Genipin protects against mitochondrial damage of the retinal pigment epithelium under hyperglycemia through the AKT pathway mediated by the miR-4429/JAK2 signaling axis

Introduction

As a common microvascular complication of diabetes, diabetic retinopathy (DR) is the main cause of vision loss in diabetic patients (1). A study has shown that in 2017, the total number of patients diagnosed with DR was about 93 million, among which about 28 million had visual impairment due to retinopathy (2). DR is a special diabetic microvascular complication. Through epidemiological studies in many places in China, it has been found that people with DR will be accompanied by low vision and even blindness. At present, it has become one of the main causes of blindness. Therefore, how to effectively treat DR to reduce the risk of blindness is particularly important.

Current scientific research is trying to find targets for DR through mechanistic studies so as to treat DR efficiently via these targets. As an important part of retinal tissue, retinal pigment epithelial (RPE) cells are closely related to the occurrence and development of DR (3). An anatomical study has shown that the retinal pigment epithelium is mainly located in the lower layer of the retina and consists of a single layer of cells (4). Its main functions are supporting photoreceptor cells, regulating blood transport and humoral conversion, and visual pigment synthesis and regeneration. Therefore, the retinal pigment epithelium plays an important role in the maintenance of the retinal microenvironment (5). In diabetic patients, due to microvascular disease, the blood supply of retinal tissue is insufficient, which ultimately leads to RPE cell damage (6). Studies have shown that the main mechanism of RPE cell damage is intracellular mitochondrial dysfunction (7). The RPE cells located in the outermost layer of the retina are susceptible to oxidative damage due to long-term exposure to light stimulation and hyperoxia, which produces chronic oxidative stress. Hyperglycemia further promotes the process of oxidative stress, thereby causing mitochondrial function damage, which ultimately leads to the impairment of cell function and activity (8). Therefore, how to effectively protect the internal mitochondrial function of RPE cells may be an effective means to treat DR lesions.

Genipin (GE) has been shown to be an effective treatment for retinal pigment epithelium injury through the development of diabetes drugs (9). Derived from the extraction and purification of gardenia jasminoides, GE has effective anti-inflammatory, antioxidant, and anti-diabetes properties (10). A previous study has shown that GE can effectively control blood glucose fluctuations in patients with type 2 diabetes (11). At the same time, a study has also shown that GE can effectively protect against RPE cell damage (12). However, there is no consensus on the molecular regulatory mechanism of GE in the protection of RPE cell function. By reviewing the literatures, a study has shown that the expression of Nrf2 signal axis, ERK signal axis and PI3K/AKT signal axis are all regulated by GE (13). As a ubiquitous intracellular regulatory pathway, AKT signaling pathway has been shown to be significantly overexpressed in retinal epithelial cells induced by high glucose. GE can significantly inhibit AKT signal pathway in retinal epithelial cells induced by high glucose (13).

In order to preliminarily predict the molecular mechanism of GE in the protection of RPE cells, bioinformatics analysis was conducted to identify the downstream proteins of differentially expressed genes located in the AKT pathway during GE treatment of RPE cells. The results showed that only RPRL and JAK2 were downstream differential expression factors of AKT. RPRL, or prolactin, is closely related to breast diseases (14). JAK2 is closely related to inflammatory responses and mitochondrial damage in the body (15). A previous study showed that JAK2 was activated in RPE cells under hyperglycemia and the inflammatory response was aggravated (16). This finding suggests that the therapeutic effect of GE may be exerted by regulating the expression of JAK2 through the AKT signaling pathway. But how does the AKT protein regulate JAK expression? This study suggests that the expression of JAK2 may be further regulated by regulating the expression of microRNAs (miRNAs). In this study, the target miRNA of JAK2 was predicted by the TargetScan, miRDB, and DIANA databases and the intersection was taken. The results showed that miR-4429 has a targeted binding effect to JAK2, and a previous study has shown that miR-4429 is a downstream regulatory molecule of AKT (17).

On this basis, the present study investigated whether GE could protect the retinal pigment epithelium from mitochondrial damage under hyperglycemic conditions through the AKT pathway mediated by the miR-4429/JAK2 signaling axis. We present the following article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-2219/rc).

Methods

Bioinformatics analysis

The Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) was searched using the keywords (“RPE”) AND (“GE”) to obtain the dataset of diabetes patients and healthy subjects for comparative analysis by gene chip technology. Differential gene analysis was performed using the GEO online analysis tool GEO2R. P value <0.01 and |log fold change (FC)| >1 were used to screen differentially expressed genes. Downstream proteins of the AKT pathway were obtained with the Kyoto Encyclopedia of Genes and Genomes (KEGG) website. TargetScan (https://www.targetscan.org/vert_71/), miRDB (http://www.mirdb.org/cgi-bin/search.cgi), and DIANA (http://diana.imis.athena-innovation.gr/DianaTools/index.php) databases were further used to determine JAK2-targeted miRNAs. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).

Reagents, antibodies, and kit sources

Dulbecco’s modified Eagle medium (DMEM; Hyclone, Logan, UT, USA; SH30021.01B), fetal bovine serum (FBS; Hyclone; SH30396.03), phosphate-buffered saline (PBS) solution containing penicillin-streptomycin (STZ) (Hyclone; SH30256.01), Cell Counting Kit-8 (CCK-8) kit (Beijing, China; CA1210), enzyme-linked immunosorbent assay (ELISA) matrix metalloproteinases-2 (MMP-2) kit (Abnova, Taipei; E-KA0393), ELISA tumor necrosis factor-β (TNF-β) kit (Shenzhen, China; ZK-H063), Reverse Transcription System (Takara, Beijing, China; RR047A), and quantitative polymerase chain reaction (qPCR) kit (Takara; RR430B) were purchased for this study.

Cell culture, treatment, and grouping

The human RPE cell line ARPE-19 was selected for this study (Stem Cell Bank of Chinese Academy of Science, GNHu45). After obtaining the cells, the cells were inoculated in DMEM medium containing 10% FBS for culture. The cells were cultured in a 5% CO₂ incubator at 37 ℃. The cells were replaced with medium every day and the cell morphology was observed under an inverted microscope. Cell passaging was carried out when the cell density grew to about 90%.

CCK-8 assay

APRE-19 cells were treated according to different grouping treatment conditions and inoculated into 96-well plates, then incubated in a constant temperature incubator. The CCK-8 kit was used to detect cell proliferation at 72 hours after inoculation. The 96-well plates were taken out of the constant temperature incubator, the medium was discarded from each well, 100 µL CCK-8 solution was added to each well, and plates were placed in the incubator for 2 hours. Finally, the absorbance of each well was measured at 450 nm.

Establishment of lentivirus knockdown/overexpression cell lines

MiR-4429-mimics and si-mimics lentiviruses were obtained from GeneChem (https://www.genechem.com.cn/index/index/index.html). The virus was constructed, sequenced, produced, titered, and transported to the laboratory in cold chain packaging. The ARPE-19 cell line was used as the research object. When the cell growth density reached 90%, the cells were digested by trypsin and suspended again. The obtained cells were inoculated in a 6-well plate at a density of 1×10⁵/well and incubated in a 37 ℃ incubator for 24 hours until they adhered to the wall. The purchased lentivirus was diluted with DMEM solution containing 10% FBS at a ratio of 1:10. After the medium in the 6-well plate was discarded, 1 ml diluent containing lentivirus was added to each well. After 24 hours of culture in a 37 ℃ incubator, the culture medium containing lentivirus was discarded, then 2 mL DMEM medium containing 10% FBS was added, and the culture was continued in the incubator.

Dual luciferase reporter assay

Starbase 2.0 was used to predict the binding site of miR-4429 to JAK2 and select the appropriate mutation site. Construction of the dual luciferase reporter gene of JAK2 contained the miR-4429 prediction site. MiR-4429-wild type (WT) + mimic control, miR-4429-WT + JAK2 mimic, miR-4429-WT + inhibitor control, miR-4429-WT + JAK2 inhibitor, miR-4429-mutant (MUT) + mimic control, miR-4429-MUT + JAK2 mimic, miR-4429-MUT + inhibitor control, and miR-4429-MUT + JAK2 inhibitor were used to transfect ARPE-19 cells after high glucose induction. At 48 hours after successful transfection, luciferase activity was measured using a dual luciferase reporter assay system (Promega, Madison, WI, USA).

Reverse transcription-PCR (RT-PCR) assay

ARPE-19 cells in different groups were treated according to different treatment conditions, and the cells were digested by trypsin, centrifuged, and collected 72 hours after treatment (15). Total RNA was extracted by the TRIzol method. The concentration and purity of RNA in 2 µL solution were determined by a microspectrophotometer. The reverse transcription reaction system was established using the Takara reverse transcription kit to reverse transcribe RNA into complementary DNA (cDNA). Using the qPCR kit to establish the cDNA reaction system, the conditions were as follows: 95 ℃ 10 min, 95 ℃ 30 sec, 60 ℃ 30 sec, 40 cycles. The dissolution curve was analyzed, and the expression levels of CYT C1, AKT, miR-4429, and JAK2 in each sample were statistically analyzed by the 2^−∆∆CT method. Primers of each gene are shown in Table 1.

Western blot (WB)

ARPE-19 cells were treated according to different grouping conditions, and the cells were trypsin digested, centrifuged, and collected 120 hours after treatment. Cell contents were obtained by lysing cells, and total proteins in cells were collected by centrifugation. The protein concentration was detected by the BCA method. After the protein concentration was quantified, SDS-PAGE electrophoresis was performed for each group of tissue/cells. After electrophoresis, the proteins separated by electrophoresis were transferred to PVDF membranes using a transfer device. The PVDF membrane was then removed and sealed with 5% skim milk for 1 hour, then rinsed with PBS. Subsequently, primary anti-AKT (1:500), CYT C1 (1:500), and JAK2 (1:500) were added and incubated overnight. The secondary antibody (sheep anti-rabbit antibody, 1:1,000) was added and incubated for 1 hour. Finally, the PVDF membrane signals were detected by the chemiluminescence imaging system.

Seahorse cellular energy metabolism analysis

First, each group of cells was inoculated into a Seahorse Xfe microplate, and the Seahorse Xfe24 cell energy metabolism analyzer was turned on for preheating the day before the experiment. At the same time, 1 mL calibration solution was added to the hydration plate in the analyzer and placed in a constant temperature cell incubator overnight. On the day of the experiment, 1.2 mL 1.0 M glucose solution, 1.2 mL 100 mM pyruvate solution, and 1.2 mL glutamine solution were added into 120 mL DMEM medium. According to the instructions, 630, 720, and 540 µL detection solutions were added to oligase, FCCP, and rotenone/antimycin reagent tubes, respectively. Cells in each group were removed from the incubator, and 500 µL detection solution was added to each well to clean cells. The optimal concentration of FCCP was obtained by FCCP titration. Then, the hydration plate and the probe plate that had been dosed were placed in the instrument and the grouping and procedure for the experiment were set up. After data acquisition, the obtained results were input into Wave software for data processing, and the results were analyzed in combination with Seahorse experimental data.

Animal feeding and grouping

A total of 45 6-week-old C57BL/6 mice (18±20 g, purchased from Nanjing Junjunke Biological Engineering Co., Ltd., Nanjing, China) were used as the research objects. The mice were raised in a specific pathogen free (SPF) environment. Animal experiments were performed under a project license (No. QMU-AECC-2021-244) granted by Animal Ethical Committee of Qiqihar Medical University, in compliance with national guidelines for the care and use of animals. The operation and treatment procedures were in accordance with the relevant regulations of the Experimental Animal Ethics Committee of Qiqihar Medical University. A protocol was prepared before the study without registration. Before the experiment, mice were fed with a standard mice diet for 1 week. The breeding environment included room temperature 2–25 ℃, relative humidity 55%±5%, and light/darkness cycle for 12 hours. The mice were randomly divided into three groups: control group, diabetic model group, and diabetic + GE group, with 15 mice in each group.

Preparation of the diabetic mouse model: after 1 week of adaptive feeding, the mice were fed with a high-fat diet for 4 weeks. At the end of the fifth week, STZ (60 mg/kg) was injected intraperitoneally for 3 days to induce type 2 diabetes. At 72 hours after induction, blood samples were collected from the tail vein, and fasting blood glucose of the mice was measured. Fasting blood glucose was more than 11.1 mmol/L, indicating that the modeling was successful.

GE treatment: after successful establishment of the diabetic mouse model, mice in the diabetic model + GE group were given GE (25 mg/kg) intragastric therapy once a day for 1 week.

Electroretinogram (ERG)

ERG detection is mainly divided into EGR detection under dark adaptation and ERG detection under light stimulation. The dark adaptation test was performed by injecting 50 mg/kg pentobarbital sodium intraperitoneally and dilating pupils with compound tropicamide drops after overnight exposure to a dark environment. After anesthesia, the mice were fixed in the prone position on the test table, and the annular electrodes were placed in front of both eyes and contacted with the cornea. Connect the electrode correctly and light response stimulation were performed. In the process of light stimulus detection, white light flashing stimuli were provided for 6 times. The intensity was 0.65 log CD·s/m, each intensity was attenuated 1 log by the neutral filter, the single light duration was 10 µs, and the stimulation interval was 12 s. Double channel synchronous sampling recording was performed, with frequency 1–1,000 Hz and amplification 10,000 times. The measurement was repeated for each light intensity at least 3 times.

Statistical analysis

SPSS 23.0 statistical software was used for analysis. The obtained data were expressed in the form of mean ± standard deviation (SD). Analysis of variance was used for comparisons between multiple groups. The independent sample t-test between two groups was performed for indicators with statistically significant results of analysis of variance. P<0.05 was considered statistically significant.

Results

GE protects the mitochondrial function of RPE cells under hyperglycemia

The CCK-8 assay showed that the activity of RPE cells was significantly decreased under hyperglycemia (P<0.05), but increased after adding GE (P<0.05; Figure 1A). RT-PCR results showed that the expression of CYT C1 in RPE cells under hyperglycemia was significantly increased (P<0.05), and the expression of CYT C1 in RPE cells was significantly decreased after adding GE (P<0.05; Figure 1B). The comparison of energy metabolism analysis results showed that the basal respiration rate, maximum respiration capacity, and adenosine triphosphate (ATP) synthesis capacity were significantly decreased under hyperglycemia (P<0.05), and all significantly increased after adding GE (P<0.05). Energy metabolism analysis showed that mitochondrial permeability increased in RPE cells under hyperglycemia (P<0.05), and decreased significantly after adding GE (P<0.05; Figure 1C-1G).

Figure 1 GE protects mitochondrial function in RPE cells induced by high glucose. (A) The CCK-8 assay was used to detect the changes in cell activity after high glucose combined with GE treatment under hyperglycemia; (B) RT-PCR was used to detect the changes in CYT C1 expression after high glucose combined with GE treatment under hyperglycemia; (C) the results of Seahorse cell energy metabolism analysis; (D-G) energy metabolism analysis results showed the changes in the basal respiration rate, maximum respiratory capacity, and ATP synthesis capacity after high glucose combined with GE treatment under hyperglycemia (***P<0.01, **P<0.05, *P<0.1). OD, optical density; GE, genipin; CCK-8, Cell Counting Kit-8; OCR, O₂ consumption rate; ATP, adenosine triphosphate; RPE, retinal pigment epithelial; RT-PCR, reverse transcription-polymerase chain reaction.

WB results showed that the expression of CYT C1 was significantly increased under hyperglycemia (P<0.05), and decreased significantly after adding GE (P<0.05; Figure 2A,2B). The expression of AKT in RPE cells was significantly increased after adding GE (Figure 2C).

Figure 2 Changes in the expression of CYT C1 and AKT in RPE cells under GE treatment and hyperglycemia. (A) WB was used to detect CYT C1 and AKT expression in cells of each group; (B,C) WB was used to detect the expression changes of CYT C1 and AKT in each group after high glucose combined with GE treatment under hyperglycemia (**P<0.05). GE, genipin; RPE, retinal pigment epithelial; WB, western blot.

GE regulates mitochondrial damage in hyperglycemia through the AKT signaling pathway

This study further clarified the specific mechanism of GE in protecting RPE cells against mitochondrial damage under hyperglycemia by adding an AKT pathway inhibitor in the environment of GE. CCK-8 assay results showed that RPE cell activity decreased under hyperglycemia, and increased after adding GE. After adding GE and AKT inhibitors, the cell activity decreased significantly (P<0.05; Figure 3A). RT-PCR results showed that the expression of CYT C1 increased in RPE cells under hyperglycemia, decreased significantly after adding GE, and increased significantly after adding GE and AKT inhibitor (P<0.05; Figure 3B). The results of energy metabolism analysis showed that the basal respiration rate, maximum respiration capacity, and ATP synthesis ability decreased significantly under hyperglycemia (P<0.05), though increased significantly after adding GE, and decreased significantly after adding GE and AKT inhibitors (P<0.05). Mitochondrial permeability increased in RPE cells under hyperglycemia (P<0.05), decreased significantly after adding GE (P<0.05), and increased significantly after adding GE and AKT inhibitor (P<0.05; Figure 3C-3G).

Figure 3 GE protects mitochondrial function in RPE cells under hyperglycemia through the AKT signaling pathway. (A) The CCK-8 assay was used to detect the changes in RPE cell activity after adding GE, AKT inhibitor, and GE combined with AKT inhibitor under hyperglycemia; (B) RT-PCR was used to detect the changes of CYT C1 expression in RPE cells after adding GE, AKT inhibitor, and GE combined with AKT inhibitor under hyperglycemia; (C) the results of Seahorse cell energy metabolism analysis; (D-G) energy metabolism analysis results showed the changes in the basal respiration rate and maximum respiration after adding GE, AKT inhibitor, and GE combined with AKT inhibitor under hyperglycemia capacity, ATP synthesis capacity (***P<0.01, **P<0.05). OD, optical density; GE, genipin; CCK-8, Cell Counting Kit-8; OCR, O₂ consumption rate; ATP, adenosine triphosphate; RPE, retinal pigment epithelial; RT-PCR, reverse transcription-polymerase chain reaction.

WB results showed that the expression of CYT C1 was increased in RPE cells under hyperglycemia, and decreased significantly after adding GE, while the expression of CYT C1 was significantly increased after adding GE and AKT inhibitor (P<0.05; Figure 4A,4B). The expression of AKT in RPE cells increased significantly after adding GE (P<0.05), and decreased significantly after adding GE and AKT inhibitor (P<0.05; Figure 4C).

Figure 4 GE regulates the expression changes of CYT C1, AKT and JAK2 in RPE cells under hyperglycemia through AKT. (A) WB was used to detect CYT C1 and AKT expression in cells of each group; (B,C) WB was used to detect the expression changes of CYT C1 and AKT in each group after adding GE and AKT inhibitor under hyperglycemia (**P<0.05, *P<0.1). GE, genipin; RPE, retinal pigment epithelial; WB, western blot.

GE regulates the mitochondrial damage of RPE cells under hyperglycemia by mediating JAK2 through the AKT pathway

To further clarify the downstream molecules regulated by GE through AKT, we obtained the GSE35934 dataset from the GEO database through bioinformatics analysis. It was mainly used to analyze the mRNA expression changes during GE treatment, and GEO2R was used to obtain differentially expressed genes (Figure 5A,5B). Finally, 76 significantly differentially expressed genes were obtained by setting the threshold. Meanwhile, 326 genes related to the KEGG-AKT pathway were obtained through KEGG analysis. By taking the intersection of Venn diagrams, we obtained 2 differentially expressed downstream proteins of AKT, namely RPRL and JAK2 (Figure 5C,5D). RPRL is mainly related to breast diseases, while JAK2 is related to cell activities such as apoptosis. Therefore, in this study, JAK2 was considered to be a downstream molecule of AKT after GE treatment of high glucose-induced RPE cell damage.

Figure 5 Bioinformatics analysis predicts the mechanism by which GE protects against RPE mitochondrial damage under hyperglycemia. (A) A cluster diagram was used to evaluate the expression changes of intracellular molecules after adding GE under hyperglycemia; (B) a volcano plot was used to display differentially expressed genes; (C) PPI network was used to select core targets in differentially expressed genes; (D) a Venn diagram was used to identify the shared genes of differentially expressed genes and AKT pathway-related genes. UMAP, Uniform Manifold Approximation and Projection; GE, genipin; FC, fold change; DEGs, differentially expressed genes; RPE, retinal pigment epithelial; PPI, protein-protein interaction.

CCK-8 assay results showed that the cell activity was significantly decreased after adding GE and AKT inhibitor (P<0.05). There was no significant decrease in cell activity after adding GE and JAK2 inhibitor. The cell activity was still higher after adding GE combined with AKT inhibitor and JAK2 inhibitor (P<0.05; Figure 6A). RT-PCR results showed that the expression of CYT C1 was significantly decreased after adding GE, though increased after adding GE and AKT inhibitors (P<0.05), and the expression of CYT C1 was significantly decreased compared with that under hyperglycemia after adding GE and JAK2 inhibitor (P<0.05). The expression of CYT C1 was significantly decreased compared with that under hyperglycemia after adding GE combined with AKT inhibitor and JAK2 inhibitor (P<0.05; Figure 6B). The results of energy metabolism analysis showed that the basal respiration rate, maximum respiration capacity, and ATP synthesis capacity of RPE cells were significantly increased after adding GE, and decreased significantly after adding GE and AKT inhibitor (P<0.05). The basal respiration rate, maximum respiration capacity, and ATP synthesis capacity of RPE cells were significantly decreased compared with that under hyperglycemia after adding GE and JAK2 inhibitor (P<0.05), and the same result was also obtained after adding GE combined with AKT inhibitor and JAK2 inhibitor (P<0.05). Mitochondrial permeability was significantly decreased after adding GE (P<0.05), while mitochondrial permeability was significantly increased after adding GE and AKT inhibitor (P<0.05). Mitochondrial permeability was significantly decreased after adding GE and JAK2 inhibitor (P<0.05), and mitochondrial permeability was also significantly decreased (P<0.05) after adding GE combined with AKT inhibitor and JAK2 inhibitor (Figure 6C-6G).

Figure 6 GE protects mitochondrial function in RPE cells under hyperglycemia via AKT/JAK2. (A) The CCK-8 assay was used to detect the changes in RPE cell activity after adding GE, AKT inhibitor, JAK2 inhibitor, and GE combined with AKT inhibitor and JAK2 inhibitor under hyperglycemia; (B) RT-PCR was used to detect the expression changes of CYT C1 in RPE cells after adding GE, AKT inhibitor, JAK2 inhibitor, and GE combined with AKT inhibitor and JAK2 inhibitor under hyperglycemia; (C) the results of Seahorse cell energy metabolism analysis; (D-G) energy metabolism analysis results showed the changes in the basal respiratory rate, maximal respiratory capacity, and ATP synthesis capacity after adding GE, AKT inhibitor, and GE combined with AKT inhibitor under hyperglycemia (***P<0.01, **P<0.05, *P<0.1). OD, optical density; GE, genipin; CCK-8, Cell Counting Kit-8; OCR, O₂ consumption rate; ATP, adenosine triphosphate; RPE, retinal pigment epithelial; RT-PCR, reverse transcription-polymerase chain reaction.

WB results showed that the expression of CYT C1 was significantly decreased after adding GE compared with that under hyperglycemia. The expression of CYT C1 was significantly increased after adding GE and AKT inhibitor, decreased after adding GE and JAK2 inhibitor, and decreased after adding GE combined with AKT inhibitor and JAK2 inhibitor (P<0.05; Figure 7A,7B). The expression of AKT in RPE cells was significantly increased after adding GE (P<0.05), and decreased after adding GE and AKT inhibitor (P<0.05). The expression of AKT was not significantly different from the GE group after adding JAK2 inhibitor, but the expression of AKT decreased significantly after adding GE combined with AKT inhibitor and JAK2 inhibitor (P<0.05; Figure 7C). The expression of JAK2 was significantly decreased compared with that under hyperglycemia after adding GE, and increased significantly after adding GE and AKT inhibitor. The expression of JAK2 was significantly decreased after adding GE and JAK2 inhibitor, and also decreased (P<0.05) after adding GE combined with AKT inhibitor and JAK2 inhibitor (Figure 7D).

Figure 7 GE regulates the changes in the expression of CYT C1, AKT and JAK2 in RPE cells under hyperglycemia through AKT/JAK2. (A) WB was used to detect CYT C1, AKT and JAK2 expression in each group; (B-D) WB was used to detect the expression changes of CYT C1 and AKT in each group after adding GE, AKT inhibitor, JAK2 inhibitor, and GE combined with AKT inhibitor and JAK2 inhibitor under hyperglycemia (**P<0.05). GE, genipin; RPE, retinal pigment epithelial; WB, western blot.

GE protects against the mitochondrial damage of RPE cells under hyperglycemia by mediating miR-4429/JAK2 through the AKT pathway

In order to further clarify the specific mechanism of GE-mediated AKT in the protection of RPE mitochondrial function under hyperglycemia by inhibiting JAK2, this study predicted JAK2-targeted miRNAs using TargetScan 7.2, MIRDB, and Starbase 3.0 and obtained the intersection. Finally, 25 miRNAs were obtained (Figure 8A). Based on the literature review, miR-4429 was shown to be a downstream miRNA molecule of AKT. The potential binding site of miR-4429 and JAK2 was predicted by online databases (Figure 8B). Therefore, this study suggested that GE protects against the mitochondrial damage of RPE cells under hyperglycemia by mediating miR-4429/JAK2 through the AKT pathway.

Figure 8 Bioinformatics analysis identifies miRNAs that have a targeted regulatory relationship with JAK2. (A) Targetscan 7.2, miRDB, and Starbase 3.0 were used to predict miRNAs that have a targeting relationship with JAK2; (B) the targeted binding site of JAK2 to miR-4429. MiRNA, microRNA.

In this study, the corresponding hypothesis was verified by constructing miR-4429-mimics and si-miR-4429 lentiviral vectors and combining inhibited or activated with AKT and JAK2 signaling. CCK-8 assay results showed that compared with adding GE under hyperglycemia, the cell activity was significantly inhibited after adding AKT inhibitor, increased after adding miR-4429-mimics, and inhibited after adding si-miR-4429 (P<0.05; Figure 9A). RT-PCR showed that the expression of CYT C1 was significantly increased after adding AKT inhibitor, decreased after adding miR-4429-mimics, and increased after adding si-miR-4429 (P<0.05; Figure 9B). RT-PCR showed that the expression of miR-4429 was significantly increased after adding GE, and inhibited after adding AKT inhibitor (P<0.05; Figure 9C). The results of energy metabolism analysis showed that the basal respiration rate, maximum respiration capacity, and ATP synthesis capacity were significantly decreased after adding AKT inhibitor (P<0.05), increased after adding miR-4429-mimics, and decreased after adding si-miR-4429 (P<0.05). Mitochondrial permeability was significantly increased after adding AKT inhibitor, decreased after adding miR-4429-mimics, and increased after adding si-miR-4429 (P<0.05; Figure 9D-9H).

Figure 9 GE protects mitochondrial function in RPE cells under hyperglycemia through AKT/miR-4429/JAK2. (A) The CCK-8 assay was used to detect the changes in RPE cell activity after adding GE, GE combined with AKT inhibitor, and GE combined with miR-4429-mimics, si-miR-4429, and JAK2 inhibitor under hyperglycemia; (B) RT-PCR was used to detect the expression changes of CYT C1 in RPE cells after adding GE, GE combined with AKT inhibitor, and GE combined with miR-4429-mimics, si-miR-4429, and JAK2 inhibitor under hyperglycemia; (C) RT-PCR was used to detect the expression changes of miR-4429 in RPE cells after adding GE, GE combined AKT inhibitor, and GE combined with miR-4429-mimics, si-miR-4429, and JAK2 inhibitor under hyperglycemia; (D) the results of Seahorse cell energy metabolism analysis; (E-H) energy metabolism analysis results showed the changes in the basal respiratory rate, maximal respiratory capacity, and ATP synthesis capacity after adding GE, GE combined with AKT inhibitor, and GE combined with miR-4429-mimics, si-miR-4429, and JAK2 inhibitor (*P<0.1). OD, optical density; GE, genipin; mir, miR-4429; CCK-8, Cell Counting Kit-8; OCR, O₂ consumption rate; ATP, adenosine triphosphate; RPE, retinal pigment epithelial; RT-PCR, reverse transcription-polymerase chain reaction.

WB was used to detect the expression of CYT C1, AKT, and JAK2 (Figure 10A). The expression of CYT C1 was significantly increased after adding AKT inhibitor, decreased after adding miR-4429-mimics, and increased after adding si-miR-4429 (P<0.05; Figure 10B). The expression of AKT was significantly decreased after adding AKT inhibitor (P<0.05). After adding miR-4429-mimics, si-miR-4429, and JAK2 inhibitor, the expression of AKT was significantly increased compared with adding AKT inhibitor (P<0.05). Meanwhile, there was no statistical difference in the GE group under hyperglycemia (P>0.05; Figure 10C). The expression of JAK2 was significantly increased after adding AKT inhibitor, decreased after adding miR-4429-mimics, and increased after adding si-miR-4429 (P<0.05; Figure 10D). The potential binding site of miR-4429 and JAK2 was predicted by online databases, and the dual luciferase gene reporter assay confirmed that miR-4429 targeted JAK2 binding (Figure 10E,10F).

Figure 10 GE regulates the expression of CYT C1, AKT and JAK2 changes in RPE cells under hyperglycemia through AKT/miR-4429/JAK2. (A) WB was used to detect the expression of CYT C1, AKT and JAK2 in each group; (B-D) WB was used to detect the expression changes of CYT C1 and AKT after adding GE, GE combined with AKT inhibitor, and GE combined with miR-4429-mimics, si-miR-4429, and JAK2 inhibitor in each group (**P<0.05); (E,F) dual luciferase gene reporter assay confirmed that miR-4429 targeted JAK2 binding. GE, genipin; mir, miR-4429; RPE, retinal pigment epithelial; WB, western blot.

Animal experiments verified the specific molecular mechanism of GE in the protection of RPE cells in diabetic mice

The results of ERG showed the intensity and amplitude of ERG wave A and wave B in each group. The results showed that the amplitudes of A and B waves in diabetic mice decreased significantly (P<0.05), while the amplitudes of A and B waves in diabetic mice increased significantly after adding GE (P<0.05; Figure 11A,11B). RT-PCR results showed that after adding GE, the expression of AKT and miR-4429 in RPE cells was significantly increased (P<0.05), while the expression of JAK2 was significantly decreased (P<0.05; Figure 11C-11E).

Figure 11 The effect of GE on RPE cells in diabetic mice. (A,B) ERG was used to detect the changes of a-wave and b-wave light intensity-amplitude in control diabetic mice and diabetic mice after GE treatment; (C-E) RT-PCR was used to detect the expression changes of AKT, miR-4429, and JAK2 in RPE cells of control diabetic mice and diabetic mice after GE treatment (***P<0.01). ERG, electroretinogram; GE, genipin; RPE, retinal pigment epithelial; RT-PCR, reverse transcription-polymerase chain reaction.

Discussion

In this study, bioinformatics analysis predicted that GE may protect RPE cells from damage by high glucose through AKT/miR-4429/JAK2. CCK-8 experiments showed that GE could regulate the miR-4429/JAK2 signaling axis through the AKT pathway to protect RPE cell activity under hyperglycemia. Cell energy metabolism detection demonstrated that GE could regulate the miR-4429/JAK2 signaling axis through the AKT pathway to protect mitochondrial function in RPE cells under hyperglycemia. Animal experiments were conducted to verify the molecular mechanism of the effect of GE on DR lesions in diabetic mice. Its specific regulatory molecular mechanism in RPE cells is shown in Figure 12.

Figure 12 Research hypothesis. GE mediates the miR-4429/JAK2 signaling axis through the AKT pathway to protect mitochondrial damage in RPE cells under hyperglycemia. GE, genipin; RPE, retinal pigment epithelial.

The retinal pigment epithelium, as the monolayer of the most important nerve cells (photoreceptors) in the adjacent retinal tissue, plays an important role in the development of DR’s characteristic vascular lesions (3). A study has shown that the retinal pigment epithelium is involved in early retinal microangiopathy (5). Oxidative stress and inflammatory responses are the main pathological changes of diabetic microangiopathy. As the organ and tissue with the highest oxygen consumption rate in the body, the retina is also more vulnerable to oxidative stress attack and thus cell damage (18). Studies have shown that oxidative stress promotes RPE cell damage mainly through affecting mitochondrial function (19). For example, mitochondrial energy metabolism changes, and the mitochondrial permeability transition pore opens, which releases CYT C1 and causes apoptosis (20). As an effective antioxidant, GE has a certain protective effect on mitochondrial function. In this study, RPE cells were cultured under hyperglycemia, and the results showed that the cell activity was significantly decreased, the expression of CYT C1 was significantly increased, mitochondrial function was significantly impaired, and mitochondrial permeability was increased. After the addition of GE, the activity of RPE cells increased, the expression of CYT C1 and inflammatory response-related proteins decreased significantly, mitochondrial function recovered, and mitochondrial permeability decreased. These results suggest that GE can effectively protect mitochondrial function, protect cell activity, and inhibit the inflammatory responses of RPE cells under hyperglycemia.

In this study, we cultured RPE cells with high glucose and added GE and AKT signaling pathway inhibitors. The results showed that the activity of RPE cells was significantly inhibited after the inhibition of the AKT signaling pathway, and the expression of CYT C1 and inflammatory response-related proteins was significantly increased. Meanwhile, mitochondrial energy metabolism was decreased, and mitochondrial permeability was significantly increased. These results showed that after the AKT signaling pathway was inhibited, the protective effect of GE on RPE cell activity, inflammatory responses, and mitochondrial function was significantly reduced in hyperglycemia. The AKT signaling pathway is closely related to the proliferation, differentiation, and apoptosis of cells in the body (21). A study has shown that the AKT signaling pathway is closely related to the progression of DR. Curcumin can protect against retinal damage in hyperglycemia through the AKT/mTOR signaling pathway (22). At the same time, a study has also shown that connective tissue growth factor can protect the functional activity of RPE cells through the AKT signaling pathway (23). Glycyrrhizin can protect RPE cells and retinal damage through the AKT signaling pathway (24). These results indicate that the AKT signaling pathway plays an important guiding role in the regulation of the functional activity of RPE cells. Meanwhile, changes in the expression of the AKT signaling pathway are closely related to changes in the mitochondrial function of RPE cells. A study has shown that pigment epithelial-derived factor (PEDF) can protect the mitochondrial function of RPE cells through the AKT signaling pathway (25). Lutein can protect cell activity through the AKT signaling pathway and effectively protect against mitochondrial energy metabolism changes (26). On this basis, this study suggests that GE mainly protects RPE cell activity, inhibits inflammatory responses, and protects mitochondrial function under hyperglycemia through the AKT signaling pathway.

As a classical signaling pathway, the AKT signaling pathway has numerous downstream proteins. The therapeutic effect of AKT on RPE cells needs to be clarified in terms of through which protein molecule it primarily achieves its therapeutic effect. With the development of information technology, we believe that bioinformatics analysis can preliminarily identify the downstream proteins mainly regulated by the AKT signaling pathway during GE treatment of RPE cells. In this study, the differentially expressed genes after GE treatment of RPE cells were screened through databases, and the downstream proteins of the AKT signaling pathway were obtained through the KEGG website. After the intersection of differentially expressed genes and the AKT signaling pathway, the results showed that RPRL and JAK2 were the downstream molecules of differentially expressed AKT. RPRL is closely related to the development of breast system diseases (13). JAK2 is closely related to the body’s inflammatory response and mitochondrial function damage (14). A study has suggested that interleukin-2 (IL-2) can regulate the expression of inflammatory cytokines in RPE cells through JAK2 and reduce the activity of RPE cells (27). Glycosylated serum protein can promote the secretion of inflammatory cytokines (IL-8) and the expression of MCP-1 by RPE cells through JAK2, thus affecting the function and activity of RPE cells (28). At the same time, a study has shown that the expression level of JAK2 is closely related to mitochondrial function, and extracellular IL-6 can affect the physiological function of intracellular mitochondria by regulating the expression of JAK2 (29). High glucose stimulation can cause mitochondrial dysfunction by promoting the expression of JAK2 (30). On this basis, this study suggests that GE may regulate the expression of JAK2 through the AKT pathway to protect RPE cells from injury under hyperglycemic conditions. How does the AKT protein regulate JAK2 expression? This study suggests that AKT may exert a certain influence on JAK expression by regulating the expression changes of intracellular miRNAs.

MiRNAs, as a class of non-coding RNAs with about 18–22 nucleotide sequences, are closely related to the functional expression of body cells (31). In order to identify the miRNAs with targeted regulation by JAK2, this study retrieved and intersected data from the TargetScan, miRDB, and DIANA databases, and finally obtained miR-4429 as a miRNA that may have a targeted relationship with JAK2 mRNA. MiR-4429 has been thoroughly studied by previous researchers as a recognized tumor suppressor gene. A study has shown that miR-4429 exists as a tumor suppressor in the progression of various malignant tumors. In glioblastoma cells, inhibiting the expression of miR-4429 can accelerate the growth of cancer cells (32). MiR-4429 is downregulated in cervical cancer and can target the DNA double-strand break repair protein (RAD51) to sensitize cervical cancer cells to radiotherapy (33). MiR-4429 inhibits cervical cancer cell proliferation, migration, and invasion by targeting FOXM1. Long non-coding RNA (lncRNA) PSMA3-AS1 promotes the migration and invasion of colorectal cancer cells by regulating miR-4429 (34). At the same time, the results also showed that miR-4429 was also closely related to intracellular energy metabolism. Overexpression of miR-4429 can reduce glucose consumption, lactic acid production, and the protein levels of PKM2 and HK2 in breast cancer cells, as well as increase the inhibition rate of cell proliferation and decrease the number of clones, suggesting that overexpression of miR-4429 can inhibit the glycolysis, lysis, proliferation, and clone formation of breast cancer cells (35,36). However, whether miR-4429 has regulatory effects on cell activity, function, and mitochondrial energy metabolism in high glucose-induced RPE cell damage has not been reported before. The results of this study showed that when miR-4429 was overexpressed during GE treatment, the activity of RPE cells was significantly increased, while the expression of inflammatory factors was significantly decreased, and the detection results of WB and mitochondrial energy metabolism showed that the mitochondrial energy metabolism function was significantly improved. Meanwhile, the expression of JAK2 was also significantly increased. However, after JAK2 inhibition during GE treatment, the activity of RPE cells was significantly increased, while the expression of inflammatory factors was significantly decreased, and the results of WB and mitochondrial energy metabolism showed that the mitochondrial energy metabolism function was significantly improved. Additionally, the expression of miR-4429 had no significant effect. On this basis, the dual luciferase reporter assay was used to further prove that miR-4429 has a targeted binding effect with JAK2. Therefore, this study suggested that GE could protect RPE cells from injury in hyperglycemia through the miR-4429/JAK2 signaling axis. In order to further clarify the regulatory relationship between the AKT signaling pathway and the miR-4429/JAK2 signaling axis, this study further compared and analyzed the changes in the function and metabolism of cells after adding AKT inhibitor and overexpression/knockdown of miR-4429. The results showed that the expression of miR-4429 was significantly reduced after adding AKT inhibitor, while there was no significant difference in the expression of AKT after overexpression/knockdown of miR-4429, suggesting that miR-4429 is a downstream molecule of AKT. In order to further verify the therapeutic mechanism of GE for DR, this study established a diabetic mouse model to further clarify the therapeutic effect of GE on DR and changes in molecular expression during treatment.

Conclusions

In this study, bioinformatics analysis was used to predict the molecular mechanism of GE in the treatment of RPE cell damage caused by high glucose. Through the addition of AKT inhibitors, knockdown/overexpression by lentivirus infection, and other methods, we demonstrated that GE can regulate the expression changes of the miR-4429/JAK2 signaling axis through the AKT signaling pathway, thus realizing the protective effect on the function, activity, and mitochondrial damage of RPE cells under hyperglycemia.

Acknowledgments

Funding: This study was funded by Science and Technology Joint Guidance Program of Qiqihar, China (No. LHYD-202012) and Basic Scientific Research Business Expenses, Research Project of Heilongjiang Provincial Undergraduate College (No. 2019-KYYWF-1253).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-2219/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-2219/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-2219/coif). All authors report that this study was funded by Science and Technology Joint Guidance Program of Qiqihar, China (No. LHYD-202012) and Basic Scientific Research Business Expenses, Research Project of Heilongjiang Provincial Undergraduate College (No. 2019-KYYWF-1253). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). Animal experiments were performed under a project license (No. QMU-AECC-2021-244) granted by Animal Ethical Committee of Qiqihar Medical University, in compliance with national guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Qi X, Mitter SK, Yan Y, et al. Diurnal Rhythmicity of Autophagy Is Impaired in the Diabetic Retina. Cells 2020;9:905. [Crossref] [PubMed]
2. Schmidt-Erfurth U, Garcia-Arumi J, Bandello F, et al. Guidelines for the Management of Diabetic Macular Edema by the European Society of Retina Specialists (EURETINA). Ophthalmologica 2017;237:185-222. [Crossref] [PubMed]
3. Potilinski MC, Ortíz GA, Salica JP, et al. Elucidating the mechanism of action of alpha-1-antitrypsin using retinal pigment epithelium cells exposed to high glucose. Potential use in diabetic retinopathy. PLoS One 2020;15:e0228895. [Crossref] [PubMed]
4. Dolinko AH, Chwa M, Atilano SR, et al. African and Asian Mitochondrial DNA Haplogroups Confer Resistance Against Diabetic Stresses on Retinal Pigment Epithelial Cybrid Cells In Vitro. Mol Neurobiol 2020;57:1636-55. [Crossref] [PubMed]
5. Kim DY, Kang MK, Lee EJ, et al. Eucalyptol Inhibits Amyloid-β-Induced Barrier Dysfunction in Glucose-Exposed Retinal Pigment Epithelial Cells and Diabetic Eyes. Antioxidants (Basel) 2020;9:1000. [Crossref] [PubMed]
6. Zhang C, Xie H, Yang Q, et al. Erythropoietin protects outer blood-retinal barrier in experimental diabetic retinopathy by up-regulating ZO-1 and occludin. Clin Exp Ophthalmol 2019;47:1182-97. [Crossref] [PubMed]
7. Tian JS, Zhao L, Shen XL, et al. 1H NMR-based metabolomics approach to investigating the renal protective effects of Genipin in diabetic rats. Chin J Nat Med 2018;16:261-70. [Crossref] [PubMed]
8. Li F, Song L, Chen J, et al. Effect of genipin-1-β-d-gentiobioside on diabetic nephropathy in mice by activating AMP-activated protein kinase/silencing information regulator-related enzyme 1/nuclear factor-κB pathway. J Pharm Pharmacol 2021;73:1201-11. [Crossref] [PubMed]
9. Zhang J, Wang YN, Jia T, et al. Genipin and insulin combined treatment improves implant osseointegration in type 2 diabetic rats. J Orthop Surg Res 2021;16:59. [Crossref] [PubMed]
10. Qiu W, Zhou Y, Jiang L, et al. Genipin inhibits mitochondrial uncoupling protein 2 expression and ameliorates podocyte injury in diabetic mice. PLoS One 2012;7:e41391. [Crossref] [PubMed]
11. Zhao H, Wang R, Ye M, et al. Genipin protects against H2O2-induced oxidative damage in retinal pigment epithelial cells by promoting Nrf2 signaling. Int J Mol Med 2019;43:936-44. [PubMed]
12. Lai JY. Biocompatibility of genipin and glutaraldehyde cross-linked chitosan materials in the anterior chamber of the eye. Int J Mol Sci 2012;13:10970-85. [Crossref] [PubMed]
13. McKinnie SMK, Wang W, Fischer C, et al. Synthetic Modification within the "RPRL" Region of Apelin Peptides: Impact on Cardiovascular Activity and Stability to Neprilysin and Plasma Degradation. J Med Chem 2017;60:6408-27. [Crossref] [PubMed]
14. Tan JK, Ma XF, Wang GN, et al. LncRNA MIAT knockdown alleviates oxygen-glucose deprivation-induced cardiomyocyte injury by regulating JAK2/STAT3 pathway via miR-181a-5p. J Cardiol 2021;78:586-97. [Crossref] [PubMed]
15. Wei J, Wang Z, Wang W, et al. Oxidative Stress Activated by Sorafenib Alters the Temozolomide Sensitivity of Human Glioma Cells Through Autophagy and JAK2/STAT3-AIF Axis. Front Cell Dev Biol 2021;9:660005. [Crossref] [PubMed]
16. Garcia-Ramírez M, Hernández C, Ruiz-Meana M, et al. Erythropoietin protects retinal pigment epithelial cells against the increase of permeability induced by diabetic conditions: essential role of JAK2/PI3K signaling. Cell Signal 2011;23:1596-602. [Crossref] [PubMed]
17. Platania CBM, Maisto R, Trotta MC, et al. Retinal and circulating miRNA expression patterns in diabetic retinopathy: An in silico and in vivo approach. Br J Pharmacol 2019;176:2179-94. [PubMed]
18. Yu B, Ma J, Li J, et al. Mitochondrial phosphatase PGAM5 modulates cellular senescence by regulating mitochondrial dynamics. Nat Commun 2020;11:2549. [Crossref] [PubMed]
19. Somasundaran S, Constable IJ, Mellough CB, et al. Retinal pigment epithelium and age-related macular degeneration: A review of major disease mechanisms. Clin Exp Ophthalmol 2020;48:1043-56. [Crossref] [PubMed]
20. Brown EE, DeWeerd AJ, Ildefonso CJ, et al. Mitochondrial oxidative stress in the retinal pigment epithelium (RPE) led to metabolic dysfunction in both the RPE and retinal photoreceptors. Redox Biol 2019;24:101201. [Crossref] [PubMed]
21. Chen Q, Tang L, Xin G, et al. Oxidative stress mediated by lipid metabolism contributes to high glucose-induced senescence in retinal pigment epithelium. Free Radic Biol Med 2019;130:48-58. [Crossref] [PubMed]
22. Qin D, Jiang YR. Tangeretin Inhibition of High-Glucose-Induced IL-1β, IL-6, TGF-β1, and VEGF Expression in Human RPE Cells. J Diabetes Res 2020;2020:9490642. [Crossref] [PubMed]
23. Zha X, Xi X, Fan X, et al. Overexpression of METTL3 attenuates high-glucose induced RPE cell pyroptosis by regulating miR-25-3p/PTEN/Akt signaling cascade through DGCR8. Aging (Albany NY) 2020;12:8137-50. [Crossref] [PubMed]
24. Wang T, Zhang Z, Song C, et al. Astragaloside IV protects retinal pigment epithelial cells from apoptosis by upregulating miR-128 expression in diabetic rats. Int J Mol Med 2020;46:340-50. [Crossref] [PubMed]
25. Su CC, Chan CM, Chen HM, et al. Lutein inhibits the migration of retinal pigment epithelial cells via cytosolic and mitochondrial Akt pathways (lutein inhibits RPE cells migration). Int J Mol Sci 2014;15:13755-67. [Crossref] [PubMed]
26. Shukal D, Bhadresha K, Shastri B, et al. Dichloroacetate prevents TGFβ-induced epithelial-mesenchymal transition of retinal pigment epithelial cells. Exp Eye Res 2020;197:108072. [Crossref] [PubMed]
27. Parmar T, Parmar VM, Arai E, et al. Acute Stress Responses Are Early Molecular Events of Retinal Degeneration in Abca4-/-Rdh8-/- Mice After Light Exposure. Invest Ophthalmol Vis Sci 2016;57:3257-67. [Crossref] [PubMed]
28. Shen CH, Hsieh CC, Jiang KY, et al. AUY922 induces retinal toxicity through attenuating TRPM1. J Biomed Sci 2021;28:55. [Crossref] [PubMed]
29. Li R, Wen R, Banzon T, et al. CNTF mediates neurotrophic factor secretion and fluid absorption in human retinal pigment epithelium. PLoS One 2011;6:e23148. [Crossref] [PubMed]
30. Chen X, Sun R, Yang D, et al. LINC00167 Regulates RPE Differentiation by Targeting the miR-203a-3p/SOCS3 Axis. Mol Ther Nucleic Acids 2020;19:1015-26. [Crossref] [PubMed]
31. Qi T, Jing R, Wen C, et al. Interleukin-6 promotes migration and extracellular matrix synthesis in retinal pigment epithelial cells. Histochem Cell Biol 2020;154:629-38. [Crossref] [PubMed]
32. Duncan RS, Rohowetz L, Vogt A, et al. Repeat exposure to polyinosinic:polycytidylic acid induces TLR3 expression via JAK-STAT signaling and synergistically potentiates NFκB-RelA signaling in ARPE-19 cells. Cell Signal 2020;66:109494. [Crossref] [PubMed]
33. Jing R, Qi T, Wen C, et al. Interleukin-2 induces extracellular matrix synthesis and TGF-β2 expression in retinal pigment epithelial cells. Dev Growth Differ 2019;61:410-8. [Crossref] [PubMed]
34. Patel AK, Syeda S, Hackam AS. Signal transducer and activator of transcription 3 (STAT3) signaling in retinal pigment epithelium cells. JAKSTAT 2013;2:e25434. [Crossref] [PubMed]
35. Li R, Maminishkis A, Banzon T, et al. IFN{gamma} regulates retinal pigment epithelial fluid transport. Am J Physiol Cell Physiol 2009;297:C1452-65. [Crossref] [PubMed]
36. Kucharska J, Del Río P, Arango-Gonzalez B, et al. Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa. J Neurochem 2014;130:227-40. [Crossref] [PubMed]

(English Language Editor: C. Betlazar-Maseh)