Piperine alleviates retinal ischemic injury by mediating the APE1 signaling

Abstract

Ischemic retinal damage is the most common cause of severe vision impairment and blindness. Anti-vascular endothelial growth factor (anti-VEGF) agents have transformed the treatment of retinal ischemic disorders and have become the cornerstone therapy for these conditions. Nonetheless, the risk for systemic and ocular adverse effects necessitates careful consideration. Meanwhile, the therapeutic potential of natural compounds for ischemic retinal injury is increasingly attracting attention. In this study, piperine (PIP), a natural compound derived from pepper, was found to reduce apoptosis by reducing the severity of retinal and optic nerve ischemic damage. However, the precise pharmacological mechanisms of PIP are yet to be fully elucidated. Molecular docking (MD) studies, MD simulations, and surface plasmon resonance experiments were conducted to determine the molecular targets of PIP. Our data revealed that PIP can bind to apurinic/apyrimidinic endonuclease 1 (APE1), thereby inhibiting apoptosis by decreasing the expression of caspase-9 and caspase-3 and regulating the mitochondrial pathway. In summary, PIP may directly targets the APE1 protein and further regulates the caspase-9/caspase-3 axis to provide neuroprotection against ischemic retinal injury.

Graphical abstract

Introduction

Ischemic retinopathy stands as the leading cause of vision impairment and blindness in the industrialized world [1]. The damage to the retina from ischemia leads to a range of ocular disorders, encompassing glaucoma, ischemic optic neuropathy, age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, and macular edema [[2], [3], [4], [5], [6], [7]]. The retina, an extension of the central nervous system (CNS), demonstrates a significant level of metabolic activity and possesses the most demanding metabolic needs [8]. Due to its elevated metabolic requirements, the retina is vulnerable to ischemic stress; a deficient blood supply results in hypoxia and ischemia, which can cause tissue damage and the apoptosis of retinal ganglion cells (RGCs) [9]. While considerable research has pinpointed potential cellular and molecular changes that lead to retinal cell loss, the precise molecular mechanisms are still not fully understood. Presently, there are only a limited number of effective pharmacological treatments available to prevent retinal ischemic injury [10]. Consequently, it is crucial to deepen our understanding of the molecular mechanisms and to identify new therapeutic strategies for treating retinal ischemic injury. Apurinic/apyrimidinic endonuclease 1 (APE1), a DNA repair enzyme, boasts a multitude of functions, its primary role is to rectify base excision DNA damage. Inadequate repair of apurinic/apyrimidinic (AP) sites can lead to DNA strand breakage, oxidative damage, apoptosis, and increased cytotoxicity [11]. A decrease or absence of APE1 in the cell nucleus indicates neuronal energy depletion, which in turn causes DNA damage and ultimately leads to neuronal apoptosis and death [12,13]. Research from both in vivo and in vitro studies has shown that reduced levels of APE1 expression are linked to oxidative damage and cellular apoptosis [[14], [15], [16]], a crucial pathological process in retinal ischemic injury. Furthermore, the upregulated expression of APE1 in certain neurons may act as a defensive mechanism against ischemic injury [17,18]. In light of these findings, it is plausible that APE1-mediated apoptotic signals play a potential role in the pathological progression of retinal ischemic damage.

Apoptosis is a complex cascade of reactions, governed by genetic regulation and initiated by both internal and external cellular signals. It involves two primary pathways: the extrinsic (death receptor) and the intrinsic (mitochondrial) pathways [19]. The mitochondrial apoptosis pathway can be initiated by various stimuli, including oxidative stress, DNA damage, and ischemia [20]. Caspases, a family of cysteine proteases, play a pivotal role in the regulation of apoptosis. Upon activation by specific signals, they catalyze the hydrolysis of cellular proteins, ultimately resulting in cell death. Activated caspases can assemble into specialized initiation-activation complexes, forming homodimers that undergo self-cleavage to become fully functional. In particular, caspase-9 is integral to mitochondria-mediated apoptosis [21,22]. Caspase-9 activates pro-apoptotic proteases, thereby executing the apoptosis program, which in turn activates caspase-3 [23]. This highlights a close interconnection between the caspase-9/caspase-3 signaling axis and the mitochondrial apoptosis pathway.

Piperine (PIP), an active natural alkaloid derived from pepper fruits, exhibits a range of pharmacological properties, including antiepileptic, anti-apoptotic, anti-inflammatory, antibacterial, antitumor, and antioxidant effects [[24], [25], [26], [27]]. Extensive research has documented the neuroprotective effects of PIP on the CNS, as it is capable of crossing the blood-brain barrier (BBB) and offering protection against various diseases, such as sclerosis, Parkinson's disease (PD), stroke, and Alzheimer's disease (AD) [[28], [29], [30], [31], [32]]. Although the retina shares similar physiological and biological characteristics with the CNS, its role and mechanism in retinal ischemic injury are yet to be fully understood. Although vascular endothelial growth factor (VEGF) is considered a standard treatment for retinal diseases as per clinical guidelines, its potential side effects should not be disregarded [33]. PIP, being a natural compound, presents minimal side effects and has shown significant efficacy in treating ischemic disorders. This study aimed to investigate the therapeutic benefits of PIP and its correlation with APE1 in retinal ischemic injury, aiming to elucidate its potential neuroprotective mechanism against retinal ischemic stress.

Material and methods

Reagents

PIP, possessing a minimum purity of 98 ​% as verified by high-performance liquid chromatography (HPLC), was sourced from Shanghai Dibai Biotechnology Co., Ltd in China. Hycanthone, with a purity of ≥99 ​% as verified by HPLC, was acquired from the MCE company.

Animals

Male Sprague-Dawley rats, aged 6–8 weeks, were obtained from Hunan Slake Jingda Experimental Animal Co., Ltd. All animal experiments strictly complied with the guidelines of the Sichuan Provincial People's Hospital Ethics Committee. Each rat was housed in designated pathogen-free facilities, ensuring consistent temperature and relative humidity levels.

Retinal ischemia model

Before initiating any experiments or surgeries, the rats were anesthetized using isoflurane. The retinal ischemia model was performed in accordance with the previously described protocol [34]. The right common carotid artery (CCA) was exposed, and the external carotid artery (ECA) was dissected. We proceeded to strip the internal carotid artery and its first branch before ligating the pterygopalatine artery. Ischemia was induced and maintained for a period of 7 ​d, after which the right eye was harvested for further experimental analysis. Rats that underwent isolation of the right CCA without ligation were designated to the sham-operated group. The remaining surgical procedures were executed as previously detailed. The efficacy of the model was confirmed via fundus and laser Doppler flow imaging examinations. The rats were subsequently divided into three groups: sham, ischemia, and ischemia ​+ ​PIP. The ischemia ​+ ​PIP group received PIP (20 ​mg/kg/d) via intragastric administration for a consecutive period of 7 ​d. Following this, all rats were euthanized to facilitate subsequent experimental procedures.

Blood flow imaging

Anesthesia was administered to the rats. Subsequently, the rats were positioned at a specific elevation beneath the Laser Speckle Contrast Imaging device, where they were photographed, video-recorded, and their blood flow was analyzed.

Hematoxylin and Eosin (H&E) and Nissl staining

The tissue slices were embedded in paraffin and sectioned at a thickness of 3–5 ​μm. Following the removal of paraffin with xylene, the slides were dehydrated through a graded series of alcohols (5 ​min per grade). Subsequently, the sections underwent staining with a H&E kit and the Nissl staining kit, gradient alcohol, and clear xylene. The images of sections were acquired through a 200x microscope and analyzed after sealing with neutral gum.

Transmission electron microscopy

The samples underwent fixation, dehydration, permeabilization, and embedding procedures. Ultrathin sections were subjected to dual staining with uranyl acetate and lead citrate. Subsequently, the sections mounted on the mesh were visualized using a JEM-1400FLASH transmission electron microscope at room temperature for observation and analysis.

In vivo imaging

The experimental rats were anesthetized, and samples and controls were prepared. The in vivo caspase activity was determined using an NIR-FLIVO 747 tracer kit. The rats were visualized using a vivo AniView600 imaging system.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

The paraffin-embedded rat eyeball was initially treated with water to remove the wax, subsequently exposed to xylene for transparency, and then dehydrated using a series of graded alcohols. In the final step, the In Situ Cell Death Detection Kit and the TUNEL Bright-Red Apoptosis Detection Kit were employed according to the manufacturer's guidelines.

Molecular docking of PIP to APE1

The protein crystal structure of human APE1 (PDB ID: 3U8U), obtained from the RCSB Protein Data Bank, was utilized for molecular docking. For the small molecule DB12582 (PIP), energy minimization was conducted using the AVOGADR 1.2.0 software with the MMFF94 force field. Subsequently, molecular docking was executed employing the AutoDock Vina 1.1.2 software.

Molecular dynamics simulation of PIP to APE1

The initial structure was selected based on the docking results. The protein position was modeled using Amber14sb, and the small molecule position was modeled using Gaff2. APE1 and PIP were solvated using the TIP3P water model to create a water box, and the system was neutralized with sodium ions. During the elastic simulation, electrostatic interactions were managed using the particle mesh Ewald method, with the Verlet and CG algorithms. The steepest descent method was used to minimize energy over 50,000 steps. The Coulomb force and the van der Waals radius cutoff distances were both set to 1.4 ​nm. The system was balanced using a canonical ensemble (NVT) and an isothermal-isobaric ensemble (NPT), and a 50-min MD simulation was conducted at normal room temperature and pressure.

Surface plasmon resonance (SPR)

The binding affinities were determined utilizing the BIAcore T200 instrument, which employs SPR technology. Recombinant human APE1 protein was diluted in a sodium acetate solution adjusted to pH 5.0, resulting in a final concentration of 50 ​μg/mL. To attain target densities of 12000 resonance units, APE1 was immobilized onto the CM5 sensor chip via amine coupling. Subsequently, immobilized EZH2 or SUZ12 was utilized to capture the chemical substance PIP. The procedure adhered to the methodology detailed in previous research [35]. Hycanthone served as the positive control. One of the sensor chip surfaces underwent a blank immobilization process to correct for non-specific binding, thereby refining the binding response-analysis fitting curve.

Immunohistochemical and immunofluorescence staining

Sections were dewaxed using xylene and subsequently dehydrated with a series of graded alcohols. The immunohistochemical (IHC) or immunofluorescence (IF) assay kit (Immunoway, #RS0033, USA) was used following instructions. These tissues were treated with primary antibodies, including anti-APE1 (CST, 1:200), anti-caspase-3 (CST, 1:400), anti-caspase-9 (Proteintech, 1:200), anti-Rbpms (HUABIO, 1:200), anti-occludin (Abcam, 1:200), and anti-ZO-1 (Abcam, 1:300). These were then incubated overnight at 4 ​°C in a humidified box. Color was developed using diaminobenzidine (DAB), and hematoxylin was used as a counterstain. The fluorescent-labeled secondary antibody was added and placed in the incubator at room temperature for 1 ​h, stained with 4′,6-diamidino-2-phenylindole (DAPI), and examined using a fluorescence microscope.

Detection of piperine by liquid chromatography-mass spectrometry (LC-MS)

Cardiac blood was collected from anesthetized rats, subjected to centrifugation, and the resultant supernatant was harvested. A 100 ​μL sample solution was then mixed with 400 ​μL of acetonitrile, followed by vigorous agitation and subsequent centrifugation at 12000 ​rpm for 30 ​min. The filtered supernatant was subsequently analyzed using ultra-performance liquid chromatography.

Serological and apoptosis assays

Peripheral blood and serum were collected from rats. Serum indicators were detected using respective kits for glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and malondialdehyde (MDA) according to the instructions. Cytokines, including tumor necrosis factor (TNF-α), interleukins (IL)-6, IL-1β, and IL-10, were detected using a cytokine detection kit. Serum APE1 was detected employing the rat APE1 quantitative assay kit. Cell apoptosis was detected by flow cytometry utilizing an Annexin V-FITC Apoptosis Detection Kit.

Flow cytometry

Adherent cells were treated following the instructions in the Annexin V-FITC kit. The samples were processed by flow cytometry within 1 ​h.

Quantitative reverse transcription polymerase chain reaction assay (qPCR)

The RT SuperMix and SYBR Green Master Mix for qPCR were procured from Vazyme Biotech Co., Ltd. Subsequently, cells or tissues were isolated, and total RNA was extracted using the TRIzol reagent. The mRNA levels were quantified using the RT Master Mix and SYBR Green qPCR Master Mix, adhering strictly to the manufacturer's recommended protocol. β-Actin served as the internal control for normalizing RNA levels. The primer sequences are detailed in Supplementary Table 1.

Western blot assay

Equal concentrations of protein samples were resolved by utilizing bis-tris-polyacrylamide (10 ​%) electrophoresis gels that were subsequently transferred onto polyvinylidene fluoride membranes. After applying a protein-free rapid-blocking solution, the membrane was incubated overnight with the primary antibody at 4 ​°C. The primary antibodies incubated, APE1 (1:1000), β-actin (1:1000), caspase-3 (1:1000), and GAPDH (1:1000) from CST, and caspase-9 (1:1000) from Proteintech. This was followed by incubation with appropriate secondary antibodies. Upon membrane exposure, the grayscale values in the photographs were analyzed.

Statistical analysis

All data were presented as the mean ​± ​standard deviation. Statistical analysis was conducted using GraphPad Prism 8 software. One-way analysis of variance was applied to compare multiple groups, followed by the least significant difference (LSD) post-hoc test for correction. The ∗ represents values with respect to the sham, ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001; ^#represents values with respect to the ischemia group, ^#p ​< ​0.05, ^##p ​< ​0.01, ^###p ​< ​0.001.

Results

PIP enhanced retinal blood flow in vivo

The potential impact of PIP on ischemic retinal blood flow was examined by administering it to rats for 7 consecutive days (Fig. 1A). As depicted in Fig. 1B, PIP treatment demonstrated a reduction in atrophy and an enhancement of reflexes in the right eye of rats. The effects on fundus vascular morphology were assessed through fundus imaging and laser speckle technology (Fig. 1C), as along with measurements of ocular blood flow (Fig. 1D and E). Following PIP treatment, blood vessels thickened and blood flow (Supplementary Fig. 1) in the retina increased significantly (Fig. 1C–E). In summary, PIP increased retinal vascular blood flow in the rat model.

Fig. 1.

Piperine (PIP) improved retinal blood flow in vivo. (A) A design chart for animal experimentation. (B) Representative changes in the eyeball phenotype are presented here. (C) A representative image shows morphological changes in retinal blood vessels. (D) Ocular blood flow imaging with laser scatter detection. (E) Quantitative analysis of blood flow. ∗Represents compared to the sham group, ∗∗p ​< ​0.01. ^#Represents compared to ischemia group, ^##p ​< ​0.01. n ​= ​3.

PIP diminished the severity of retinal and optic nerve damage

The effect of PIP on retinal and optic nerve injury was evaluated via pathological staining experiments. Fig. 2A shows a schematic of the experiment. Fig. 2C shows the structure of each layer of the retina. PIP administration resulted in an increased count of RGCs cells, a reduction in the intercellular gap within the nuclear layer, and a marked alleviation of optic nerve injury vacuolization in comparison to the ischemia group (Fig. 2D–F, G). Subsequently, Nissl staining confirmed the effect of PIP on the reduction of Nissl bodies in RGCs and demonstrated a significant inhibition by PIP on the depletion of Nissl bodies (Fig. 2E). Moreover, LC-MS analysis identified PIP as the principal active compound in the retinal tissue of the treatment group (Fig. 2B, Supplementary Fig. 2). These findings indicate that PIP substantially mitigated the extent of retinal damage, improved the retention of Nissl bodies in RGCs, and lessened optic nerve injury.

Fig. 2.

Piperine (PIP) reduced the extent of retinal and optic nerve injury. (A) Animal experimental design chart. (B) PIP was the main detected compound. (C) This schematic diagram shows the structure of the layers of the retina. (D) Morphological analysis of retinal histopathological staining (magnification: ​× ​200, scale bar: 60 ​μm). (E) Nissl staining for retinal histopathologic analysis. (Original magnification: ​× ​200, scale bar: 60 ​μm; red arrows, Nissl bodies). (F, G) Morphometric analysis of optic nerve histopathology. (F, horizontal section; G, longitudinal section, magnification: ​× ​200, scale bar: 60 ​μm; red arrows, vacuolization). n ​= ​3.

APE1 is a direct target of PIP

PIP is one of the significant active ingredients in the Chinese medicinal plant Piper nigrum. Various pharmacological characteristics of PIP have been reported, including anti-inflammatory, anti-apoptotic, and antioxidant effects [[36], [37], [38]]. We utilized the protein crystal structure 3U8U (APE1) from the RCSB Protein Data Bank for molecular docking with the small molecule DB12582 (PIP). The binding free energy between 3U8U and DB12582 was estimated to be −5 ​kcal/mol (see Supplementary Table 2) (Fig. 3A), suggesting the potential activity of the DB12582 molecule with the 3U8U protein. Subsequently, molecular dynamics (MD) simulation experiments were conducted using the small molecule PIP and the APE1 protein, and hycanthone was used as a positive control. As shown in Figure 3B the atomic root mean square difference (RMSD) between PIP and APE1 molecules indicated the stability of the binding system. However, the RMSD perturbation of hycanthone was more pronounced. The turning radius of PIP was larger compared to that of hycanthone (see Supplementary Fig. 3). Furthermore, small-molecule drugs, including naringenin, curcumin, dihydroartemisinin, and sinomenine, were randomly selected along with the reference small positive molecule, hycanthone. These were examined in terms of their binding to the APE1 protein. Subsequently, the results of PIP binding to the APE1 protein were evaluated in comparison to other compounds. The surface plasmon resonance (SPR) assay was performed to determine the binding affinity of different small molecules to the APE1 protein (see Supplementary Data and Supplementary Table 3). A high affinity of PIP to APE1 was observed, with a KD value of 2.555E-5(μM) (Fig. 3C and D). These data indicate that PIP has a direct target action for APE1.

Fig. 3.

Apurinic/apyrimidinic endonuclease 1 (APE1) as a direct target of piperine (PIP). (A) Overall and local binding perspectives of the 3U8U DB12582 complex (hydrogen bonding: the yellow dashed line; amino acids forming hydrogen bonds with small molecules: the green line indicates). This diagram shows a small protein molecule interaction. (B) Stability of the root-mean-square difference system of PIP binding to hycanthone and APE1 proteins. (C, D) The interaction between PIP and APE1 demonstrates high affinity (see supplementary).

PIP mitigated retinal apoptosis by modulating the APE1 signaling pathway

To evaluate the impact of PIP on retinal cell apoptosis and its potential to safeguard against retinal ischemic injury (refer to Fig. 4A), we conducted transmission electron microscopy, in vivo caspase activity labeling, and TUNEL assays. Apoptosis was induced by retinal ischemic injury (refer to Fig. 4B–D, F, K). Fig. 4C clearly depicts the progression from mid to late-stage apoptosis in RGCs in comparison to cells from the sham group. Fig. 4D shows the damage to retinal pigment epithelial (RPE) cells relative to those in the sham group. Furthermore, we noted an expansion of tissue gaps, vacuolization of intracellular mitochondria, rupture of nuclear membranes, and loss of organelles. Notably, the damage to both RGCs and RPE cells was reduced in the PIP-treated group (refer to Fig. 4C and D). A substantial loss of RGCs was observed in the ischemic group, as indicated by the red dashed box in Fig. 4E and L, in stark contrast to the significant preservation observed in the PIP-treated group.

Fig. 4.

Piperine (PIP) attenuated the retinal apoptosis by mediating the APE1 signaling pathway. (A) A schematic showing animal experimental design. (B) In vivo imaging of caspase. (C, D) Retinal ganglion cells (RGCs) and retinal pigment epithelial (RPE) cells were detected by transmission electron microscopy (RGC magnification: ​× ​15000, scale bar: 1 ​μm; RPE magnification: ​× ​8000, scale bar: 2 ​μm; red arrows: mitochondrion, tissue gap). (E, L) Specific markers for RGCs were detected through immunohistochemical (IHC) staining and quantitative RGCs analysis (magnification: ​× ​200, scale bar: 60 ​μm, red arrows, RGCs). (F, K, M, Q) IHC and immunofluorescence (IF; red) staining were performed to assess apoptotic cells and quantitative apoptotic cells analysis (magnification: ​× ​200, scale bar: 60 ​μm, 50 ​μm, red arrows, apoptotic cells). (I, K, J, P) The expression of retinal APE1 was determined by western blotting and IF (green) staining (magnification: ​× ​200, scale bar: 50 ​μm). (R) Assessment of APE1 factor content in retinal tissue supernatant via enzyme-linked immunosorbent assay. (G, H, N, O) IHC staining to assess caspase 9 and caspase 3 in cells (magnification: ​× ​200, scale bar: 60 ​μm; red arrows, positive cells). (S) Quantitative polymerase chain reaction was conducted to determine the mRNA content of retinal APE1. ∗Represents relative to the sham group. ^#Represents compared to the ischemia group. ∗p, ^#p ​< ​0.05, ∗∗p, ^##p ​< ​0.01, ∗∗∗p, ^###p ​< ​0.001, n ​= ​3.

As depicted in Fig. 4B, the treatment for ischemic injury markedly elevated caspase expression in the right eye of rats when compared to the normal left eye, suggesting caspase activation. Moreover, the effect of PIP on the APE1/caspase cascade was evaluated using various techniques, such as fluorescence microscopy, western blotting, IHC, qPCR, and enzyme-linked immunosorbent assay (Fig. 4). Subsequently, the TUNEL assay was conducted to detect cellular apoptosis. The outcomes indicate a substantial reduction in apoptosis post-PIP treatment (Fig. 4F, M, K, Q). We also examined the mRNA, tissue factor, and protein levels involved in APE1 signaling. Following PIP treatment, the level of APE1 showed a significant increase compared to that in the ischemic group (Fig. 4I–K, P, R, and Supplementary Fig. 4). Additionally, PIP notably boosted the mRNA expression of APE1 relative to the ischemic group (as illustrated in Fig. 4S). Correspondingly, we observed that the total levels of caspase-9 and caspase-3 proteins also surged significantly post-ischemic injury, which diminished following PIP treatment (Fig. 4G, N, H, and O). These findings suggest that PIP mitigated retinal apoptosis by modulating the APE1/caspase-9/caspase-3 axis.

PIP has the potential to alleviate optic nerve injury by modulating the APE1 signaling pathway

We explored whether PIP also influences optic nerve ischemic injury via the APE1/caspase cascade pathway, thereby reducing the severity of optic nerve damage (Fig. 5A). We performed transmission electron microscopy, fluorescence microscopy, and qPCR to assess the effects of PIP on optic nerve tissue (Fig. 5). Initially, optic nerve samples from sham, ischemic, and PIP-treated groups were collected to evaluate ultrastructural damage. The ischemic group exhibited optic nerve vasospasm and intima-media rupture (Fig. 5B) to a greater extent than the sham group. Additionally, myelin sheaths varied in size, were demyelinated, and lysed. However, PIP treatment significantly mitigated optic nerve damage, particularly concerning vascularization and myelination (Fig. 5B). Subsequently, we examined the expression of the caspase-9/caspase-3 cascade.

Fig. 5.

Piperine (PIP) improved optic nerve injury by mediating the APE1 signaling pathway. (A) Animal experimental design chart. (B) Detection of blood vessels and myelin was performed by transmission electron microscopy (magnification: ​× ​15000, scale bar: 1 ​μm, red arrows: endothelium and myelin). (C, F) Immunofluorescence (IF; green) staining assessed cellular caspase-3 expression (magnification: ​× ​200, scale bar: 10 ​μm). (D, G) IF (red) staining assessed cellular caspase-9 expression (magnification: ​× ​200, scale bar: 10 ​μm). (E, H) IF (red) staining was performed to assess apoptotic cells (magnification: ​× ​200, scale bar: 10 ​μm). (E, I) IF (green) staining assessed cellular APE1 content (magnification: ​× ​200, scale bar: 10 ​μm). (J) Quantitative polymerase chain reaction (q-PCR) was conducted to determine the optic nerve APE1 mRNA content. ∗Represents relative to the sham group. ^#Represents relative to the ischemia group. ∗p, ^#p ​< ​0.05, ∗∗p, ^##p ​< ​0.01, ∗∗∗p, ^###p ​< ​0.001, n ​= ​3.

Consistent with Fig. 5C, D, 5F, and 5G, the ischemic group showed a significant rise in both the quantity and fluorescence intensity of caspase-9 and caspase-3 expression within the optic nerve, relative to the sham group. Conversely, the PIP group displayed a marked decrease in caspase-9 and caspase-3 protein levels (Supplementary Fig. 5A and 5B). Moreover, as depicted in Fig. 5E, H–I, and (Supplementary Fig. 5C), the number of apoptotic cells (red) was considerably higher in the ischemic group, and the expression intensity of APE1 was notably diminished (Supplementary Figs. 5C, 5D, 5E). In contrast, PIP treatment significantly elevated the level of APE1 mRNA compared to its levels in the ischemic group (Fig. 5J). Compared to the ischemic group, PIP treatment reduced ischemia-induced apoptosis and markedly enhanced protein fluorescence expression intensity, aligning with the outcomes presented in Fig. 4F, I, 4K, and 4S. These results underscore PIP's involvement in the APE1/caspase-9/caspase-3 pathway during retinal ischemia injury, highlighting its crucial role in modulating this pathway in optic nerve injury scenarios.

PIP mitigated ischemia-induced peripheral blood inflammation, apoptosis, and oxidative stress

To evaluate PIP's effects on apoptosis, oxidative stress, and inflammation in peripheral blood, assessments, including flow cytometry apoptosis assays, reagent kit detection experiments, and LC-MS assays (Fig. 6A). As depicted in Fig. 6B, LC-MS identified PIP as a significant active metabolite component in the serum. Subsequently, the flow cytometry assay indicated a markedly elevated ratio of apoptotic cells in the ischemia group. Nevertheless, the PIP group exhibited a significant reduction in this ratio (Fig. 6C and D). As illustrated in Fig. 6E, the concentration of APE1 was notably lower in the ischemic group compared to the sham group. Following PIP treatment, there was a significant elevation in APE1 concentration. Furthermore, assays for inflammation-related factors demonstrated a substantial increase in levels of IL-6, IL-1β, and TNF-α, and a marked decrease in IL-10 concentrations in the ischemia group compared to the sham group (Fig. 6F–I). In contrast, the PIP group experienced a significant reduction in IL-6, IL-1β, and TNF-α levels and a notable increase in IL-10 concentration.

Fig. 6.

Piperine (PIP) attenuated peripheral blood inflammation, apoptosis, and oxidative stress in the rat ischemic retinal injury model. (A) A schematic of the experimental design of the animal. (B) The main compound detected was PIP. (C, D) Apoptosis detection by flow cytometry. (F–I) Detection of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and IL-1β factors according to cytometric bead array. (J–L) Determination of glutathione peroxidase (GSH-PX), superoxide dismutase (SOD), and malondialdehyde (MDA) levels using specific kits. ∗Represents relative to the sham group. ^#Represents relative to the ischemia group. ∗p, ^#p ​< ​0.05, ∗∗p, ^##p ​< ​0.01, ∗∗∗p, ^###p ​< ​0.001, n ​= ​6.

Similarly, measurements of oxidative stress-related factors indicated that SOD and GSH-PX levels were significantly diminished in the ischemic group, while MDA levels were notably elevated (Fig. 6J-L). However, the PIP group showed a remarkable improvement in SOD, GSH-PX, and MDA levels (Fig. 6J-L). These findings suggest that PIP alleviates apoptosis, inflammation, and oxidative stress levels in the peripheral blood of rats.

Discussion

Previous studies have shown that PIP, a natural compound derived from pepper, has been shown to possesses neuroprotective properties [37,39]. Nonetheless, the precise mechanism by which PIP alleviates retinal ischemic injury is not yet fully understood. This study delves into the influence of PIP on ischemia-induced cell apoptosis, utilizing in vivo experiments. The findings indicate that PIP is capable of inhibiting cell apoptosis, reducing retinal and optic nerve damage, and mitigating peripheral blood inflammatory responses and oxidative stress levels. The data analysis uncovers that PIP modulates the APE1/caspase-9/caspase-3 axis, thereby exerting a protective effect against retinal ischemic injury.

Pepper, a vine plant, is widely cultivated in Southeast Asian countries, subtropical regions, and China. After being dried or roasted, its fruit is commonly used as a culinary spice and seasoning [40]. Pepper's principal chemical component, PIP, has garnered significant interest due to its extensive pharmacological properties, encompassing anti-inflammatory, anti-oxidant, anti-apoptotic, anti-ischemic, and antihyperglycemic effects [41]. PIP has shown potential in offering protection against ischemic diseases, such as cerebral ischemia [32]. Recent studies have indicated that PIP exerts neuroprotective effects on diabetic retinopathy by modulating the HIF-1α/VEGFA signaling pathway [42]. During ischemia and hypoxia, the apoptotic signaling pathway is activated, and mitochondrial damage disrupts the equilibrium between pro-apoptotic and anti-apoptotic proteins. This disruption leads to the release of cytochrome c (Cyt-c), which binds to Apaf-1 to form apoptotic bodies, initiating a cascade of caspase reactions and inducing apoptosis of RGCs [21,43]. Aberrant expression of APE1 following DNA damage is one of the mechanisms behind neuronal apoptosis [44]. Utilizing LC-MS analysis, we investigated the direct effects of PIP on retinal tissue and serum. The analysis indicated that PIP was the primary active compound in the rat model of retinal ischemic injury. This presents a potential therapeutic strategy for retinal diseases through APE1 modulation. Consequently, SPR experiments were conducted to confirm the high affinity between PIP molecules and the APE1 protein. MD and molecular dynamics simulations validated the interaction and stability of PIP with APE1, suggesting that PIP may influence retinal protection against ischemic damage by directly targeting APE1 activation.

Ischemia stands as a primary cause of DNA damage, particularly evident in CNS injuries. The existence of deficiencies in repair mechanisms is linked to the neurodegenerative processes seen in stroke, brain injury, and cardiovascular disorders [45,46]. DNA damage can initiate cell apoptosis, potentially contributing to tissue atrophy in injury and neurodegenerative conditions. DNA repair is pivotal in sustaining the vitality of brain cells and neurological function. Concurrently, neurodegenerative diseases are correlated with defects in DNA repair or double-strand break repair. APE1 plays a crucial role in the base excision repair pathway, where unrepaired AP sites can lead to adverse outcomes such as DNA strand breaks, oxidative damage, and cell apoptosis [11]. Furthermore, phosphorylation of APE1 at Thr232 reduces its AP endonuclease activity, resulting in the accumulation of DNA damage, neuronal apoptosis, and cell death [12]. Brain ischemia rapidly induces DNA oxidative damage, coupled with decreased APE1 expression, impeding DNA repair and ultimately causing cell apoptosis [17,47]. In this study, by employing the laser speckle technique, we examined the ocular blood flow in a rat model of retinal ischemic injury. Protein expression of APE1 in retinal tissues was examined, alongside the detection of DNA fragmentation during apoptosis using TUNEL staining. Our results showed an increase in TUNEL-positive cells and a decrease in APE1 expression levels with prolonged ischemia. Following PIP treatment, a notable increase in retinal blood flow was observed, indicating PIP's potential in alleviating circulatory disturbances. Our data suggest that PIP targets APE1 activation, enhances its expression levels, and thereby alleviates cellular apoptosis.

In the tissues of the CNS, the expression of caspase-9 and caspase-3 escalate during ischemia and hypoxia [8,48], a trend corroborated by studies showing similar increases during the apoptotic process of retinal ischemic damage [49,50]. The onset of intrinsic apoptotic pathways is intimately associated with the activity of caspases-3 and -9 [51]. In our study, following retinal ischemia, RGCs displayed DNA damage at the ultrastructural level, evidenced by membrane disruption, nuclear fragmentation, mitochondrial swelling and vacuolization, a diminished number of ribosomes, and other hallmarks of late-stage apoptosis. To measure the activation of caspase, we initially utilized caspase reagents to label its expression in vivo in rats. Our findings revealed a significant elevation in the expression of caspase at the site of ocular injury, with no discernible changes in the corresponding uninjured eye, suggesting a marked activation of the caspase cascade subsequent to ischemic retinal damage. Additionally, heightened levels of caspase-9 and caspase-3 expression in retinal tissue affirmed the initiation of the apoptotic signaling pathway in response to retinal ischemic injury. The protective role of PIP against apoptosis in CNS diseases is well-established. In our results, PIP markedly suppressed the expression levels of caspase-9 and caspase-3. Furthermore, the diminished expression of APE1 following retinal ischemic injury correlates with apoptosis, suggesting the involvement of DNA repair function od APE1 in the mitochondrial apoptotic pathway and the modulation of caspase-9/caspase-3 signaling. Our research reveals that ischemic injury leads to diminished APE1 expression in retinal tissues, which is paralleled by a rise in apoptotic cell counts. Post-PIP intervention, APE1 expression is elevated, whereas the expression of caspase-9 and caspase-3 is suppressed. The activation of caspases is pivotal in the inflammatory response and neuronal apoptosis triggered by ischemia, as evidenced by studies [[52], [53], [54]]. Oxidative stress is also known to contribute to the cell apoptosis signaling pathway that involves caspase-3 in cerebral ischemic injury [55,56]. In our retinal ischemic injury model, a significant reduction in the expression of TNF-α, IL-6, and IL-1β was noted, along with a substantial elevation in IL-10 expression following PIP intervention. Additionally, PIP treatment markedly enhanced the levels of GSH-PX and SOD, while diminishing MDA expression. Significantly, PIP intervention led to a reduction in cell apoptosis within the peripheral blood of rats subjected to ischemic retinal injury. Consequently, this study highlights the potential of PIP in modulating APE1 protein levels and suggests that PIP exerts a protective effect against ischemic retinal injury in rats by modulating the caspase-9/caspase-3 axis.

Retinal ischemic injury in its early stages is characterized by a loss of RPE barrier function [57]. RPE damage can be linked to severe choroidal ischemia [58]. Studies on animals have revealed a significant reduction in tight junction proteins within the RPE under ischemic conditions, suggesting considerable damage to the choroid and BRB [59]. Hypoxia and ischemia decrease the expression of tight junction-related proteins occludin and ZO-1, resulting in increased BRB permeability and subsequent vascular leakage [60]. After ischemic-hypoxic injury, endothelial cells display cytoplasmic vacuolation and vacuolar mitochondria [60]. In this study, ultrastructural analysis disclosed significant vasoconstriction and mitochondrial damage in the optic nerve subsequent to ischemic injury, with a notable reduction in vascular damage post-treatment with PIP. Oxidative stress and apoptosis can trigger retinal inflammation, leading to the impairment of retinal vessels and RGCs [61]. Moreover, mitochondrial dysfunction may be pivotal in the demise of RGCs [62]. Ischemia-induced mitochondrial DNA damage can cause cell apoptosis in the retina [63]. As a result, ischemia-induced damage to RGCs results in axonal injury, further aggravating retrograde degeneration of the optic nerve [64]. Previous research has demonstrated that ischemic injury induces optic nerve demyelination, vascular damage, and axonal degeneration, consequently causing severe retinal damage [65]. Our ultrastructural analysis revealed significant vascular spasm and mitochondrial damage in the optic nerve pathology, coupled with widespread demyelination and heterogeneous axon diameters within the myelin sheath, indicating axonal dysfunction in the optic nerve. Following PIP intervention, a considerable reduction in optic nerve demyelination was observed, suggesting that PIP therapy can effectively mitigate severe axonal injury at this site. Neuronal damage is intimately connected with the apoptotic pathway of mitochondria [66,67]. The death of RGCs leads to optic nerve injury, simultaneously triggering a cascade reaction of caspases [68,69]. Caspase-9 plays a pivotal role in RGC apoptosis in a rat model [70]. Gene therapy utilizing caspase-3 small interfering RNA nanoparticles exhibits neuroprotective effects against optic nerve injury [71]. Moreover, the death of RGCs is closely associated with DNA damage [72]. Our data indicated an increase in apoptotic cells within the optic nerve, along with a decrease in APE1 expression and elevated levels of caspase-9 and caspase-3 subsequent to optic nerve ischemic injury. These observations were consistent with ischemic damage seen in retinal tissue. However, post-treatment with PIP notably alleviated the extent of optic nerve injury, upregulated APE1 expression, and significantly diminished the expression of caspase-9 and caspase-3. These findings suggest that PIP may exert a protective effect on optic nerve injury via the APE1/caspase-9/caspase-3 pathway.

In conclusion, a correlation exists between decreased APE1 expression and retinal ischemic injury. PIP can directly exert a protective effect through interaction with APE1. Animal experiments further confirm that targeting APE1 with PIP effectively reduces cell apoptosis and caspase cascade reactions in retinal ischemic injury, while may further regulating the caspase-9/caspase-3 axis to exert neuroprotective effects in retinal ischemic injury (Fig. 7).

Fig. 7.

A scheme showing the mechanism of neuroprotective action of piperine in retinal ischemic injury.

Limitations and future prospects

Although this study confirms the direct protein interaction between PIP and APE1, which can lead to significant upregulation of APE1 mRNA and protein levels, the specific molecular mechanisms upstream remain to be elucidated. At present, the potential pathways for this upregulation may include PIP acting as a transcription cofactor that, upon entering the nucleus, synergizes with other transcription factors and binds to the promoter region of the APE1 gene, directly enhancing its transcriptional activity. The binding of PIP to APE1 may indirectly affect the activity or expression of an RNA binding protein that can stabilize APE1 mRNA, thereby increasing its abundance by prolonging the half-life of APE1 mRNA. The interaction between PIP-APE1 may activate specific intracellular signaling pathways, and downstream effector molecules of these pathways promote the transcription of APE1 genes. In future research, we will further utilize methods such as reporter gene experiments, RNA immunoprecipitation, and specific signaling pathway inhibitors to distinguish the above possibilities and fully reveal the core role of PIP in regulating cellular DNA damage repair ability.

Author contributions

Qianxiong He: Data charting, conceptualization, investigation, methodology, animal experiments, writing–original draft. Yannan Chen: Methodology, data collation, investigation. Peiwen Chen: Methodology, data collation, and investigation. Yi Wang: Methodology, data collation, data charting, cell culture. Rong Zou: Methodology, animal experiments, data collation. Feng Zhao: Data charting, investigation, methodology. Guangqun Zeng: Data collation, formal analysis. Lin Zhang: Data curation, investigation. Haiping Liu: Data curation, investigation. Yuanjiang Shi: Data collation, software. Liuyi Xiao: Software, formal analysis. Xiaorong Xin: Supervision, conceptualization, investigation, revising–original draft. All authors have read and agreed to the published version of the manuscript.

Declaration of competing interests

These authors have no conflicts of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article.