Combination Therapy with N-Acetylserotonin and Aflibercept Activated the Akt/Nrf2 Pathway to Inhibit Apoptosis and Oxidative Stress in Rats with Retinal Ischemia–Reperfusion Injury

Abstract Purpose N-acetylserotonin (NAS) can reduce retinal ischemia–reperfusion injury (RIRI) by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway. Aflibercept is an anti-VEGF drug used to treat a variety of eye diseases. This study was performed to investigate the effect of combination therapy with N-acetylserotonin and aflibercept on RIRI and its mechanism. Methods The RIRI model was established by elevating the intraocular pressure. H&E staining was used to observe the pathological changes in the retinal tissue. Cell apoptosis was evaluated by TUNEL. The expression of cleaved caspase-3 in the retina was detected by immunofluorescence and western blotting. The levels of SOD, GSH-Px, and MDA in retinal tissue were measured by ELISA. The protein expression of cytoplasmic Nrf2, nuclear Nrf2, HO-1, Akt, and p-Akt was determined by western blotting. Results The results showed that combination therapy with NAS and aflibercept significantly alleviated retinal histopathological damage, decreased retinal thickness (from 335.49 ± 30.50 µm to 226.16 ± 17.20 µm, p < 0.001) and the rate of retinal apoptosis (from 28.27 ± 0.39% to 7.87 ± 0.19%, p < 0.001), and downregulated protein expression (from 2.42 ± 0.03 to 1.39 ± 0.03, p < 0.001) and positive expression (from 31.88 ± 0.52 to 25.36 ± 0.58, p < 0.001) of cleaved caspase-3. In addition, combination therapy with NAS and aflibercept also upregulated the levels of SOD (from 20.31 ± 0.18 to 29.66 ± 0.83, p < 0.001) and GSH-Px (from 13.62 ± 0.36 to 19.31 ± 0.82, p < 0.001) and downregulated the level of MDA (from 0.51 ± 0.01 to 0.41 ± 0.01, p < 0.001) to inhibit oxidative stress. Finally, combination therapy with NAS and aflibercept increased the protein expression of cytoplasmic Nrf2 (from 0.10 ± 0.002 to 0.85 ± 0.01, p < 0.001), nuclear Nrf2 (from 0.43 ± 0.01 to 0.88 ± 0.04, p < 0.001), and HO-1 (from 0.45 ± 0.03 to 0.91 ± 0.04, p < 0.001) and the p-Akt/Akt ratio (from 0.45 ± 0.02 to 0.81 ± 0.07, p < 0.001). Conclusions Overall, combination therapy with NAS and aflibercept attenuated RIRI, and its mechanism may be related to inhibiting apoptosis and oxidative stress and activating the Akt/Nrf2 pathway.


Introduction
Retinal ischemia-reperfusion injury (RIRI) is a common pathological process of many ophthalmic diseases, such as glaucoma, retinal vascular occlusion, and diabetic retinopathy, which often leads to retinal cell death and irreversible damage to the visual function of the patient and is currently the focus of clinical ophthalmology research. 1,2The pathological mechanism underlying RIRI is very complicated and includes oxidative stress, inflammation, and neuronal apoptosis and necrosis. 3,4Further study of the specific mechanism underlying RIRI occurrence and development can provide a theoretical basis for the treatment strategy of RIRI.
N-acetylserotonin (NAS) is a precursor substance of melatonin and can be converted to melatonin for neuroprotective effects. 5It was found that NAS could reduce the apoptosis of neuronal cells in rats with ischemic brain injury and had neuroprotective effects. 6In RIRI, NAS plays an important role in neuroprotection by inhibiting apoptosis, oxidative stress, and inflammatory reactions. 7It has been shown that NAS can reduce IL-1b expression, reduce retinal edema and promote the survival of retinal ganglion cells by inhibiting the TLR4/NF-jB/NLRP3 signaling pathway, thereby reducing retinal damage and exerting neuroprotective effects. 80][11][12] Research has shown that ranibizumab combined with aflibercept prolongs the best corrected visual acuity in neovascular age-related macular degeneration. 13However, studies on NAS combined with aflibercept for RIRI treatment are less commonly reported.
Reactive oxygen species (ROS) can activate a variety of chain reactions that cause damage to the cells themselves and to the retinal tissue through different pathways.Proteins, DNA and lipids can undergo oxidative reactions with ROS, damaging cell structure and function.Studies have shown that withaferin attenuates retinal ischemiareperfusion injury via Akt-dependent inhibition of oxidative stress. 14Nuclear Factor E2-related Factor 2 (Nrf2) is a key transcription factor that regulates the antioxidant defense system and mainly activates the expression of downstream genes, such as HO-1 (heme oxygenase 1) and NAD(P)H: quinone oxidoreductase 1 (Nqo1).Among these downstream genes, HO-1 is an antioxidant enzyme that plays a key role against endogenous sources.In addition, acetylation of Nrf2 promotes its transcriptional activity and activates the expression of downstream target genes, which has been demonstrated in animal models of oxidative stress-associated diseases. 15It has been shown that tetrahedral framework nucleic acids prevent retinal ischemia-reperfusion injury from oxidative stress by activating the Akt/Nrf2 pathway. 16n this study, a RIRI model was established by increasing IOP, and the effects of combination therapy with NAS and aflibercept on apoptosis, oxidative stress, and the Akt/Nrf2 pathway were discussed to provide a theoretical basis for the treatment of RIRI.

Animals
Forty-two male SD rats (aged 2-3 months and weighing 180-200 g) were purchased from Chengdu Dashuo Biological Co., Ltd.(Sichuan, China).The rats were placed in an isolated environment with a temperature of 22-24 � C and humidity of 40-60% in a 12 h light þ dark cycle and were provided with clean drinking water and chow.In this study, all animal experiments were conducted according to the ethical standards of experimental animals (Ethics Committee of West China Hospital, Sichuan University, ID: 20220114001).

RIRI model
The RIRI model was established by raising the intraocular pressure. 17Rats were injected intraperitoneally with 3 mL/kg pentobarbital sodium and fixed on the operating table after successful anesthesia.Compound tropicamide eye drops were added to the right eye to dilate the pupil, and one drop of 0.4% oxybuprocaine hydrochloride was added for corneal anesthesia.The microsyringe (including normal saline) was inserted into the anterior chamber from the temporal corneal limbus of the right eye of the rat, and the infusion bottle was elevated 165 cm from the plane of the eye by fixing the needle so that the intraocular pressure reached 60 mmHg (1 kPa ¼ 7.5 mmHg).At this point, the conjunctiva was observed to be pale, and fundoscopy showed that retinal blood flow was blocked, indicating retinal ischemia formation.After 1 h, the height of the infusion bottle was slowly lowered to the level of the eye, and the needle was removed.Then, the retinal color gradually returned to orange, and the blood flow gradually returned to normal, indicating the success of the RIRI model.In the control group, only a microinjector was used to pierce the anterior chamber of the rats without raising intraocular pressure.

Animal grouping
The rats were randomly divided into 4 groups: control group (n ¼ 6), model group (n ¼ 12), NAS group (n ¼ 12), and NAS þ aflibercept group (n ¼ 12).Rats in the model and control groups were injected intraperitoneally with equal amounts of saline.Rats in the NAS group were injected intravenously with 10 mg/kg NAS (Sigma-Aldrich, USA) 30 min before modeling.The rats in the NAS þ aflibercept group (combination therapy with NAS and aflibercept) were injected with aflibercept (specifications: 40 mg/mL, Vetter Pharma-Fertigung GmbH & Co. KG, Germany) in the vitreous cavity at a distance of 3.5 mm between the superior temporal and corneoscleral margins at a dose of 0.05 mL.After the injection, a cotton swab was pressed, and antibiotic eye ointment (tobramycin) was applied to the eyes.Aflibercept was administered every 4 weeks for 3 months.Rats were sacrificed by cervical dislocation, and retinal tissues were collected for subsequent experiments.

H&E staining
Retinal tissues were fixed in 10% formalin, and paraffin sections (4 mm) of retinal tissues were routinely prepared.First, the sections were dewaxed in xylene, dehydrated in gradient ethanol, and rinsed with tap water.Next, the sections were stained with hematoxylin for 5 min, fractionated in 1% hydrochloric acid for 3 s, and returned to blue for 30 min.Then, eosin was restained for 2 min, hydrated in gradient ethanol, treated with xylene transparency, and sealed with neutral gum.Finally, the retinal histomorphology was observed under a light microscope, and the retinal thickness of each group of rats was evaluated.

TUNEL staining
Paraffin sections of retinal tissue were dewaxed, dehydrated and incubated with Proteinase K working solution (20 mg/ ml) for 20 min at room temperature.The sections were washed three times with PBS, incubated in TUNEL working solution for 1 h at room temperature protected from light and then washed three times in PBS.Finally, the sections were sealed with an anti-fluorescence quencher containing DAPI (Thermo, USA) and observed under a fluorescence microscope, and the apoptosis rate was analyzed by ImageJ software.

Immunofluorescence
Retinal paraffin sections were dewaxed, hydrated, repaired with antigen (H 2 O 2 ), and blocked with sheep serum at room temperature.Sections were incubated with primary antibody (Biorbyt, UK) overnight at 4 � C in a wet chamber protected from light and rinsed three times with PBS.Then, the sections were incubated with secondary antibody for 2 h at 37 � C in a wet chamber protected from light and rinsed three times with PBS.Finally, an anti-fluorescence quenching agent containing DAPI was used to seal the slices, and images were collected for fluorescence microscopy.

Western blotting
Retinal tissues were lysed with RIPA lysis buffer (Abcam, Ab156034, UK) and centrifuged, total protein was extracted, and the protein concentration was determined by a Pierce TM BCA Protein Assay Kit (Thermo Scientific).The target protein was separated by SDS-PAGE electrophoresis and then transferred onto a PVDF (polyvinylidene fluoride) membrane for the transmembrane reaction.The membrane was blocked with 5% skim milk powder (diluted with Tris buffer) for 2 h and then incubated overnight at 4 � C with primary antibodies against Nrf2 (ab31163, Abcam, USA), HO-1 (ab1324, Abcam, USA), Akt (ab132384, Abcam, USA), cleaved caspase-3 (ab32351, Abcam, USA), Akt (ab132384, Abcam, USA) and p-Akt (ab38449, Abcam, USA).Subsequently, a secondary goat antirabbit IgG (HRP) antibody (ab205718, Abcam, USA) was incubated with the membranes at room temperature for 1 h.The blots were assayed by using the Novex TM ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific TM , WP20005, USA).b-actin and histone H3 were used as internal reference proteins.ImageJ software was used to quantify the relative expression of the target protein bands.

ELISA
The retinal tissues of rats in each group were prepared as homogenates and centrifuged at 3500 r/min for 10 min, and the supernatants were taken.The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were measured using ELISA kits (Nanjing Jiancheng Bioengineering Institute, China) according to the instructions.

Statistical analysis
The experimental data were statistically analyzed with SPSS 17.0 statistical software and GraphPad Prism 7 software.All the data are expressed as the mean ± standard error (mean ± SD).One-way ANOVA (one-way analysis of variance) was used for comparisons between more groups, the LSD t test (least significant difference) was used for homogeneity of variance, and the Dunnett t test was used for heterogeneity of variance.P values < 0.05 were considered to be significant.

Combination therapy with NAS and aflibercept weakened retinal pathological lesions in RIRI rats
To observe the effect of combination therapy with NAS and aflibercept on retinal pathological changes after RIRI, retinal histopathological changes were detected by H&E staining.The results showed that the retinal cells were arranged orderly and regularly in the control group and were sparsely and disorderly, irregular in shape, and thinner in the inner retinal layer in the model group (Figure 1A).NAS treatment alleviated the pathological injury of RIRI, and the effect of combination therapy with NAS and aflibercept was better.The retinal thickness was significantly increased in the model group, NAS treatment reduced the retinal thickness, and the effect of combination therapy with NAS and aflibercept was more significant (Figure 1B).

Effect of combination therapy with NAS and aflibercept on retinal cell apoptosis in RIRI rats
In RIRI, apoptosis is one of the major forms of injury.Cleaved caspase-3 is an important indicator of the severity of apoptosis.To assess the effect of combination therapy with NAS and aflibercept on apoptosis in RIRI, TUNEL staining was used to measure the cell apoptosis rate, and western blotting and immunofluorescence staining were used to measure the expression of cleaved caspase-3.TUNEL staining results showed that the apoptosis rate was significantly increased in the model group and that NAS treatment decreased the apoptosis rate.When NAS was combined with aflibercept, the inhibitory effect on the cell apoptosis rate was more significant (Figure 2A).The western blotting and immunofluorescence staining results (Figure 2B,C) suggested that the protein expression and positive expression of cleaved caspase-3 were significantly increased in the model group, and NAS treatment decreased the expression of cleaved caspase-3.Compared with that in the NAS group, cleaved caspase-3 expression was significantly reduced in the NAS þ aflibercept group.Thus, combination therapy with NAS and aflibercept inhibited apoptosis in RIRI rats.

Effect of combination therapy with NAS and aflibercept on oxidative stress in RIRI rats
To elucidate the effect of combination therapy with NAS and aflibercept on oxidative stress in RIRI, the levels of SOD, GSH-Px and MDA were measured by ELISA.The results showed that the levels of SOD and GSH-Px were significantly decreased and that the level of MDA was significantly increased in the model group, while NAS treatment upregulated SOD and GSH-Px levels and downregulated MDA levels.Compared with those in the NAS group, SOD and GSH-Px levels were significantly upregulated and MDA levels were markedly downregulated in the NAS þ aflibercept group (Figure 3).These data suggested that combination therapy with NAS and aflibercept has a stronger antioxidant effect on RIRI.

Effect of combination therapy with NAS and aflibercept on the Akt/Nrf2 pathway in RIRI rats
The protein expression of cytoplasmic Nrf2, nuclear Nrf2, HO-1, Akt, and p-Akt was measured by western blotting to investigate the effect of combination therapy with NAS and aflibercept on the Akt/Nrf2 pathway in RIRI rats.The results showed that the protein expression of cytoplasmic Nrf2, nuclear Nrf2 and HO-1 and the p-Akt/Akt ratio were significantly decreased in the model group.Nevertheless, NAS treatment upregulated the protein expression of cytoplasmic Nrf2, nuclear Nrf2 and HO-1 and the p-Akt/Akt ratio.Compared to those in the NAS group, the protein expression of cytoplasmic Nrf2, nuclear Nrf2 and HO-1 and the p-Akt/Akt ratio were significantly upregulated in the NAS þ aflibercept group (Figure 4A,B).These data suggested that combination therapy with NAS and aflibercept could activate the Akt/Nrf2 pathway in RIRI rats.

Discussion
Retinal ischemia-reperfusion injury (RIRI) is the pathological basis of many ophthalmic diseases, and timely restoration of blood supply to the retina, after a short period of ischemia, can prevent visual impairment, while prolonged ischemia can aggravate RIRI and bring serious health risks to people. 18The elevated intraocular pressure method is an animal model frequently used to study RIRI. 19,20This process causes insufficient blood flow to the eye or complete blockage of blood supply in the eye, resulting in RIRI. 21his model is highly manipulable, can completely block retinal blood flow, has a stable process, has a high success rate, and causes less damage to other tissues of the retina.In this study, the retinal thickness of RIRI rats was significantly increased, and the retinal cells were sparsely arranged, disordered, and irregularly shaped, suggesting successful model construction.Nevertheless, combination therapy with NAS and aflibercept alleviated retinal morphological damage.
Apoptosis is considered to be the main mode of neuronal cell injury in RIRI. 22Cleaved caspase-3 is a major executive protein involved in the regulation of apoptosis and plays an important role in the pathogenesis of ophthalmology-related diseases. 23,24Huoxue-Tongluo-Lishui-Decoction treatment significantly reduced cleaved caspase-3, cleaved PARP and caspase-3 activity at 48 h after reperfusion. 25Ribonuclease therapy can effectively attenuate retinal damage by reducing the inflammatory response and apoptosis. 26Our results agreed with previous research.Both the apoptotic rate and expression of cleaved caspase-3 were significantly increased in retinal tissue after RIRI.Combination therapy with NAS and aflibercept significantly reduced the apoptosis rate and cleaved caspase-3 expression.Thus, combination therapy with NAS and aflibercept inhibited apoptosis in RIRI rats.
Several studies have also suggested that oxidative stress is an important pathophysiological mechanism of retinal I/R injury. 27These three indicators (SOD, GSH-Px and MDA) determine the degree of oxidative stress and further reflect the severity of tissue damage.SOD removes superoxide anions by catalyzing their generation of O 2 and H 2 O 2 .GSH-Px catalyzes the reduction of hydrogen peroxide by glutathione.MDA is an end product of lipid oxidation.Studies have demonstrated that resveratrol protects against I/R-induced retinal ganglion cell loss and endothelial dysfunction in the murine retina by reducing nitro-oxidative stress, possibly via suppression of NOX2 upregulation. 28Our results suggested that combination therapy with NAS and aflibercept upregulated SOD and GSH-Px levels and downregulated MDA levels.Accordingly, combination therapy with NAS and aflibercept had a significant antioxidant effect.
Many reports have revealed that Nrf2 is a new resistant oxidant stress factor. 29A considerable number of studies have demonstrated that Nrf2 promotes the expression of phase II detoxification and antioxidant response elements, such as glutathione synthase, HO-1, and catalase, under oxidative stress and activates the PI3K/Akt pathway when oxidative stress occurs. 30Gastrodin protects retinal ganglion cells from ischemic injury by activating the PI3K/AKT/Nrf2 signaling pathway. 31In this study, combination therapy with NAS and aflibercept dramatically increased the protein expression of cytoplasmic Nrf2, nuclear Nrf2, and HO-1 and the p-Akt/Akt ratio.In summary, combination therapy with Values are expressed as the mean ± SD. ��� p < 0.001 vs. control group; ### p < 0.001 vs. model group; @ p < 0.05, @@@ p < 0.001 vs. NAS group.
NAS and aflibercept activated the Akt/Nrf2 pathway in RIRI rats.
In conclusion, combination therapy with NAS and aflibercept activated the Akt/Nrf2 signaling pathway to inhibit apoptosis and oxidative stress in RIRI rats.However, the limitation of this study was that it did not use an Akt/Nrf2 pathway inhibitor to further validate that combination therapy with NAS and aflibercept regulated the Akt/Nrf2 pathway to attenuate RIRI.In a follow-up study, we will further explore this aspect (using Akt/Nrf2 pathway inhibitors).