Exendin-4, a GLP-1 receptor agonist, suppresses diabetic retinopathy in vivo and in vitro

Abstract

Diabetic retinopathy (DR) is a complication of diabetes and a leading cause of blindness in adults. Studies have shown that glucagon-like peptide-1 (GLP-1) exerts a protective effect on patients with DR. Here, we investigated the protective effects of Exendin-4, a GLP-1 analogue, on DR. We established a high-glucose-induced HREC cell model and an STZ-induced rat DR Model to study the effect of Exendin-4 in DR in vitro and in vivo. The qRT-PCR, CCK-8, TUNEL, western blotting, tube formation assays, and ELISA were performed. In addition, we overexpressed TGFB2 to observe whether the protective effect of Exendin-4 was reversed. Our results showed that Exendin-4 inhibited the progression of DR. Furthermore, the protective effect of Exendin-4 was suppressed in cells overexpressing TGFB2. Our findings suggest that Exendin-4 may be involved in the regulation of TGFB2 expression levels to inhibit DR. These results indicate that Exendin-4 could be an effective therapy for DR.

Keywords:

• Diabetic retinopathy
• GLP-1
• Exendin-4

Introduction

Diabetic retinopathy (DR) is one of the most common and serious complications of diabetes mellitus (DM) and can lead to blindness in patients (Leasher et al. 2016, Roy et al. 2017, Hu and Jia 2018). The prevalence of DR increases with the growing number of diabetic patients and their extended lifespans (Cheung et al. 2010, Solomon et al. 2017). DR ranks first among diseases causing blindness in the working population; however, the mechanism of DR pathogenesis is complex and remains unclear (Jenkins et al. 2015). DR includes lesions in retinal microcirculation, retinal nerve degeneration, and optic neuropathy (Vujosevic et al. 2020, Lin et al. 2021, Fung et al. 2022). Therefore, retinal neurodegeneration must be prevented and treated in addition to improving retinal microcirculation and hypoxia. DR is characterised by retinal neovascularization, and inflammatory processes largely contribute to the early stages of DR. Currently, several therapeutic drugs for DR have been reported, with their primary effects being anti-VEGF and anti-inflammatory actions (Lazzara et al. 2019).

Glucagon-like peptide-1 (GLP-1) is an endogenous approximately 30 amino-acid polypeptide secreted by gastrointestinal L-cells, which can be used for the treatment of DM (Bethel et al. 2021). GLP-1 stimulates insulin secretion by pancreatic β-cells. However, it inhibits glucagon secretion by α-cells by binding to the GLP-1 receptor on α-cells, reducing the blood sugar concentration (D'Alessio 2016). GLP-1 also increases insulin sensitivity of the liver and adipose tissues and reduces pancreatic resistance to control blood sugar (Kim et al. 2022). In addition to the pancreas, GLP-1 receptors are expressed in the heart, intestine, kidney, brain, and retinal tissues (Drucker 2001, Holst 2002, List et al. 2006, Schlatter et al. 2007, Zhai et al. 2020). GLP-1 receptor has been reported to be expressed in rat retinal capillary endothelial cells and cultured HRECs (Zhai et al. 2020). Therefore, GLP-1 and its receptor agonists are believed to play an extra-pancreatic role and are used for the treatment of DM. Liraglutide, a GLP-1 receptor agonist, has been reported to protect the optic nerve by preventing glutamate accumulation and reversing the downregulation of glutamate/aspartate transporters induced by diabetes (Xie et al. 2012). In addition, there was no significant difference in the electroretinography results of diabetic mice following treatment with exenatide, another GLP-1 agonist. Moreover, the retinal thickness and cell count of diabetic mice treated with exenatide showed notable improvement compared to those of untreated diabetic mice (Zhang et al. 2009).

Exendin-4 (Ex4), a GLP-1 receptor agonist found in the saliva of the Gila monster venomous lizard, shares a 53% sequence homology with GLP-1 (Guerci and Martin 2008, Zhang et al. 2009). The majority of research into the protective mechanisms of Ex4 has centred around neurodegenerative diseases, including Alzheimer’s disease and diabetic peripheral neuropathy (Song et al. 2022). Ex4 has demonstrated its neuroprotective properties in vitro by protecting neurons against glutamate-induced apoptosis (Perry and Greig 2005). Ex4 alleviates H₂O₂-induced oxidative damage in ARPE-19 cells by activating the NRF2-signalling pathway. Therefore, it can be used as a potential drug to treat AMD (Cui et al. 2020). In addition, Ex4 stimulates pancreatic insulin secretion, enhances islet sensitivity, and improves islet cell function. These promising effects hold great potential for the treatment of DM (Mayendraraj et al. 2022). Currently, several studies have confirmed the promise of Ex4 in treating DR. Research has indicated that in diabetic mice, GLP-1 receptor agonists reduce blood glucose, lipid levels, and body weight, while simultaneously improve retinal thickness, morphology, and vascular ultrastructure; thereby improving the manifestations of DR (Zhang et al. 2009). Intravitreal administration of Ex4 can prevent retinal damage in diabetic rats both functionally and morphologically (Zhang et al. 2011). Although Ex4 has been confirmed to have a protective effect in rats (Fan et al. 2014), the molecular and cellular mechanisms mediating this effect are not fully understood. Therefore, we aimed to explore the molecular and cellular mechanisms of Ex4 on DR and analyse its potential therapeutic effects.

The transforming growth factor-β (TGF-β) family plays a significant role in various cellular biological processes, including inflammation (Caruso et al. 2019). Up-regulation of TGFB2, one of TGF-βmembers, has been reported to inhibit the proliferation of retinal endothelial cells (Yafai et al. 2014). In addition, knocking down TGFB2 expression reduce the secretion of pro-inflammatory cytokines (Tang et al. 2017) and lower endothelial angiogenesis activity. Endothelial angiogenesis is directed by (Liao et al. 2017). In patients with DR, overexpression of TGFB2 correlates with the levels of matrix metalloproteinase-2 (MMP2), MMP9, and vascular endothelial growth factor (VEGF), suggesting that TGFB2 may be a potential treatment for DR (Van Geest et al. 2013).

It has been previously reported that Ex4 reverses the retinal function in diabetic rats, prevents the death of retinal cells, and maintains the normal thickness of the retina, proposing that Ex4 could be an effective approach for the early treatment of DR (Zhang et al. 2009). However, further studies are required to test this hypothesis. Moreover, the mechanism by which Ex4 protects against DR is not fully understood. This study aimed to elucidate the protective role of Ex4 in DR and its mechanism of action using both in vivo and in vitro model systems. These findings will improve our understanding of the fundamental mechanisms of Ex4 action during DR treatment.

Materials and methods

Cell culture and transfection

Human retinal microvascular endothelial cells (HRECs) were purchased from the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences and cultured in an endothelial cell medium (ScienCell, USA) supplemented with 1% endothelial cell growth supplement (ScienCell) and 5% FBS, 5% CO₂, and 37 °C. In the treatment group, HRECs were cultured in the medium containing high glucose (HG) (30 mM), while the medium in the control group contained normal glucose (NG) levels (5 mM). Full-length TGFB2 was cloned into the pcDNA3.1 vector (GenePharma, Shanghai, China); an empty pcDNA3.1 vector was used as a control. HRECs were transfected with plasmids using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. All cells were cultured overnight in 60 mm culture dishes. Subsequently, HG cells were treated with 0, 0.1, 1, 10, 20, and 40 µM Ex4. The Ex4 was purchased from AnaSpec (Fremont, CA, USA).

RT-qPCR

Total RNA was extracted from the collected peripheral blood samples or cultured HRECs using the TRIzol reagent according to the manufacturer’s instructions. Amplification was performed using the SYBR Green PCR Master Mix Kit (Applied Biosystems, USA) on a 7900HT Fast Real-Time System (Applied Biosystems). The relative expression of TGFB2 was calculated using the 2^−ΔΔCt method, and U6 was used as an endogenous control. The primer sequences used were as follows:

TGFB2 F: 5′-TCCATCTGTGAGAAGCCACA-3′,

R: 5′-GGGTCATGGCAAACTGTCTC-3′;

U6 F: 5′-GCTTCGGCAGCACATATACTAAAAT-3′, R: 5′-CGCTTCACGAATTTGCGTGTCAT-3′

CCK-8 assay

Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was used to evaluate the viability of HRECs in response to different treatments. Briefly, treated HRECs were plated in 96-well plates at approximately 3000–5000 cells/well. CCK-8 solution (10 µL) was added to the cells cultured for 0, 24, 48, and 72 h. Absorbance was measured at 450 nm after 2 h of incubation.

ELISA

IL-1β, IL-6, and TNF-α levels were determined using ELISA kits (R0015c, R0567c, and R2856c, Elabscience Biotechnology, Wuhan, China) following the manufacturer’s instructions. Each sample was independently analysed three times.

Tube formation assay

The tube formation assay was performed according to the manufacturer’s instructions (CBA-200, Cell Biolabs, USA). Cells were plated on the extracellular matrix component gel and incubated for 4–18 h, and tube formation parameters, such as length and node number, were determined using an optical microscope.

TUNEL staining

A cell death in situ assay kit (code:11684795910, Roche, Basel, Switzerland) was used to evaluate apoptosis in the HRECs. Fitc-labelled nuclei appeared positive (green), whereas DAPI-stained nuclei appeared positive (blue) under a forward microscope. Apoptosis was assessed by counting the number of positive (green) and total (blue) nuclei in five random fields using the Image-Pro Plus 6.0 software.

Animals

Male Sprague–Dawley rats (6–8 weeks old) were purchased from Shanghai Slack Laboratory Animal Co., Ltd. (Shanghai, China). All the surgeries were performed under aseptic conditions. The experimental and animal care procedures were approved by Shangyu People’s Hospital of Shaoxing City (Approval Number. 2022YB032) and conformed to the Guide for the Care and Use of Laboratory Animals produced by the National Institutes of Health.

Establishment of animal model

Streptozotocin (STZ; Sigma, USA) was dissolved in a freshly prepared sodium citrate buffer (0.1 mM, pH 4.5). Fifteen SD rats were randomly divided into a control group (injected with citrate buffer, n = 5) and a model group (intraperitoneally injected with STZ (60 mg/kg, n = 10)). Diabetes was diagnosed when blood glucose exceeded 250 mg/dL for 3 consecutive days after STZ induction. After 10 weeks of induction, all rats within the model group were diagnosed with diabetes accompanied by DR. The model group rats were randomly divided into DR (n = 5) and DR + Ex4 (n = 5) groups. All model groups of rats continued STZ induction as previously established, while rats in the DR + Ex4 group were administered a subcutaneous injection of Ex4 at 60 mg/kg weekly for six weeks (Zhang et al. 2009, Zhai et al. 2020, Wei et al. 2022), while control and DR groups were injected with saline solution. Blood samples were obtained from the tail veins of rats, and serum was collected and stored at −80 °C for qRT-PCR detection. At the end of the experiment, the rats were euthanised in a CO₂ chamber. The retina was dissected and a section of the tissue was fixed with 10% neutral-buffered formaldehyde for 24 h for pathological evaluation, while the remainder was used for western blotting.

Immunohistochemistry

The collected retinae were embedded in paraffin, sectioned, and 5 µm retinal sections were dewaxed and rehydrated as previously described (Zhai et al. 2020). The sections were washed with PBS and incubated overnight at 4 °C with an anti-VEGF-164 monoclonal antibody (1:100; ab52917; Abcam; China). Subsequently, the sections were washed three times in PBS for 5 min and incubated with biotinylated goat anti-rabbit IgG (1:1000; ab150077; Abcam, China) at room temperature for 20 min. Sections were washed with PBS, and the avidin-horseradish peroxidase complex was added (SABC Kit; Bost, Wuhan, China). The 3,3-diaminobenzidine (DAB) was used as a chromogen. The reaction was terminated by adding water. Finally, the sections were washed, and dehydrated, and the cells were visualised under a light microscope (LM).

Western blot

Proteins from HRECs or rat retinae were extracted and separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). A Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used to quantify protein concentrations in the supernatants. Equal amounts (20 μl) of protein were loaded onto the gels, and separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in tris buffered saline containing 5% Tween 20 (TBST) and 5% skim milk at room temperature for 1 h and incubated overnight at 4 °C with a primary antibody (anti-TGF-β2, 1:1000, Abcam, China; anti-VEGF, 1:1000, ab52917, Abcam, China; anti-MMP-2, 1:1000, ab51075, Abcam, China; anti-MMP-9, 1:1000, ab76003, Abcam, China). Primary antibodies were detected using a secondary anti-mouse or anti-rabbit IgG antibody coupled with HRP (1:5000, ab6728, Abcam, China). The target proteins were visualised using an EZ-ECL chemiluminescence detection kit (Pierce, Rockford, IL, USA).

Statistical analysis

Data were analysed using GraphPad Prism 8.0 software (GraphPad Software, Inc.). Results are presented as the mean ± standard deviation (SD). Student’s t-test was used for comparisons between the two groups. Differences among multiple groups were analysed using a one-way analysis of variance (ANOVA). Differences were considered statistically significant at P < 0.05.

Results

Exendin-4 alleviates HG-induced cell injury

HRECs were treated with different concentrations of Ex4, and CCK-8 assay results showed that cell viability declined in response to HG. However, Ex4 rescued the viability of HG-treated cells in a dose-dependent manner, with 20 µM Ex4 exerting the strongest effect. Therefore, Ex4 (20 µM) was used in subsequent experiments (Figure 1(A)). Subsequently, we used the TUNEL assay to evaluate apoptosis in HRECs. Our results showed that apoptosis was induced in HRECs treated with HG for 24 h, while Ex4 partially reversed this effect (Figure 1(B)). The tube formation assay showed that in the HG group, the tube-forming ability of cells was enhanced; however, it was partially decreased after Ex4 treatment (Figure 1(C)). ELISA results demonstrated that TNF-α, IL-1β, and IL-6 concentration in cells increased after HG induction and decreased after Ex4 treatment (Figure 1(D)). Western blotting results showed that VEGF protein levels were upregulated in HG-induced cells and downregulated after Ex4 treatment (Figure 1E).

Figure 1. Exendin-4 alleviates HG-induced cell injury. (A) HRECs were treated with different concentrations of Ex4, and cell viability was determined using the CCK-8 assay. (B) The apoptosis of HRECs was evaluated using the TUNEL assay. DAPI staining indicates the cell nucleus (blue); Fitc-labelled nuclei appeared positive (green). (C) The tube formation ability of HRECs was determined using the tube formation assay. (D) The levels of inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, in HRECs, were detected using ELISA. (E) VEGF protein levels in HRECs were detected using western blotting. * Comparison with NC, P < 0.05.

Exendin-4 alleviates injury in DR rats

First, we established a diabetic rat model using STZ induction and measured body weights and blood sugar levels at 0, 2, 4, 6, 8, and 10 weeks. The results showed that the rats in the DR group had significantly lower weights and higher blood glucose levels than those of the control group, confirming that the rat model was successfully established (Figure 2(A)). Immunohistochemistry results demonstrated that VEGF expression was significantly increased in the retinal tissues of DR rats. However, it partially decreased after Ex4 treatment (Figure 2(B)). In addition, the TNF-α, IL-1β, and IL-6 levels in the peripheral blood of DR rats were elevated, while Ex4 treatment partially reduced the expression levels of these inflammatory cytokines (Figure 2(C)).

Figure 2. Exendin-4 alleviates STZ-induced effects in DR rats. (A) Body weight and blood glucose levels in normal and STZ-induced diabetic rats were measured at 0, 2, 4, 6, 8, and 10 weeks to confirm the successful establishment of the DR model. (B) The expression levels of VEGF in the rat retina were detected using immunohistochemical staining. (C) The expression of inflammatory cytokines TNF-α, IL-1β, and IL-6 in rat peripheral blood was evaluated using ELISA. n = 5, comparison with NC,* P < 0.05.

Ex4 involves the expression of TGFB2 in DR

We evaluated whether Ex4 alters TGFB2 expression in vivo and in vitro. The results showed that TGFB2 expression increased after HG induction, while Ex4 treatment decreased TGFB2 expression (Figure 3(A)). The expression levels of TGFB2 in the peripheral blood and retinae were detected using qRT-PCR and western blotting, respectively. Our results demonstrated that both protein and gene expression levels of TGFB2 were increased in DR rats, whereas Ex4 treatment reversed this effect (Figure 3(B,C)).

Figure 3. Exendin-4 is involves in the TGFB2 expression regulation. (A) The protein expression level of TGFB2 in HRECs was detected using western blotting. (B) and (C) The expression of TGFB2 in the rat peripheral blood and retinae was evaluated using qRT-PCR and western blotting. n = 5, comparison with NC,* P < 0.05.

TGFB2 reverses the protective effect of exendin-4 on DR

It was reported that transforming TGFB2 was involved in the pathogenic process of DR. Therefore, we wanted to investigate whether Ex4 is involved in regulating TGFB2 in altering the progression of DR at the cellular level. To confirm the association between Ex4 and TGFB2, we performed TGFB2-overexpression experiments. The CCK-8 assay results showed that TGFB2 reversed the effect of Ex4 treatment on the HG-induced cell viability (Figure 4(A)). The inhibition of HG-induced apoptosis by Ex4 is also reduced by TGFB2 overexpression (Figure 4(B)). Moreover, the effect of Ex4 treatment on HG-induced tube-formation ability was reversed by TGFB2 overexpression (Figure 4(C)). Finally, the TNF-α, IL-1β, and IL-6 levels in HG-induced cells decreased in response to Ex4 reversed by TGFB2 overexpression (Figure 4(D)). A similar effect was observed for VEGF protein expression levels (Figure 4(E)).

Figure 4. Exendin-4 inhibits DR progression via TGFB2. (A) The viability of HRECs overexpressing TGFB2 and treated with Ex4 was determined using the CCK-8 assay. (B) The apoptosis of HRECs were evaluated using the TUNEL assay. DAPI staining indicates the cell nucleus (blue); Fitc-labelled nuclei appeared positive (green). (C) The tube formation of HRECs was determined using the tube formation assay. (D) The expression of inflammatory cytokines TNF-α, IL-1β, and IL-6 in HRECs was evaluated using ELISA. (E) VEGF protein expression in HRECs was detected using western blotting. Comparison with NC, * P < 0.05.

Discussion

One of the key interventions for DR is controlling blood sugar levels. However, even with the blood sugar control at the normal level, the disease will continue progressing due to the “metabolic memory” of DM. Therefore, it is important to identify a treatment method that reduces blood sugar levels and delays or even reverses the progression of DR. GLP-1 can inhibit glucagon secretion by α-cells by binding to the GLP-1 receptor on these cells, thereby reducing blood sugar concentration (Meier 2012). As a long-acting agonist of the GLP-1 receptor, Ex4 has been shown to have neuroprotective properties in vitro and to be involved in retinal repair in rats with retinal injury (Zhai et al. 2020). Previous studies have reported that Ex4 can prevent retinal cell death in diabetic rats and maintain normal retinal thickness. Therefore, have proposed that Ex4 could be a candidate for the treatment of DR (Zhang et al. 2009). In this study, we further tested the effect of Ex4 on the development of DR using both in vivo and in vitro model systems.

First, we explored the effect of Ex4 on the viability and apoptosis of HRECs cultured under HG conditions. Our results showed that Ex4 alleviated cell viability and apoptosis affected by HG treatment. Vascular dysfunction induced by retinal vascular remodelling and inflammation is thought to be the primary cause of DR (Ding and Wong 2012, Gong et al. 2022). In our study, we observed that Ex4 reduced HG-induced cell-tube formation and the expression levels of TNF-α, IL-1β, and IL-6. These results suggest that Ex4 has a positive therapeutic effect on hyperglycaemia-induced retinal endothelial cells in vitro and has the potential to treat DR. Second, we investigated the effects of Ex4 on DR using a rat model. It has been reported that Ex4 has a significant hypoglycaemic effect (Zhang et al. 2009). Consistent with previous studies, we observed the hypoglycaemic properties of Ex4 in our rat model. In addition, Ex4 reduced the expression levels of inflammatory and angiogenic factors, such as VEGF, in the retinal tissues of DR rats. These results confirm that Ex4 has potential therapeutic properties in vivo.

Several reports describe the mechanism by which Ex4 inhibits the development of DR. Therefore, this study aimed to identify possible Ex4 target proteins in HRECs. TGFB2 has been reported to be associated with DR (Gong et al. 2022) and involved in fibrosis, vascularisation, and apoptosis induction (Grant et al. 2004; Simó et al. 2006). In this study, TGFB2 expression was elevated in HG-induced HRECs and decreased after Ex4 treatment. Similar results were observed in a rat model of DR, suggesting that Ex4 could affect TGFB2 expression during the inhibition of DR. To test this hypothesis, we overexpressed TGFB2, and our results demonstrated that the effect of Ex4 on HG-induced cell viability, apoptosis, angiogenesis, and the expression of inflammatory factors was inhibited by TGFB2 overexpression. These results could provide a possible explanation for the mechanism by which Ex4 inhibits DR development.

This study has some limitations. First, prolonged intraocular hyperglycaemia triggers the development of DR. At present, the conclusion of this study is that Ex4 inhibits DR by lowering blood glucose. Whether Ex4 directly affects the progression of DR and its comprehensive molecular mechanism needs to be explored in further experiments. Second, we found that the protective effect of Ex4 against DR was related to the regulation of TGFB2 expression. Another member of the TGF family, TGFB1, is abnormally expressed in the serum of patients with DR and may also be an important protein in the progression of DR (Bonfiglio et al. 2020). Whether the molecular mechanism of Ex4 in DR therapy is related to TGFB1 requires further exploration. In addition, the optimal concentration of Ex4 will need to be explored more accurately in the future.

In conclusion, our study provides evidence suggesting that Ex4 treatment inhibits the development of DR. Moreover, we investigated the association between Ex4 and TGFB2 in DR and showed that Ex4 is involved in the regulation of TGFB2 expression, which could potentially contribute to the inhibition of DR. However, additional in vivo and in vitro experiments are required to confirm this mechanism of action. Collectively, these findings could provide additional potential options for the treatment of DR.

Disclosure statment

No potential conflict of interest was reported by the authors.

Acknowledgements

Not applicable.

Data sharing statement

The data sets used and analysed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.