Liraglutide Up-regulation Thioredoxin Attenuated Müller Cells Apoptosis in High Glucose by Regulating Oxidative Stress and Endoplasmic Reticulum Stress

ABSTRACT Purpose: Diabetic retinopathy (DR) has become one of the most important complications of diabetes which is the leading cause of vision impairment and blindness all over the world. Increasing evidence shows that reactive gliosis are basic pathological features of early DR. The study was aimed to explore the protective effect and mechanism of Liraglutide (LIRA) which has similar properties to Glucagon-like peptide-1 (GLP-1) on Müller cell damage induced by diabetes. Materials and methods: In vitro, the Müller cell was cultured in high glucose (HG) to establish the model of diabetic retinopathy. The apoptosis was detected using flow cytometry. Western blot and immunofluorescence were used to detect the expression of related proteins. DCFH-DA probe was used to detect the ROS generation. Results: The data showed that the apoptosis and the expression of GFAP were increased significantly with HG treatment. However, the apoptosis percentage and the expression of GFAP were decreased after LIRA treatment. Moreover, the expression of p-Erk/Nrf2/Trx-signaling pathway proteins was also up-regulated and the generation of ROS was decreased after LIRA treatment which was inhibited after treatment with U0126 (Erk inhibitor). Besides, endoplasmic reticulum stress (ER stress) related proteins were up-regulated after Trx down-regulation by transfection with sh-RNA. Conclusions: LIRA could protect Müller cells from HG-induced damage via activating p-Erk pathway through increasing Trx expression which attenuated oxidative stress and ER stress. Trx could play a key role in the process.


Introduction
Diabetic retinopathy (DR) has become the leading cause of blindness in people of working age. 1 Retina degeneration and related loss of vision in DR induced by hyperglycemia are known to be irreversible. 2Recent studies have showed that retina neurodegeneration is irreversible and may precede and participate in the vascular lesions. 3One important link between vascular abnormalities and neurodegeneration may be the changes in the glial cell which envelops blood vessel and neuronal cell bodies, forms the blood-retinal barrier (BRB) and modulates neuronal activity and blood flow.Increasing evidence shows that reactive gliosis are basic pathological features of early DR. 4 Müller cells are the principal glial cells in the retina.6][7] Glial fibrillary acidic protein (GFAP) is the most sensitive reactive gliosis marker in DR.][10] Current prevention and therapies for DR are performed in advanced stages of the disease.Therefore, new pharmacological interventions for early stages are needed to prevent the onset and the progression of the DR.
Glucagon-like peptide-1 (GLP-1) is a gut hormone that is released from endocrine L-cells in the intestinal epithelium in response to food ingestion, and facilitates glucose-dependent insulin secretion, and it is evaluated as a potential treatment for diabetes. 11,12However, just a little intact GLP-1 reaches the systemic circulation due to immediate degradation by dipeptidyl peptidase-4 (DPP-4). 13,14Liraglutide (LIRA) is a GLP-1 analogue that shares 97% homology with human native GLP-1.It exhibits biologic actions similar to GLP-1 and has a longer half-life against degradation by DPP-4. 15Besides the effects in diabetes, growing evidence demonstrated that LIRA has a neuroprotective effect in some neurodegenerative disease. 16,17However, it remains elusive about the related mechanisms.
Oxidative stress (OS) refers to a disease state caused by the imbalance between oxidation and antioxidation in vivo, resulting in a large number of oxidation intermediate products such as in vivo highly active molecules such as reactive oxygen species (ROS), organisms to resist OS damage through a series of antioxidant systems, including thioredoxin Trx system.More and more studies have shown that OS plays an important role in the occurrence and development of diabetes, especially in the process of DR. [18][19][20] OS produces too much ROS, can damage retinal blood vessels and nerves, at the same time, it can activate other metabolic pathways, thus aggravating the injury of the disease.The endoplasmic reticulum (ER) is a critical intracellular organelle, which has several vital functions such as protein synthesis, protein transport. 21,22The accumulation of unfolded or misfolded protein in the ER will activate the ER stress response.The ER stress response is mediated by at least three types of ER transmembrane proteins: inositolrequiring enzyme 1 (IRE1), protein kinase-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6).ER stress can activate several cell death pathways. 23any studies have indicated that ER stress are involved in the pathogenesis of DR. 24 Therefore, inhibited ER stress might become one therapeutic strategy to prevent the onset and progression of DR.
Thioredoxin (Trx) is a 12-kDa multifunctional protein, which regulates the cellular redox balance by a redox-active disulfide/ dithiol in the active site: -Cys-Gly-Pro-Cys-. 23,25It is widely expressed in many organs.The pathogenesis of DR is relatively complex and its pathogenesis is not fully understood at present.But many studies have shown that oxidative stress is the central link in the development of DR. 18 The main component of oxidative stress is ROS, which can cause cellular metabolic disorders, cell apoptosis, and so on.Trx which existed in cytosolic and nuclear can effectively scavenge ROS in vivo, 19 and delay the occurrence and development of the disease.It also has been noted that in the development of DR. 26 It has been reported that Trx is a modulator of ER stress, which has a protective role in ER stress-induced apoptosis. 27,28Based on the former research, our aim was to explore the effect of LIRA on high glucose-induced Müller cells damage and related underlying mechanism.

Cell culture and regents
The Müller cell obtained from the college of basic medical science of Sun Yat-sen University (GuangZhou, China). 29The cells were maintained in DMEM medium (Gibco, Invitrogen, CA) with 10% fetal bovine serum (FBS, Gibco) at 37°C in a humid atmosphere containing 5% CO 2 .The medium was replaced every 1 or 2 days.When the cells grew to logarithmic growth, they were washed 2-3 times with PBS according to the cell growth state and digested with Trypsin-EDTA containing 0.25% for cell passage.The concentration of HG medium was 200 mmol/L for storage at 4°C.LIRA (Novo Nordisk®, Denmark) was dissolved at 1 μl/L in the DMEM medium without FBS and stored at 4°C.U0126 (Biouniquer, Nanjing, China) was dissolved in 10 mM dimethyl sulfoxide (DMSO, Sigma) for storage at −20°C.The cells were treated with a concentration of HG (50 mM) for 48 h.The cells were treated with HG (50 mM) with/without LIRA (100 nM) and U0126 in the following experiments in vitro.

Flow cytometry analysis
Cells were washed several times with PBS before the experiment.The cells were collected by centrifuging 1000 rpm for 5 min, and then resuspended by binding buffer, stained with AnnexinV (5 μl)/PI (10 μl) staining at room temperature incubated for 15 min according to the manufacturer's instructions (Vazyme, China).After mixing with binding buffer, the samples were analyzed using a flow cytometer.The data was analyzed by soft of BD Accuri C6 (BD Bioscience, San Diego, CA, USA).

Immunofluorescence
The cells were incubated with a blocking solution (10% bovine serum albumin (BSA) in PBS) for 30 min, followed by incubation with primary anti-GFAP antibody (Proteintech Cat# 16825-1-AP, RRID:AB_2109646) overnight at 4°C.The cells were then washed with PBS 3 times (3 min/wash).It was incubated with secondary antibodies in a 1:1000 dilution of Alexa Fluor 488-labeled goat anti-rabbite antibody and incubation at room temperature for 2 h.The cells were then washed with PBS 3 times (3 min/wash), counterstained with DAPI, and imaged using fluorescence microscope (Nikon, Japan).

Transfection
The cells were seeded into a 6-well plate at a density of 2 × 10 5 /ml.The Trx-shRNA plasmid (Dr.Hiroshi Masutani, Japan) and Lipofectamine 2000 were diluted separately in serum-free Opti-MEM for a final volume of 250 μl, gently mixed, and incubated at room temperature for 10 min.Then, the two mixtures were mixed gently and incubated at room temperature for 10 min.The complex was added into each well of the plates.After transfection for 48 h, prepared for the subsequent studies.

Measurement of intracellular ROS
Collected logarithmic cells washed with PBS for 2 times, diluted DCFH-DA.According to 1:1000, the final concentration of DCFH-DA was 10 μmol/L diluted with serum-free medium, and an appropriate amount of DCFH-DA diluent

Statistical analysis
The results were expressed as the mean ± S.E.M and analyzed by GraphPad Prism version 5.0.One-way analysis of variance (ANOVA) followed by Dunnett's test was used for multiple comparisons.Differences resulting in a P-value of .05 or less were considered to be statistically significant.

Effect of HG on Müller cells reactive gliosis and apoptosis
We used flow cytometry to analyze the HG-induced apoptosis in Müller cells.Figure 1 shows that the percentage of apoptosis and expression of GFAP in the HG group were all increased significantly compared with the control group (NC).Besides, we also use mannitol to treat the cells to confirm the effect of osmotic pressure during the process (the data was not showed here).These results indicated that HG can induce retinal reactive gliosis leading to apoptosis in Müller cells.

LIRA decreased HG-induced reactive gliosis and apoptosis in Müller cells
As shown in Figure 2, the expression of GFAP was detected by immunofluorescence staining which was down-regulated after LIRA-and HG-treatment compared with only the HG treatment group.At the same time, we also detected the expression of TXNIP which played an important role in the oxidative stress process.The expression of TXNIP was decreased after LIRA treatment in HG.Moreover, we also detect the expression of GLP-1R which affected the function of LIRA.The data was shown in the supplementary data section.Besides, the percentage of apoptosis in HG+LIRA group was decreased dramatically compared with the HG group.

The protection of LIRA on HG-induced Müller cells apoptosis was through p-Erk/Nrf2/Trx pathway activation
To confirm the related mechanism of the LIRA protection on the apoptosis of Müller cell in HG, we pretreated Müller cells with U0126 (an inhibitor of Erk) for 6 h.As shown in Figure 3, the expression of p-Erk, Nrf2, and Trx was increased in the HG+LIRA group compared to the HG group, the generation of ROS products was decreased.However, the expression level of p-Erk, Nrf2, and Trx was decreased in the HG+LIRA+U0126 group compared to the HG+LIRA group and the generation of ROS products was increased.(Figure 3a-g).The apoptotic percentage of Müller cells was decreased in the HG+LIRA group compared with that of the HG group.However, the apoptotic percentage of Müller cells was increased in the HG+LIRA+U0126 group compared with the HG +LIRA group (Figure 3h,i).

LIRA activated p-Erk/Nrf2/Trx pathway regulated HGinduced ER stress in Müller cells
The expression of GRP78, ATF6, p-PERK, and IRE1 expression was evaluated by western blot.As shown in Figure 4, the expression of GRP78, ATF6, p-PERK, and IRE1 was decreased in the HG+LIRA group compared to the HG group, however, it increased respectively in HG+LIRA+U0126 group compared to the HG+LIRA group.These results indicated that LIRA activated p-Erk/Nrf2/Trx pathway regulated HGinduced ER stress in Müller cells.

Trx plays a key role in the protective effect of LIRA on HGinduced apoptosis by regulating ER stress in Müller cells
To further confirm the role of Trx in this process, we transfected shRNA-Trx plasmid to knock down Trx expression in Müller cells and the expression of Trx and ER stress components was detected by western blot.After transfection, we used western blot to detect the expression of Trx to confirm the transfection experiment condition.We found that the expression of the Trx was decreased about 30 percent after transfection 24 h.Moreover, the expression of the Trx was decreased about 80 percent after transfection 48 h.These data was not shown here.The results showed that in Figure 5, the apoptotic percentage of Müller cells was increased in the HG +LIRA+sh-Trx group compared with that in the HG+LIRA +MOCK group.Meanwhile, GRP78, ATF6, p-PERK, and IRE1 expression level were increased in the HG+LIRA+sh-Trx group compared to the HG+LIRA+MOCK group, respectively.These results indicated that Trx plays a key role in the protective effect of LIRA on HG-induced apoptosis by regulating ER stress in Müller cells.Moreover, the underlying mechanism was summarized in Figure 6.

Discussion
DR is a neurodegenerative disease of the eye that includes microangiopathy, blood-retina barrier breakdown, neurodegeneration, and glial changes may be related to both neuronal and vascular abnormalities at the early stage of DR. 30,31 GFAP is the most sensitive reactive gliosis marker. 32Up-regulation GFAP is indicative of reactive gliosis in retina impairment. 33,34In our former study, we used STZ-induced diabetic mice as our diabetic animal model to display retinal morphologic and functional changes at the early stage of DR. 35 Our experiment validated the fact that retinal cell loss in the GCL, INL, ONL, and GFAP up-regulation was obviously observed in the STZ-induced diabetic mice.Müller cells are the principal glial cell of the retina that plays a crucial role in supporting retinal function. 36,37Reactive Müller cells may directly (e.g., via the release of toxic molecules) and indirectly (by impairing neuron-supportive functions) contribute to retinal damage. 31Müller cell gliosis may impair neural-vascular relationships and contribute to neurodegeneration in the retina of DR patients. 38,39Moreover, Müller cells gliosis also impedes regenerative processes in retinal tissue via the formation of a glial scar. 38ur present study showed that the percentage of apoptosis and expression of GFAP in Müller cells were all increased in high glucose in vitro.Based on these findings, it is very important to understand HG-induced apoptosis and some methods of prevention in Müller cells.
They exert multiple anti-diabetogenic effects, such as the stimulation of glucose-dependent insulin release, inhibition of glucagon secretion, and attenuate the elevation of blood glucose concentration. 13There were evidences showed that GLP-1 and LIRA can provide neuroprotection against excitotoxicity caused by neurodegeneration. 40,41In the peripheral nervous system, LIRA has relatively smaller molecules, and both can diffuse readily across the blood-retina barrier and access the retina directly. 42,43In this study, we put forward the concept pertaining to the involvement of LIRA in the relieve of diabetes-induced retinal glial cell apoptosis.Our study showed that LIRA significantly reduced GFAP up-regulation and inhibited the percentage of apoptosis induced by HG.These results indicate that LIRA has anti-apoptosis property and relieve retina gliosis induced by HG in Müller cells.
The binding of GLP-1 on GLP-1 receptor (GLP-1R) stimulates adenylate cyclase and enhances the production of cAMP. 13urthermore, increased levels of cAMP activate PKA or cAMPregulated guanine nucleotide exchange factor II (Epac2), and contribute to mediating various physiological actions including insulin secretion. 44Given the evidence indicating that cAMP and PKA pathways link to antioxidative effects. 45In our study, we found that HG can decrease the expression of GLP-1R and it can be increased after LIRA treatment in HG.Trx, a ubiquitous and multifunctional protein, helps maintaining the balance of the thiol-related redox environment in cells by two cysteine residues (Cys) and also plays pivotal roles in the regulation of redox signaling. 32It can inhibit the production of excessive ROS in the body and participate in the in-vivo redox balance.There were evidences suggested that Trx has neurotrophic factor-like activity and plays a crucial role in maintaining neuronal cell integrity. 46Dysregulation of Trx system affects various neuronal cellular functions and cell fate such as cell survival and apoptosis, leading to human diseases including neurodegeneration in DR. 47 Our data showed that the expression of Trx was decreased and it also can be increased after LIRA treatment in HG.Besides, we also detected the expression of TXNIP which can be decreased after LIRA treatment in HG.Extracellular receptor kinase (Erk) is involved in the regulation of many activities in differentiated cells.Nuclear factor erythroid 2 (NFE2)-related factor-2 (Nrf2) is a transcription factor that regulates the expression of Trx and other antioxidants. 48As shown in data, LIRA can active p-Erk pathway, reduce the expression of ROS, and decrease HGinduced apoptosis in Müller cell.Numerous studies have indicated that ER stress is involved in neurodegeneration in DR 49 , and Trx is a modulator of ER stress, which has a protective role in ER stress-induced apoptosis, especially the transmembrane thioredoxin-related molecule (TMX). 30We detected the expression of ER stress-related protein which decreased after LIRA treatment and then increased after U0126 treatment.Downregulation of Trx by shRNA further inhibited the protection of LIRA on ER stress-induced by HG.
In summary, our data demonstrated that LIRA inhibits HG-induced Müller cells apoptosis.LIRA activated p-Erk/ Nrf2/Trx signaling pathway regulating ER stress and oxidative stress, Trx plays an important role in the process.

Conclusion
LIRA could protect Müller cells from HG-induced apoptosis and the underlying mechanism was related to activate p-Erk /Nrf2 pathway inducing up-regulation Trx expression which inhibited ER stress and OS.

Figure 1 .
Figure 1.The effect of HG on Müller cells reactive gliosis and apoptosis.(a,b) Apoptosis analysis of Müller cells after treatment with/without HG by flow cytometry.(c,d) Immunostaining of GFAP in Müller cells after treatment with/without HG.The data are presented as the mean ± SD (n = 3 in each group) and are representative of three independent experiments.**p < .01,***p < .001.

Figure 2 .
Figure 2. Reduction in the retinal reactive gliosis and apoptosis by LIRA treatment in HG. (a,b) Apoptosis analysis of Müller cells induced by HG with/without LIRA treatment by flow cytometry.(c,d) Immunostaining of GFAP in Müller cells treated with/ without LIRA in HG.The data are presented as the mean ± SD (n = 3 in each group) and are representative of three independent experiments.*p < .05,**p < .01,***p < .001.

Figure 3 .
Figure 3. Inhibition of Erk by U0126 affects the expression of p-Erk, Erk, Nrf2, Trx, ROS, and apoptosis in HG-induced Müller cells treated with LIRA.(a,b) DCFH-DA probe was used to detect the generation of ROS.Western blots (c) was used to evaluate the expression levels of p-Erk, Erk, Nrf2, Trx.Densitometric analysis showed that the descend of p-Erk (d), Erk (e), Nrf2 (f) and Trx (g) is statistically significant when standardized to actin.(h,i) HG-induced apoptosis in Müller cells treated with LIRA in HG with/without U0126.The data are expressed as the mean ± SD (n = 3 in each group) and are representative of three independent experiments.*p < .05,**p < .01,***p < .001.

Figure 4 .
Figure 4.The effect of U0126 on ER stress-related proteins treated with LIRA in HG.Western blots (a) were used to evaluate the expression levels of ER stress-related proteins.Densitometric analysis showed that the elevation of GRP78/Bip (b), ATF6 (c), p-PERK (d) and IRE1 (e) is statistically significant when standardized to β-actin.The data are expressed as the mean ± SD (n = 3 in each group) and are representative of three independent experiments.*p < .05,**p < .01,***p < .001.

Figure 5 .
Figure 5. Inhibition of Trx by sh-Trx affects ER stress-related proteins treatment with LIRA in HG-induced Müller cells apoptosis.(a,b) Apoptosis analysis of Müller cells after treated with sh-Trx in HG with/without LIRA treatment.Western blots (c) were used to evaluate the expression levels of ER stress-related proteins.Densitometric analysis showed that the elevation of GRP78 (d), ATF6 (e), p-PERK (f) and IRE1 (g) is statistically significant when standardized to β-actin.The data are expressed as the mean ± SD (n = 3 in each group) and are representative of three independent experiments.*p < .05,**p < .01,***p < .001.

Figure 6 .
Figure 6.Summary of the effect of LIRA and the potential mechanism of HGinduced Müller cells apoptosis.