. ML221 hindered the acti on of apelin-13 compared with the apelin-13 + H₂O₂ group. Furthermore, GLUT1 protein expression was examined (illustrated in Fig. 6c), and its expression trend was similar to that of the G6PD protein.

Fig. 6.

Apelin-13 regulated the intracellular PPP under H₂O₂ conditions. (a) The protein levels of G6PD and GLUT1 were measured using western blotting analyses. The full-length blots/gels are presented in Supplementary Figs. 12–14. (b, c) The relative protein expression levels of G6PD and GLUT1. (d) The specific activity of G6PD was detected using the glucose 6-phosphate dehydrogenase assay kit. Representative images of three replicated experiments were presented. The significance marked at the top of the columns refers to comparison with the control group. Data are shown as mean ± SD (n = 3)

Apelin-13 regulation of intracellular redox state in H₂O₂ conditions

NADPH and glutathione play essential roles in providing the reducing power for the antioxidant defense of cells and participating in the detoxification of H₂O₂ [23]. We measured the levels of GSH/GSSG and NADPH/NADP + to investigate the redox status of cells. The findings demonstrated that the H₂O₂ group exhibited decreased levels of GSH/GSSG compared with the control group (P = 0.0029), and the levels of NADPH/NADP + was decreased compared with the control group (P = 0.0004) as illustrated in Fig. 7. However, treatment with apelin-13 increased the levels of GSH/GSSG (P = 0.0039) and NADPH/NADP+ (P = 0.0029), compared with the H₂O₂ group. And the levels of GSH/GSSG in apelin-13, ML221, and H₂O₂ group was higher than the ML221 and H₂O₂ group (P = 0.0019). These findings demonstrate that apelin-13 maintained the intracellular redox balance by enhancing NADPH and GSH levels and reducing intracellular oxidative stress.

Fig. 7.

Apelin-13 regulated the intracellular redox state in H₂O₂ conditions. (a) GSH/GSSG of HLE-B3 cells was detected using the GSH/GSSG kit. (b) NADPH/NADP + of HLE-B3 cells was detected using the NADPH/NADP + kit. Representative images of three replicated experiments were presented. The significance marked at the top of the columns refers to a comparison with the control group. Data are shown as mean ± SD (n = 3)

Discussion

Oxidative stress plays a significant role in the pathogenesis of various eye diseases, including cataracts and glaucoma [8, 24]. Glaucoma, which is associated with increased oxidative stress, is a risk factor for cataract formation [25]. Apelin exerts a protective effect against oxidative stress [17]. However, the effects of apelin-13 on HLECs and their regulatory mechanisms have not been studied. In eyes, the most metabolically active part of the entire lens is LECs, and most metabolites, including antioxidants, are synthesized or actively transported in LECs. Apoptosis of LECs may be the common cellular basis of noncongenital cataract formation, and inhibiting apoptosis may prevent cataract formation [26]. Additionally, oxidative stress is a crucial determinant of cataract development [27]. We confirmed that the protein expression of APJ, GLUT1, G6PD, and Bcl-2 in the anterior lens capsule of APCAG was lower compared to that in the anterior capsule of cataract patients, while the protein expression of Bax and Caspase-3 was higher. Thus, this study represents the first investigation into the impact of apelin-13 on H₂O₂-induced injury in HLECs.

Considering the significant role of oxidative stress in the pathogenesis of glaucoma, which can further exacerbate cataract progression, reducing oxidative stress may be a potential therapeutic target for glaucoma complicated by cataracts. Apelin/APJ has numerous biological effects, such as regulation of cell survival and proliferation, antioxidative effects, inflammation inhibition, and apoptosis suppression [18, 22]. Apelin-13, an amino acid peptide, has been demonstrated to be safe for human use in several studies [22, 28]. Yuki et al. [29] discovered that exogenous [Pyr1]-apelin-13 protected mouse retinal ganglion cells from NMDA-induced death in an APJ-dependent manner and that endogenous apelin also exhibited a protective effect against glutamate-induced retinal cell death. Additionally, the systemic application of the APJ agonist ML233 prevented retinal neuron loss associated with NMDA receptor-mediated excitatory toxicity [30, 31]. Furthermore, apelin-13 reduces lung ischemia-reperfusion injury by mitigating oxidative stress and mitochondrial damage [5].

High intraocular pressure in glaucoma can result in oxidative stress [32]. Apoptosis of LECs, alterations in membrane permeability [5], and reprogramming of energy metabolism resulting from oxidative stress are likely contributors to the accelerated cataract growth in glaucoma, and apelin-13/APJ may be involved. Thus, to validate this hypothesis, we conducted this study. Furthermore, our findings demonstrated that apelin-13 exhibited no significant cytotoxicity toward HLECs and effectively prevented cell damage and apoptosis caused by H₂O₂. Apelin-13/APJ may be employed as a therapeutic target for glaucoma complicated with cataracts because apelin-13 is nontoxic (experimentally proven but not demonstrated) and has a good antioxidant effect.

In patients with glaucoma, the significant reduction in circulating glutathione levels may be the basis of enhanced oxidative stress caused by the deterioration of overall antioxidant defense function [33]. Wang et al. [19] demonstrated that the increased GSH/GSSG ratio during electroacupuncture pretreatment reduced oxidative stress and myocardial injury during cardiopulmonary bypass. Imbalances in intracellular redox homeostasis can result in oxidative stress in cells. GSH is a crucial antioxidant and free radical scavenger in the body that protects the lens, cornea, and retina from ROS-induced oxidative damage, whereas NADP + provides reducing power for ROS clearance [27]. The ratio of GSSG/GSH to NADP+/NADPH serves as an indicator of the redox state of cells, and an increase in this ratio reflects a decrease in the antioxidant level of the organism relative to oxidation levels. Here, we cultured the cells using H₂O₂ and compared the differences in the above-related indices between the model and control groups. The PPP pathway was inhibited, the NADP+/NADPH and GSSG/GSH ratios increased, and the cells were in an oxidized state after H₂O₂ treatment. Apelin-13 pretreatment based on H₂O₂ reduced the expression of these indices, weakened the oxidative state of cells, and demonstrated antioxidant effects. However, after ML221 treatment, the ratios of NADP+/NADPH and GSSG/GSH were lower than those of the apelin-13 pretreatment and H₂O₂ groups, which was inconsistent with the expected findings.

Oxidative stress disrupts the balance between reactive oxygen radical production and radical scavenging effects, resulting in cell apoptosis through the mitochondrial apoptosis pathway [34]. Our experiment demonstrated that apelin-13 inhibited apoptosis in H₂O₂-induced HLECs. Apelin-13 is a bioactive peptide known for its free radicals scavenging properties, which can effectively reduce ROS and superoxide anions production, inhibit DNA damage, and thus suppress apoptosis [35]. Furthermore, the mitochondrial pathway of apoptosis is the primary intrinsic pathway, which can induce various reactions, including impairment of the mitochondrial electron transport chain system and loss of MMP. Previous studies have demonstrated that apelin-13 acts on the retention of mitochondrial membrane potential, prevents cytochrome C release from mitochondria, reduces ROS production, and protects bone marrow mesenchymal stem cells from apoptosis cell death [18, 36]. Pouresmaeili-Babaki et al. [37] demonstrated that apelin-13 protects SH-SY5Y cells from apoptotic death by preventing mitochondrial depolarization, cytochrome C release, and Caspase-3 activation. In this study, HLECs pretreated with apelin-13 downregulated Bax and Caspase-3 expression and considerably upregulated Bcl-2 expression and MMP compared with those treated with H₂O₂ alone. However, the effect of apelin-13 on these indexes was attenuated in H₂O₂-induced HLECs treated with both apelin-13 and the APJ inhibitor ML221. These findings demonstrate that apelin-13 inhibits H₂O₂-induced oxidative damage through the APJ receptor and inhibits cell apoptosis. Bcl-2 is an anti-apoptotic protein localized in the mitochondria, which can inhibit the release of cytochrome C and protect against oxidative stress-induced apoptosis. Bax promotes the release of cytochrome C, thereby facilitating apoptosis. Furthermore, the downregulation of Bcl-2 and upregulation of Bax are closely associated with the apoptosis of HLECs [38, 39].

Conclusion

This study shows that apelin-13 can effectively protect LECs from H₂O₂-induced cytotoxicity. Apelin-13, a neuroprotective peptide, controls the redox state of cells and prevents cell death by influencing the activity of the PPP pathway. In addition, glaucoma may enhance LEC apoptosis by inhibiting the PPP pathway through the apelin/APJ system, resulting in cataracts. These results have potential clinical significance and show that apelin-13 plays a preventive role in the process of glaucoma by accelerating the process of cataracts.

Electronic supplementary material

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Abbreviations

APJ

Angiotensin receptor like 1

HLECs

Human lens epithelial cells

PACG

Primary angle-closure glaucoma

ROS

Reactive oxygen species

H₂O₂

Hydrogen peroxide

GSH

Glutathione

GSSG

Glutathione disulfide

NADPH

Nicotinamide adenine dinucleotide phosphate

PPP

Pentose phosphate pathway

G6PDH

Glucose-6-phosphate dehydrogenase

GLUT1

Glucose transporter 1

Bcl-2

B-cell lymphoma-2

Bax

BCL2-Associated X

Author contributions

Ling Yu designed the study, Xue Li and Chao Gu contributed to experiment and analyze the data. Qiumei Hu contributed to culture the lens epithelial cells. Liqin Wang and Ya Zhang collected the anterior lens capsule tissues. Ling Yu, Xue Li, and Chao Gu wrote the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (NO.82070962 and NO.82201164).

Data availability

The data that support the findings of this study are available from the corresponding author.

Declarations

Ethical approval and consent to Participate

All procedures involving human material in this study were reviewed and approved by the Ethics Committee of the Army Medical Center, Chongqing, China (No.82070962), and performed in accordance with the Declaration of Helsinki. And we obtained the informed consent and the written informed consent of all patients in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.