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. 2025 Apr 2;77(3):84. doi: 10.1007/s10616-025-00744-4

Metformin inhibits subretinal fibrosis by activating Klotho by miR-126-5p

Zhijuan Hua 1,#, Qin Zhu 1,#, Jingfei Yang 2,#, Yuxiang Zheng 3, Wenchang Yang 3, Dongli Li 3, Yixin Cui 3, Lu Shen 3, Lingna Rao 3, Xiaofan Zhang 1,✉, Ling Yuan 3,✉
PMCID: PMC11965049  PMID: 40190424

Abstract

Subretinal fibrosis is a main cause of visual loss in patients with neovascular age-related macular degeneration (nAMD), for whom there has been a lack of effective medication. Metformin can improve inflammation and angiogenesis in eye diseases. This study aimed to investigate the mechanism by which metformin inhibits subretinal fibrosis. A subretinal fibrosis cell model was induced by treating human retinal pigment epithelial cells (ARPE-19) with TGF-β1, a subretinal fibrosis mouse model was induced by a laser, and both cells and mice were treated with metformin. Cell proliferation, migration, and invasion were detected by CCK-8, scratch, and Transwell assays. Western blotting and immunofluorescence were used to evaluate protein expression levels, and RT‒qPCR was used to detect gene expression levels. HE and Masson staining were used to observe the morphological changes in retinal and choroidal tissues. Metformin treatment inhibited the TGF-β1-induced proliferation, migration, invasion and epithelial‒mesenchymal transition (EMT) of ARPE-19 cells and effectively ameliorated laser-induced subretinal fibrosis in mice. Mechanistically, metformin inhibits the expression of miR-126-5p, promotes Klotho synthesis, slows the progression of subretinal fibrosis, and miR-126-5p targets and negatively regulates Klotho. Metformin activates Klotho by inhibiting miR-126-5p, thereby reversing TGF-β1-induced ARPE-19 cell EMT and improving laser-induced subretinal fibrosis in mice.

Keywords: Subretinal fibrosis, Metformin, miR-126-5p, Klotho

Introduction

Age-related macular degeneration (AMD) is a degenerative condition that occurs in the macula and is one of the leading causes of vision loss in elderly individuals (Thomas et al. 2021). AMD can be divided into neovascular age-related lesions (nAMDs) and nonneovascular age-related lesions (nnAMDs) (Patel and Sheth 2021). In nAMD, subretinal fibrosis occurs, leading to subretinal scarring and damage to choroidal capillaries, resulting in loss of central vision (Wong et al. 2008). The pathogenesis of nAMD subretinal fibrosis involves the epithelial‒mesenchymal transition (EMT) process that occurs in the retinal pigment epithelium (RPE) (Liu et al. 2024). Therefore, the treatment of progressive fibrosis, including the reversal of EMT and the restoration of the epithelial phenotype, is highly important for treating AMD.

Vascular endothelial growth factor (VEGF) not only plays a crucial role in choroidal neovascularization but also leads to the development of subretinal fibrosis by activating fibroblasts and inducing EMT (Gill et al. 2024). Currently, intravitreal injection of antivascular endothelial growth factor (anti-VEGF) is widely used as a first-line therapy to reduce choroidal and retinal neovascularization, thereby improving visual acuity in patients with nAMD (Yang et al. 2016). However, patients with subretinal fibrosis often do not benefit from anti-VEGF therapy, and even after treatment, subretinal fibrosis occurs in approximately 45–70% of eyes (Daniel et al. 2014; Wolff et al. 2020). Therefore, it is necessary to continue to explore effective treatments for subretinal fibrosis in AMD patients.

Metformin is the first-line drug for the treatment of type 2 diabetes mellitus (T2D) and lowers blood glucose primarily by inhibiting hepatic gluconeogenesis and improving insulin sensitivity (LaMoia and Shulman 2021). In recent years, metformin has been identified as a candidate for a novel treatment for AMD. Urooba Nadeem et al., using bioinformatics tools to identify novel therapeutic candidates for AMD, found that metformin was not only the drug most genetically associated with wet AMD but was also one of the top candidates among all dry AMD subtypes (Nadeem et al. 2022). Metformin has also been found to be associated with a reduced risk of developing AMD (Blitzer et al. 2021) and may prevent the development of AMD (Aggarwal et al. 2024). These observations all indicate that metformin has a potential therapeutic effect on AMD, and further in-depth research on the mechanism of the effect of metformin on AMD is needed. Recent studies have shown that metformin prevents retinal cell death and choroidal neovascularization (CNV) formation caused by various pathological factors (Dang et al. 2021). It also has antiangiogenic effects that may help inhibit neovascularization in retinopathy (Amin et al. 2022; Wang et al. 2021). Therefore, we hypothesize that metformin can reverse the EMT phenotype and has great potential for the treatment of subretinal fibrosis.

MicroRNAs (miRNAs) are a highly conserved class of noncoding RNAs that are widely involved in a variety of signaling pathways and have regulatory roles in many important biological processes (Wen et al. 2022). In particular, miRNAs have been shown to be key regulators in the field of vascular development. For example, miR-23, miR-27, and miR-21 have been shown to be important regulators of CNV in laser injury-induced CNV models (Zhou et al. 2011; Sabatel et al. 2011). In addition, overexpression of miR-126-5p significantly enhances angiogenesis in endothelial cells (ECs) (Zhou et al. 2016). At the same time, miR-126-5p has also been found to play an important role in the regulation of cellular EMT (Meng et al. 2019; Lv et al. 2022). Based on these findings, we hypothesize that miR-126-5p may influence the progression of nAMD by mediating the EMT process in RPE. On the other hand, α-klotho (Klotho) is a protein known for its anti-aging properties (Tang et al. 2023). Studies have found that Klotho levels are significantly lower in patients with AMD and are inversely correlated with the size of macular degeneration (Ma et al. 2022). Further studies have shown that Klotho can attenuate the EMT process of RPE in subretinal fibrosis by inhibiting the ERK1/2 and Wnt/β-catenin signaling pathways (Jiang et al. 2025). Given that both miR-126-5p and Klotho are closely related to the EMT process, we predicted the presence of a target binding site between the two using StarBase, which further confirmed the close relationship between them. Based on these observations, we investigated whether miR-126-5p affects the progression of nAMD by targeting the EMT of Klotho-mediated RPE.

This study aimed to investigate whether metformin can improve AMD-related subretinal fibrosis and further explore the downstream mechanisms involved.

Materials and methods

Cell culture and transfection

Human retinal pigment epithelial cells (ARPE-19) were purchased from Procell Life Science & Technology Co., Ltd. (CL-0026, Wuhan, China). ARPE-19 cells were cultured in DMEM/F12 medium (mixed at a ratio of 1:1) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin‒streptomycin. The cells were cultured in a constant-temperature incubator at 37 °C with 5% CO2. Later, when the cellular density reached 70%, the cultured cells were used for further experiments after starvation for 12 h in serum-free medium. TGF-β1 (10 ng/mL) was added to the cells for 24 h to create the fibrotic group. The fibrotic model group was treated with different concentrations of metformin (0.5, 1.0, or 2.0 mM) for 24 h. The miR-126-5p mimic (SS sequence: 5′-CAUUAUUACUUUUGGUACGCGGU-3′; AS sequence: 5′-CGCGUACCAAAAGUAAUAAUGAU-3′) and its negative control (NC-mimic: SS sequence: 5′-UUGUACUACACAAAAGUACUG-3′; AS sequence: 5′-GUACUUUUGUGUAGUACAAUU-3′) were purchased from RiboBio (RiboBio, China), and the sh-Klotho (SS Sequence: 5′-GTTGATGAATGTTGTTTAA-3′; AS Sequence: 5′-TTAAACAACATTCATCAAC-3′) plasmid and its negative control (sh-NC: SS Sequence: 5′-TTCTCCGAACGTGTCACGT-3′; AS Sequence: 5′-ACGTGACACGTTCGGAGAA-3′) were purchased from Promega (Promega, USA). Transfection was performed using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.

Laser-induced CNV

Male C57BL/6 J mice aged 6–8 weeks were purchased from the Laboratory Animal Center of Kunming Medical University. A laser-induced model of subretinal fibrosis was constructed according to previous methods (Liu et al. 2024). The mice were first anesthetized via an intraperitoneal injection of 5% pentobarbital sodium (60 mg/kg), and then compound tropicamide eye drops were used to dilate the eyes twice. Both eyes were anesthetized with one spot of oxybuprocaine hydrochloride eye drops. The mice were then placed in front of a laser machine, and laser points were applied using a VISULAS 532 s laser (Carl Zeiss, Germany) (laser power: 120 mW; duration: 100 ms; spot size: 50 μm). The formation of air bubbles upon laser application indicates rupture of Bruch’s membrane and successful laser injury, and mice with intraocular vitreous hemorrhage are excluded. On the day of laser-induced injury, 5 μL of the miR-126-5p mimic (SS sequence: 5′-CAUUAUUACUUUUGGUACGCGGU-3′; AS sequence: 5′-CGCGUACCAAAAGUAAUAAUGAU-3′) or sh-Klotho (SS sequence: 5′- GGATAGATCTGGAATATAA -3′; AS sequence: 5′- TTATATTCCAGATCTATCC -3′) plasmid was injected into the vitreous cavity of the mice. Low-dose metformin (50 mg/kg), medium-dose metformin (120 mg/kg) and high-dose metformin (300 mg/kg) were then administered by gavage every day according to the groups, the mice were sacrificed after 5 weeks of treatment, and samples were taken for follow-up experiments.

HE and Masson staining

The mouse eyes were removed and fixed in FAS eye fixative for more than 24 h. After dehydration, the eyes were embedded in paraffin. The paraffin sections were dewaxed and stained in hematoxylin staining solution for 5 min, differentiated with 5% acetic acid for 1 min, treated with returning blue solution for 2 min, stained with eosin staining solution for 1 min, dehydrated in ethanol, and sealed with neutral gum for observation and analysis. Masson staining was performed using a three-color staining kit (ab150686, Abcam, USA).

CCK-8 assay

ARPE-19 cell viability was measured with a CCK-8 kit. A total of 2 × 104 cells/well were seeded in a 96-well plate. After treatment with TGF-β1 for 1 h or metformin for 24 h, CCK-8 solution (10 μL/well) was added, and the mixture was incubated at 37 °C for 24 h. Eventually, the absorbance value was measured at 450 nm with a microplate reader (Bio-Rad, USA).

Report analysis of double luciferase

The cDNA fragment of the Klotho 3′-UTR was subsequently cloned and inserted into the pmirGLO double-luciferase vector (Promega, USA). ARPE-19 cells were cotransfected with Klotho wild-type (WT) or mutant (MUT) 3′-UTR vectors and a miR-126-5p mimic using Lipofectamine 3000 (Invitrogen, USA). After 24 h, luciferase activity was measured using a dual-luciferase reporting gene assay system (Promega, USA) according to the manufacturer’s instructions.

Immunofluorescence assay

ARPE-19 cells (4 × 104) were cultured in a 24-well plate for 1 day and pretreated with 10 ng/mL TGF-β1 for 24 h. Then, various concentrations of metformin (0.5, 1.0, and 2.0 mM) were added, and the mixture was incubated for 24 h. The cells were diluted in 3% Triton at room temperature and fixed with 4% paraformaldehyde for 20 min. Next, the mixture was blocked with 5% goat serum albumin (Solarbio, China) for 30 min at 37 °C, and the cells were covered with the primary antibody α-SMA (1:200, Abcam, UK) and incubated overnight at 4 °C. The membrane was subsequently incubated with secondary antibody (1:200, Abcam, UK) at room temperature for 1 h, after which DAPI was added for 5 min. Finally, the cells were washed with PBS and sealed. Immunofluorescence signals were observed using a fluorescence microscope (AMF5000, Invitrogen, USA).

Scratch wound assay

A total of 2 × 105 cells were seeded and incubated in each well of 12-well plates for 24 h. A 200 μL pipette tip was used to create a scratch on the bottom of the wells. The cells were washed with PBS 3 times. TGF-β1 or metformin was added to each well, and the samples were incubated for 24 h for observation under a microscope (Zeiss Observer A1, Oberkochen, Germany).

Transwell assay

A total of 1 × 105 cells/well were suspended in serum (5% FBS) medium and inoculated into the upper compartment of the Matrigel-coated Transwell chamber. Complete medium (with 10% FBS) was added to the lower chamber, and TGF-β1 or metformin was added to each group. After 12 h, the cells attached to the surface of the filter membrane were removed, and the cells that migrated to the surface of the lower chamber were stained with 5% crystal violet for 5 min. Finally, images were captured using an inverted light microscope (Zeiss Observer A1, Oberkochen, Germany) for observation.

Western blotting assay

Proteins were extracted from tissues and cells using RIPA buffer containing 1% protease inhibitors (R0278, Sigma‒Aldrich, USA). Equivalent amounts of protein from cells or tissues were added to each well of an SDS‒PAGE gel for electrophoresis. The proteins were transferred to a PVDF membrane and blocked with 10% mild medium for 30 min. Primary antibodies, including α-SMA (1:1,000), E-cadherin (1:1,000), vimentin (1:5,000), N-cadherin (1:1,000), and internal reference β-actin (1:1,000), were incubated separately overnight at 4 °C. The washed membrane was incubated with secondary antibody and HRP (1:5,000). Color was developed with an enhanced chemiluminescence (ECL) kit (Millipore, USA). Finally, the bands were semiquantitatively analyzed by ImageJ software.

RT‒qPCR

Total RNA was extracted from cells or tissues using TRIzol reagent. The RNA was reverse transcribed into cDNA using a first-strand cDNA synthesis kit, and a SYBR Green real-time PCR kit (Solarbio, China) was used for real-time PCR. Gene expression levels were calculated using the 2−ΔΔCt method, and β-actin and U6 were used as internal controls. The primer sequences are shown in Table 1.

Table 1.

Primer sequences

Target gene Primer sequence (F: Forward primer; R: Reversed primer)
miR-126-5p F: 5′-CGGCGCGTACCAAAAGT-3′
R: 5′-GTGCAGGGTCCGAGGT-3′
has-Klotho F: 5′-GAAGCGGTAGTGAGTGACCC-3′
R: 5′-TAGCCAGCGACAGCTACAAC-3′
mmu-Klotho F: 5′-GGGACACTTTCACCCATCACT-3′
R: 5′-ACGTTGTTGTAACTATCGCTGG-3′
has-β-actin F: 5′-CATGTACGTTGCTATCCAGGC-3′
R: 5′-CTCCTTAATGTCACGCACGAT-3′
mmu-β-actin F: 5′-TATAAAACCCGGCGGCGCA-3′
R: 5′-TCATCCATGGCGAACTGGTG-3′
U6 F: 5′-CTCGCTTCGGCAGCACA-3′
R: 5′-AACGCTTCACGAATTTGCGT-3′
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Statistical analysis

The data were analyzed in GraphPad Prism8.0 (GraphPad Software Inc., USA). The quantitative data are presented as the means ± standard deviations. A t test was used for comparisons of differences between two groups, and one-way analysis of variance was used for comparisons of differences between multiple groups. P < 0.05 was considered statistically significant.

Results

Metformin inhibits the TGF-β1-induced proliferation, migration, and EMT of ARPE-19 cells

The results of the CCK-8 assay revealed that the proliferation of ARPE-19 cells was significantly greater after 10 ng/mL TGF-β1 treatment (Fig. 1A); therefore, 10 ng/mL TGF-β1 was selected for the subsequent experiments. We treated the TGF-β1-induced ARPE-19 cells with different concentrations of metformin (0.5, 1.0, and 2.0 mM) to explore the effects of metformin on the EMT of the ARPE-19 cells. The CCK-8 results showed that metformin significantly inhibited the TGF-β1-induced proliferation of ARPE-19 cells (Fig. 1B). In addition, Western blot results revealed that the expression of the EMT-related protein E-cadherin decreased after TGF-β1 induction, the expression of N-cadherin and vimentin increased, and the expression of the fibrosis protein α-SMA increased. Treatment with metformin reversed the changes in the expression of these proteins (Fig. 1C). Immunofluorescence assays revealed that the expression of α-SMA increased after TGF-β1 induction but decreased after the addition of metformin (Fig. 1D). Transwell and scratch experiments revealed that TGF-β1 significantly promoted the invasion and migration of ARPE-19 cells, whereas the addition of metformin inhibited the effect of TGF-β1 (Fig. 1E and F). In addition, light microscopy revealed that TGF-β1 stimulated the morphologic transformation of ARPE-19 cells from typical cobblestone cells to slender, fusiform mesenchymal cells, and metformin significantly inhibited this transformation (Fig. 1G). The above results indicate that metformin can alleviate the TGF-β1-induced proliferation, migration, invasion, and EMT of ARPE-19 cells, with the best inhibitory effect observed at a dose of 2.0 mM metformin, which was used for subsequent experiments.

Fig. 1.

Fig. 1

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Metformin inhibits the TGF-β1-induced proliferation, migration, and EMT of ARPE-19 cells. A The proliferative activity of ARPE-19 cells treated with different concentrations of TGF-β1 (0, 2.5, 5, 10, 20, or 40 ng/mL) was detected by CCK-8. B Effects of metformin (0.5, 1.0, or 2.0 mM) on the proliferative activity of TGF-β1 (10 ng/mL)-induced ARPE-19 cells were detected by CCK-8. C Western blotting was used to detect the expression of E-cadherin, N-cadherin, α-SMA, and vimentin. D Immunofluorescence detection of α-SMA (red) expression distribution and nuclear DAPI staining (blue). E Transwell assay for detecting cell invasion. F Cell migration was detected by a scratch test. G Morphological changes in the cells were observed under an optical microscope. Compared with the NC group, ***P < 0.001; compared with the TGF-β1 group, #P < 0.05, ##P < 0.01, ###P < 0.001. Met: metformin

Effects of metformin on the expression of miR-126-5p and Klotho and confirmation of their targeting relationships

First, we detected the expression of miR-126-5p and Klotho in retinal epithelial cells. The results revealed that the induction of TGF-β1 promoted the expression of miR-126-5p and that metformin treatment reversed the effect of TGF-β1 (Fig. 2A). However, the expression of Klotho was opposite to that of miR-126-5p (Fig. 2B). Next, we predicted via StarBase that there was a targeted binding site between miR-126-5p and Klotho (Fig. 2C). Dual-luciferase gene reporting experiments further confirmed the targeted binding relationship between miR-126-5p and Klotho (Fig. 2D). Finally, we transfected a miR-126-5p mimic into ARPE-19 cells to detect the expression of Klotho. The RT‒qPCR results revealed that the expression of Klotho was significantly downregulated in ARPE-19 cells after transfection with the miR-126-5p mimic (Fig. 2E).

Fig. 2.

Fig. 2

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Effects of metformin on the expression of miR-126-5p and Klotho and confirmation of their targeting relationships. A The expression level of miR-126-5p was detected by RT‒qPCR; B The expression level of Klotho was detected by Western blot; C: Prediction of binding sites between miR-126-5p and Klotho; D: Double luciferase was used to verify the targeting relationship between Klotho and miR-126-5p; E: RT‒qPCR was used to detect the expression of Klotho. Compared with the NC or miR-NC-mimic group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the TGF-β1 group, ##P < 0.01. Met: metformin

Metformin alleviates TGF-β1-induced ARPE-19 cell proliferation, migration, and EMT by downregulating miR-126-5p and activating Klotho

The relationships between the effects of metformin on ARPE-19 cell biology and the molecular axis of miR-126-5p/Klotho were further investigated. First, the transfection efficiency was confirmed by transfecting cells with the miR-126-5p mimic or sh-Klotho. The results revealed that the expression of miR-126-5p in the miR-126-5p mimic group was significantly upregulated and that the expression level of Klotho in the sh-Klotho group was significantly downregulated, confirming successful transfection (Fig. 3A and B). Subsequently, Western blot analysis revealed that TGF-β1 inhibited the expression of E-cadherin and promoted the expression of N-cadherin, α-SMA and vimentin, whereas metformin reversed the changes in the expression of these proteins. On this basis, the expression of E-cadherin decreased, and the expression of N-cadherin, α-SMA and vimentin increased after the cells were transfected with the miR-126-5p mimics. Similarly, Klotho knockdown reversed the effects of metformin on E-cadherin, N-cadherin, α-SMA and vimentin protein expression (Fig. 3C). The distribution of α-SMA immunofluorescence was consistent with the Western blot results (Fig. 3D). Scratch and Transwell experiments revealed that TGF-β1 promoted ARPE-19 cell migration and invasion, whereas the addition of metformin inhibited TGF-β1-induced migration and invasion. On this basis, transfection of the miR-126-5p mimic led to increased cell migration and invasion. Similarly, sh-Klotho transfection increased invasion and migration (Fig. 3E and F). In addition, the optical microscopy results revealed that TGF-β1 induced ARPE-19 cells to transform into spindle-shaped mesenchymal-like cells and that metformin inhibited TGF-β1-induced transformation, but the transfection of the miR-126-5p mimic inhibited the effect of metformin; similarly, the transfection of sh-Klotho inhibited the effect of metformin (Fig. 3G).

Fig. 3.

Fig. 3

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Metformin alleviates the TGF-β1-induced ARPE-19 cell proliferation, migration, and EMT by downregulating miR-126-5p and activating Klotho. A Transfection efficiency of the miR-126-5p mimic was detected via RT‒qPCR. B Western blot detection of sh-Klotho transfection efficiency. C Western blotting was used to detect the expression of EMT- and fibrosis-related proteins in ARPE-19 cells. D Immunofluorescence observation of the expression and distribution of α-SMA in ARPE-19 cells. E Transwell experiments were used to evaluate the invasiveness of the cells in each group. F Cell migration ability was evaluated by a scratch test. G Morphological changes in the cells were observed under an optical microscope. Compared with the NC group, ***P < 0.001; compared with the TGF-β1 group, ##P < 0.01, ###P < 0.001; compared with the TGF-β1 + Met group, @P < 0.05, @ @P < 0.01, @ @ @ @P < 0.001. Met: metformin

Metformin has a positive effect on subretinal fibrosis in mice

To investigate the therapeutic effect of metformin in animal models, we investigated the effects of different doses (50, 120, 300 mg/kg) of metformin on the progression of laser-induced subretinal fibrosis in mice. Compared with those in the NC group, many fibroblasts were visible under the retina in the model group, and the arrangement of cells in the pigment epithelium layer was disordered. After treatment with metformin, the number of fibroblasts under the retina was significantly reduced (Fig. 4A). Compared with those in the NC group, the number of collagen fibers in the retinal tissue of the model group increased, and the number of collagen fibers decreased after metformin treatment (Fig. 4B). Finally, Western blotting was used to detect the expression of EMT and fibrosis marker proteins in mouse retinal choroid tissue, and the results revealed that, compared with those in the Model group, the expression levels of N-cadherin, α-SMA, and vimentin were reduced and that of E-cadherin was upregulated after metformin treatment (Fig. 4C).

Fig. 4.

Fig. 4

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Effects of metformin on subretinal fibrosis. A HE staining was used to observe the morphological changes in the retina and choroid in mice after the administration of different doses of metformin. B Masson staining was used to observe the subretinal number of collagen fibers in mice treated with different doses of metformin (blue). C Western blotting was used to detect the expression of EMT and fibrosis marker proteins. Compared with the NC group, ***P < 0.001; compared with the Model group, #P < 0.05, ##P < 0.01, ###P < 0.001. Met: metformin

Metformin inhibits subretinal fibrosis in vivo by activating Klotho through miR-126-5p

Finally, we validated the molecular mechanism by which metformin improves subretinal fibrosis in mice at a dose of 300 mg/kg. RT‒qPCR revealed that miR-126-5p expression was significantly upregulated in the Model group compared with the NC group. Metformin effectively suppressed miR-126-5p expression, which was reversed by miR-126-5p mimic treatment, whereas sh-Klotho treatment had no impact on the effect of metformin (Fig. 5A). Western blot analysis revealed that Klotho expression was significantly lower in the Model group than in the NC group. Metformin treatment effectively increased Klotho expression, which was reversed by either the miR-126-5p mimic or sh-Klotho treatment (Fig. 5B). HE and Masson staining results revealed that overexpression of miR-126-5p or knockdown of Klotho after metformin administration promoted the proliferation and collagen deposition of subretinal fibroblasts (Fig. 5C and D). Western blotting was used to detect the expression of fibrosis marker proteins. Compared with those in the metformin-treated group, the protein expression levels of N-cadherin, α-SMA, and vimentin increased after treatment with the miR-126-5p mimic or sh-Klotho, whereas the protein expression level of E-cadherin decreased (Fig. 5E). These findings confirmed the negative effects of the miR-126-5p mimic or sh-Klotho on metformin-mediated inhibition of subretinal fibrosis.

Fig. 5.

Fig. 5

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Metformin inhibits subretinal fibrosis by activating Klotho through miR-126-5p. A The expression of miR-126-5p was detected via RT‒qPCR. B The expression of Klotho was detected by Western blotting. C HE staining was used to observe the morphological changes in the retina and choroid of the mice. D Masson staining was used to observe the deposition of subretinal collagen in the mice. E Western blotting was used to detect the expression of EMT and fibrosis marker proteins. Compared with the NC group, ***P < 0.001; compared with the Model group, #P < 0.05, ##P < 0.01, ###P < 0.001; compared with the Met group, @P < 0.05, @ @P < 0.01, @@@P＜0.001 . Met: Metformin

Discussion

Metformin was originally derived from isopentene guanidine, which was found in French eugenol guanidine (Triggle et al. 2022). Studies have confirmed that the therapeutic effect of metformin on age-related macular degeneration also involves a variety of mechanisms (Liang et al. 2022). However, few studies have reported its effect on retinal fibrosis. Our study revealed that metformin inhibits the proliferation of ARPE-19 cells in the early stage of disease. Moreover, it prohibits epithelial mesenchymal transition in the subretinal layer. This research is consistent with previous in vivo studies.

Metformin can inhibit cell and tissue fibrosis through TGF-β1-related signaling pathways (Hurley et al. 2021). Transforming growth factor-β (TGF-β) belongs to the TGFβ superfamily of growth factors, and TGF-β has been reported to be a key cytokine involved in coordinating EMT. Therefore, we used TGF-β1, a subtype of TGF-β, to induce ARPE-19 cells to construct a fibrotic cell model (Pérez et al. 2017). We found that metformin can inhibit the occurrence of TGF-β1-induced migration, invasion and EMT. Previous studies have confirmed that paeoniflorin decreases cell viability through the miR-126-5p/ZEB2 axis to inhibit migration, invasion, and TGF-β1-induced EMT (Lv et al. 2022). Other studies have shown that PRNCR1 modifies EMT in non-small cells through the miR-126-5p/MTDH axis (Guo et al. 2020). Our study revealed that metformin can significantly inhibit the expression of miR-126-5p. The results of this study also revealed that miR-126-5p mimics can inhibit the expression of E-cadherin and increase the levels of N-cadherin, α-SMA, and vimentin. E-cadherin is a core protein that maintains lateral contact between adjacent epithelial cells through adherent junctions, and the first step of EMT involves the downregulation of E-cadherin (Shu and Lovicu 2017). N-cadherin, α-SMA, and vimentin are mesenchymal markers, and the increased expression of these mesenchymal markers is accompanied by this cellular transition to a mesenchymal phenotype (Saito 2013). In addition, miR-126-5p can enhance the migration and invasion ability of ARPE-19 cells. These findings suggest that metformin inhibits ARPE-19 cell migration, invasion and EMT by inhibiting miR-126-5p.

Our research revealed that miR-126-5p can negatively regulate the expression of Klotho, which is a membrane-binding protein expressed in RPE cells and is involved in the regulation of retinal fibrosis (Zhou et al. 2023). In its shedding form, Klotho plays an antifibrotic role in kidney tissue and myocardial fibrosis (Martín-Carro et al. 2023; Chen et al. 2022). In addition, the absence of Klotho in mice can accelerate aging or premature aging syndrome and significantly shorten lifespan (Zhu et al. 2022). In this study, we also found that, compared with those in the control group, the Klotho levels in the ARPE-19 cells (TGF-β1 treatment) and subretinal layer in the laser-damaged mice were obviously lower. Klotho knockdown increased the expression of α-SMA (fibrosis marker protein), N-cadherin and vimentin (EMT marker proteins), but the expression of E-cadherin decreased to promoted subretinal fibrosis.

Subretinal fibrosis is a difficult and hot topic in the current treatment of AMD and is characterized mainly by excess extracellular matrix (ECM) proteins, such as RPE-derived collagen and fibronectin, and myoblastic fibroids (Llorián-Salvador et al. 2022; Resnikoff et al. 2022). Under pathological conditions, RPE cells differentiate into myofibroblast-like cells through the EMT, which facilitates ECM deposition and fibrosis formation (O’Driscoll et al. 2023; Yin et al. 2023). The results of this study suggested that the decrease in collagen fibers in retinal and choroidal tissues caused by metformin was clear. Compared with the model group, the groups in which miR-126-5p was overexpressed or Klotho was knocked down after metformin administration exacerbated the pathological damage to the retinal and choroidal tissues of the mice. Moreover, the inhibitory effects of metformin on the N-cadherin, α-SMA and vimentin proteins and the promotion of the E-cadherin protein were weakened by the overexpression of miR-126-5p or the knockdown of Klotho. These results indicate that metformin inhibits the expression of miR-126-5p, activates Klotho, and alleviates the occurrence and progression of subretinal fibrosis.

Conclusion

In summary, our study revealed that metformin inhibits the proliferation, migration, and EMT of ARPE-19 cells through the miR-126-5p/Klotho axis and inhibits the development of laser-induced subretinal fibrosis in mice. The results of this study contribute to the understanding of the mechanism of TGF-β1-induced EMT in RPE cells and indicate the potential therapeutic value of metformin in the treatment of subretinal fibrosis. However, this study has certain limitations. The analysis is constrained by the lack of evaluation across varying timepoints, levels of fibrosis, and seeding densities in cell culture experiments. Additionally, murine eye experiments do not account for differences in the degree of laser-induced damage, assuming a singular disease state. Future studies should incorporate diverse experimental conditions, such as multiple timepoints, varying fibrosis levels, and different degrees of tissue damage, to further elucidate the scope and limitations of the antifibrotic properties of metformin.

Acknowledgements

Not applicable.

Author contributions

All authors contributed substantially to this manuscript. Conceptualization: Zhijuan Hua, Qin Zhu, Jingfei Yang; Data Curation: Yuxiang Zheng, Wenchang Yang; Formal Analysis: Dongli Li, Yixin Cui; Funding Acquisition: Ling Yuan; Investigation: Ling Yuan; Methodology: Lu Shen, Lingna Rao; Project Administration: Zhijuan Hua, Qin Zhu, Jingfei Yang; Resources: Ling Yuan; Software: Dongli Li, Yixin Cui; Supervision: Yuxiang Zheng, Wenchang Yang; Validation: Xiaofan Zhang, Ling Yuan; Visualization: Xiaofan Zhang; Writing-Original Draft: Zhijuan Hua, Qin Zhu, Jingfei Yang; Writing – Review & Editing: Xiaofan Zhang, Ling Yuan. All authors read and approved the final manuscript.

Funding

This study was supported by the Applied Basic Research Foundation of the Department of Science and Technology of Yunnan Province (Grant No. 202201AY070001-036); National Natural Science Foundation Project (Grant No. 82260207); and Scientific Research Fund of Education Department of Yunnan Province (Grant No. 2023J0036).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interest

The authors declare no competing interests.

Ethical approval

All animal experimental protocols were approved by the Ethics Review Committee of Animal Experiments, Kunming Medical University (kmmu20211570). All methods were performed in accordance with the relevant guidelines and regulations and in accordance with ARRIVE guidelines.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Zhijuan Hua, Qin Zhu, and Jingfei Yang have contributed equally to this work.

Contributor Information

Xiaofan Zhang, Email: 18088311081@163.com.

Ling Yuan, Email: yuanling@kmmu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Citations

  1. Guo R, Hu T, Liu Y et al (2020) Long non-coding RNA PRNCR1 modulates non-small cell lung cancer cell proliferation, apoptosis, migration, invasion, and EMT through PRNCR1/miR-126-5p/MTDH axis. Biosci Rep. 10.1042/bsr20193153 [DOI] [PMC free article] [PubMed]

Data Availability Statement

No datasets were generated or analysed during the current study.

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