The Relationship between Expression of Nuclear Factor I and the Progressive Occurrence of Diabetic Retinopathy.

The loss of nuclear factor I (NFI) function can lead to defects in Muller's glial differentiation, abnormalities of retinal morphology, and changes in retinal neurons numbers, which are highly involved in diabetic retinopathy (DR). In this study, we addressed the roles of NFIA and NFIB gene expression in the development of DR by using diabetes mellitus (DM) rat models. Retinal histologies were examined, and the expression of NFIA and NFIB at mRNA and protein levels was detected. The results showed that retinal edema and disordered cell arrangement frequently occurred in DR rats. The expressions of NFIA and NFIB in retinal tissue were significantly decreased in DM rats with DR complications. After further inhibiting the expression of NFIA gene in DM rats by using RNA-silencing, majority of DM rats occurred retinopathy and lens fibrosis, which indicated the relationship between decreased expression of NFI and occurrence of retinopathy in DM.

1. Introduction

Diabetes mellitus (DM) is a metabolic disease caused by defects in insulin secretion and/or utilization and characterized by chronic hyperglycemia [1]. The incidence of DM is increasing yearly worldwide, becoming one of the major diseases threatening human health. According to reports, the global prevalence of DM exceeded 400 million in 2017, and it is estimated that nearly 200 million people have undiagnosed DM [1]. The prevalence of DM in high-income countries is more than double that of low-income countries [2]. The main manifestations of DM are polydipsia, overeating, polyuria, and weight loss, with complications, such as diabetic retinopathy (DR), diabetic nephropathy, diabetic neuropathy, and diabetic foot [3]. Among these, DR, the most familiar chronic complication of DM [4], has become an important cause of visual impairment and blindness in DM patients [5]. DR accounts for 4.8% of the total global blindness cases (37 million), and the overall prevalence of DR in DM patients is 34.6% [6]. Due to high sugar content in blood vessels of DM patients, capillary cells will shrink and degenerate, leading to circulatory disturbances [7]. The retina has abundant blood circulation and is more prone to microvascular disease. Studies have confirmed that the biochemical pathways related to hyperglycemia, such as oxidative stress, polyol and hexosamine pathway activation, advanced glycosylation end product formation, and activation of protein kinase C isoforms, are related to the pathogenesis of DR [8, 9]. There are also studies showing that changes in retinal neurons are related to DR development [10], but its exact molecular mechanism is still unclear. Therefore, studying the molecular mechanism of DR is of great significance for its treatment.

Nuclear factor I (NFI) is an important regulator of mouse and human astrocytogenesis and neurogenesis [11, 12]. The NFI family contains four genes (NFIA, NFIB, NFIC, and NFIX). In the spinal cord, NFIA and NFIB promote the regulation of astrocytes by synergistically activating the transcription of NFIX, and the lack of NFIA, NFIB, or NFIX function leads to Muller glial differentiation defects and retinal morphology destruction [13, 14]. In addition, NFI is also necessary for the cessation of the late retinal neural progenitor cell cycle. Therefore, the expression of NFI is of great interesting to elucidate the mechanism of retinopathy development. In current studies, we aimed to explore the relationship between expression of NFIA/NFIB in neuroretinal cells and development of DR.

2. Material and Methods

2.1. Experimental Rats

Ninety clean normal SD rats weighing between 180 g and 200 g were selected. SD rats were brought from the Experimental Animal Center of Guangxi Medical University. The rats used in the study were adapted to feeding. The animal experiment conducted in this study meets the requirements of the Animal Experiment Ethics committee of involved institutions.

2.2. Establish DM Rat Models

Seventy rats were randomly selected for modeling, and 1.2 mL streptozotocin solution (10 mg/mL; Shanghai Lianmai Bioengineering Co., Ltd.) was injected intraperitoneally. The remaining 20 rats served as healthy controls and were injected with 1.2 mL of saline (concentration: 0.9%). After 4 weeks of high fat diet feeding (common feed 78.8%, cholesterol 1%, bovine bile salt 0.2%, egg yolk powder 10%, lard 10%), the fasting blood glucose content of rats was measured. When the fasting blood glucose is greater than the mean value of normal blood glucose +3 standard deviations, the model of diabetes is successful; 65 rats are successfully modeled, and 20 rats were selected as the DM group, while 45 rats continued to be fed with ordinary feed (corn 73.5%, wheat bran 20%, fish meal 5%, grain meal 1%, salt 0.5%) for 12 weeks. Twelve weeks later, 8 mL ketamine hydrochloride injection (drug specification: 0.1 g/2 mL, Jiangsu Hengrui Pharmaceutical Co., Ltd.) was given to sedate the rats. One drop of tropicamide (Jiangxi Zhenshiming Pharmaceutical Co., Ltd.) was dripped onto the eyes to dilate the pupils, and then, proparacaine hydrochloride eye drops (Nanjing Ruinian Best Pharmaceutical Co., Ltd.) were instilled to anesthetize the ocular surface. Fluorescein dextran isothiocyanate (Shanghai Yuanye Biotechnology Co., Ltd.) was injected into the tail vein for fundus angiography. If there are visible microvascular tumors in the fundus of the rat, it is judged as DM with retinopathy, of which 25 were successfully identified and defined as the DR group. The remaining 20 DM without retinopathy were used for NFIA gene interference experiment.

2.3. Histological Examination of the Retina

Ten rats were randomly taken from each of the healthy control, DM, and DR groups. The rats were intraperitoneally injected with 1% pentobarbital sodium 30 mg/kg and sacrificed by cervical push dislocation. The eyeball was removed, and the neuroretinal tissue was separated. Rats were dissected under an anatomical microscope along the limbus of the cornea to remove the cornea, lens, and vitreous and to strip the neuroretinal tissue, which was fixed in 10% formaldehyde. The fixative was rinsed with running water. The tissues were dehydrated by different concentrations of alcohol (low concentration to high concentration) and transparentized by xylene. The transparent tissue block was embedded in paraffin and sliced continuously with a thickness of 5 μm (HS-S7120-A paraffin slicing machine, Shenyang Hengsong Technology Co., Ltd.). The paraffin in slices was removed with xylene and then hydrated with high to low concentration of alcohol. HE staining (HE kit purchased from Solebo Biotechnology Co., LTD.). Tissue sections were placed in hematoxylin for staining for 5 min and then rinsed with water for 3 s. Then differentiated in 1% hydrochloric acid alcohol solution for 3s, rinsed with running water for 30s. Return 0.6% ammonia to blue for 30 s, and rinse with water for 15 min. The sections were dyed in eosin solution for 3 min. After dehydration and drying, seal the sheet with neutral gum. The morphology and structure of tissue were observed under microscope.

2.4. Isolation of Rat Retinal Cells

Ten rats were extracted from each of the healthy control, DM, and DR groups and sacrificed to extract the retinal tissue. Papain solution was added to separate the cells, incubated at 37°C for 10 min, and lightly ground every 2 min. An amount of 600 μL of 10% FBS neural basal medium was added to every 400 μL of dissociation solution. The sample was separated with a pipette, followed by DNAse processing every 5 min. The cells were centrifuged at 300 RCF for 5 min to pellet the cells. Depending on the required cell concentration in the suspension, the liquid was carefully aspirated from the cell pellets. Then, the pellet was resuspended in 1–5 mL neural basal medium (containing 1% FBS). The cells were filtered through a 50 μm filter to remove the cell aggregates. Retinal cells were analyzed by PCR and western blot.

2.5. PCR Detection

Nanomaterial PCR is a gene amplification technology assisted by nanomaterials. Gold nanomaterials can enhance the overall efficiency of gene amplification. The sensitivity and stability are relatively good. The total RNA of the cell was extracted using the RNA extraction kit (Invitrogen). Then, reverse transcription of RNA into cDNA was done using reverse transcription kit (Thermo Fisher). Using cDNA as a template, transcription was done according to All-in-one miRNA Q-PCR Detection Kit (GeneCopoeia), and qRT-PCR experiment was performed according to the procedure of SYBR-GreenMaster Mix kit (Beijing Unicom Biotechnology Co., Ltd.). The PCR reaction system was composed of 10 μL SYBR Green 1 dye, 1 μL upstream primer, 1 μL downstream primer, 1 μL dNTP, 2 μL Taq polymerase, 5 μL test sample cDNA, 30 μL ddH2O, and 1 nM gold nanoparticles (0.4 nM/20 μL). The size of the gold nanoparticles prepared was about 20 nm (Figure 1(a)), and the gold nano absorption peak was 518 nm (Figure 1(b)). The PCR reaction program was 95°C for 1 min, 1 cycle; 45 cycles: 95°C for 20 sec, 55°C for 20 sec, and 72°C for 40 s for amplification. The relative expression was calculated by the 2−△△Ct method.

Figure 1

Gold nanoparticles: (a) scanning electron microscope (SEM) of gold nanoparticles; (b) visible spectrums of gold nanoparticles.

2.6. Western Blot

The cells were prepared into a cell suspension (2 × 105 cells/mL) and inoculated in a 24-well plate. After cultivation for 48 hours, the total cell protein was extracted. The BCA method determines whether the total cell protein concentration was qualified. Then, the protein was separated by SDS-PAGE electrophoresis and then transferred to nitrocellulose membrane. The nitrocellulose membrane was sealed with 5% skim milk for 1.5 hours. Anti-NFIA (1 : 200, Shanghai Ruiqi Biotechnology) and NFIB antibodies (1 : 200, Shanghai Ruiqi Biotechnology) were added and incubated for 1 hour; the corresponding secondary antibody (1 : 1,000) was added and incubated for 1 hour. The hypersensitive ECL liquid was added to react with the blot in the dark for 5 min. The gel imaging system was used to take pictures, and the ImageJ software (NIH, USA) was used to analyze the level of each protein band's intensity. The relative level of protein was calculated with β-actin as the internal control.

2.7. Interference with NFIA Gene in Diabetic Rats

The corresponding NFIA mRNA sequence was searched in the NCBI database, and NFIA siRNA sequence was designed with siDirect online tool (http://sidirect2.rnai.jp/). The siRNA sequence synthesis was completed by Shanghai Jima Pharmaceutical Technology Co., Ltd. siRNA was dissolved in normal saline treated with nuclease-free water (DEPC), and 20 μmol/L solution was prepared for later use. Twenty diabetic rats without retinopathy were selected as the interference group and were slowly injected with 1 μL of NFI-A siRNA (20 μmol/L) and 1 μL of transfection reagent into the vitreous cavity of both eyes. The lens and retinal blood vessels were carefully avoided during injection. Gatifloxacin ophthalmic gel was applied to both eyes after injection. The above operation was repeated every 2 weeks for a total of five injections. The pathological changes in the retina of rats were observed. The rats were sacrificed after 12 weeks, and the eyeballs were removed. The lens was observed under a microscope.

2.8. Statistical Analysis

Each experiment was repeated thrice. The comparison between the results was performed using the SPSS 21.0 (IBM, USA) and GraphPad Prism 8.0 statistical software (GRAPHPAD, San Diego, California, USA). All data were shown as mean ± standard deviation. t-test was used in comparing two groups, and ANOVA was used for multigroup comparisons. The difference was found to be statistically significant if P < 0.05.

3. Results and Discussion

3.1. Histopathological Changes in DM and DR Rats

As shown in Figure 2(a), the surface of the retina in the normal group is flat, the cells in each layer are arranged evenly and neatly, and the tissue structure is clear. The surface of the retina of DM group was similar to that of normal rat, with no obvious abnormalities (Figure 2(b)). In the DR group, the cells in each retinal layer are not neatly arranged, inner limiting membrane has obvious edema, vascular endothelial cells proliferated, basement membrane was thickened, and tissue structure was unclear (Figure 2(c)).

Figure 2

Hematoxylin eosin staining to examine the retinal tissues of rats in each group (200x): (a) normal rat retina; (b) rat retina in the diabetic group; (c) retina of rats with diabetes and retinopathy.

3.2. NFIA and NFIB Expression in Retinal Tissue of DM and DR Rats

The gene expression of NFIA and NFIB was decreased in some of DM rats comparing to normal rats, but with no statistically significant difference between DM and normal group was observed. The NFIA expression was significantly decreased in the retinal cells of the DR group comparing to the normal and DM group (Figure 3(a)). The NFIB expression in the retinal cells had similar results as NFIA (Figure 3(b)). We further explored the NFIA and NFIB expression at protein levels, and the results showed that NFIA and NFIB proteins extracted from the retinal cells were significantly lower in the DR group than that in the normal and DM groups (Figures 3(c) and 3(d)).

Figure 3

The level of NFIA and NFIB in rat retina: (a) retinal NFIA gene expression in three groups of rats; (b) retinal NFIB gene expression in three groups of rats; (c) NFIA and NFIB protein expressions in three groups of rat retinal progenitor cells; (d) relative expression levels of NFIA and NFIB proteins, ∗P < 0.05 comparison with control group.

3.3. The Effect of Interference with NFIA Gene of DM Rats on Retinopathy

In order to verify that the decreased level of NFI transcription factors is likely to account for retinopathy in patients with DM, we selected 20 DM without retinopathy to interfere NFIA expression by using RNA-silencing. The gene expression of NFIA in DM rats was 4.60 ± 1.04. After NFIA RNA-silencing, the expression of NFIA was declined to 0.8 ± 0.63.We found that 18 (90%) had diabetic eye diseases after interfering NFIA gene in DM rats. Among them, 7 (35%) of retinopathy and 11 (55%) of the lens fibers disordered were observed (Figure 4). It is suggested that abnormal expression of NFIA is relevant to diabetic eye diseases DM modeled animals.

Figure 4

Changes in lens after interference with NFIA gene in diabetic rats. (a) The lens is normal, with no fibrosis (400x). (b) The lens is abnormal, with disordered fibers (400x).

4. Discussion

DR is a frequently observed complication in DM patients, and its pathological changes manifest as retinal exudation and edema, neovascularization, hemorrhage, and proliferative membrane formation [15]. The patient's vision was consequently impaired. However, the pathogenesis of DR is not yet fully understood. A number of studies have shown that DR is the result of multiple factors [16, 17]. The course of DM and poor blood sugar control are risk factors for retinopathy [18]. The longer the illness, the higher the incidence of retinopathy. The effect of blood sugar control is related to the severity of DR. Studies have also mentioned that changes in retinal neurons are also closely related to retinopathy [10]. Therefore, studying the mechanism of DR is of great significance in formulating corresponding therapeutic intervention.

Under the action of chronic hyperglycemia, various metabolic pathways in the DM patients are disordered. The retinal capillaries, affected by hyperglycemia, are prone to atrophy and degeneration, leading to retinopathy. In this study, we used streptozotocin induction and fat emulsion gavage to generate rat model of DM and DR. In 70 rats, 65 diabetic models were successfully modeled after 4 weeks of injection with streptozotocin. Forty-five DM rats were randomly selected for intragastric administration of fat emulsion. After 12 weeks, 25 DM rats successfully developed retinopathy. We used HE staining to examine the retinal histology of normal, DM, and DR rats. The results demonstrated that the retinal cells of normal and DM rats were arranged neatly, and there was no obvious abnormality in blood vessels; the retinal cells of DR rats were disorderly arranged, and vascular endothelial cells protruded from the inner limiting membrane. It shows that the course of diabetes is a risk factor for retinopathy.

The knockout of the NFI gene in mice will have a variety of phenotypes, including corpus callosum hypoplasia in NFIA knockout [19], lung hypoplasia in NFIB knockout [20], tooth defects in NFIC knockout [21], and nerve and bone defects in NFI-X knockout [22]. Therefore, each NFI gene has a nonredundant role. In order to explore the relationship between NFI and DR occurrence, we examined the expression of NFIA and NFIB in the retinal cells of various modeled rats. Our results demonstrated that the expressions of NFIA and NFIB were significantly decreased in DR rats comparing to normal or DM rats without retinopathy. In order to further verify this observed relationship between expression NFI and retinopathy, we selected 20 DM rats without retinopathy to interfere their NFIA gene expression. Our results showed that 90% of rats had retinal or lens disease after NFIA gene expressions were significantly silenced, indicating that abnormal expression of NFIA can cause either retinal or len diseases in DM rats. Clark et al. [14] analyzed the effect of NFIA/B/X mutants on retinal progenitor cells, and their results showed the mechanism by which NFI transcription factors control the proliferation and arrest of retinal progenitor cells. If the expression of NFI transcription factors in diabetic patients is abnormal, retinal cells will proliferate excessively, retinal morphology will be destroyed, and pathological changes will occur.

5. Conclusion

This study demonstrated the decreased expressions of NFIA and NFIB at mRNA, and protein levels are highly related to the occurrences of DM with DR complications in the DM rat models. The observations were verified by silencing the expression of NFIA gene, which showing disrupted expression of NFIA gene increasing occurrence of retinopathy in DM rats. The findings of NFI in development of diabetic eye complications in this study provide a new basis for the treatment of diabetic retinopathy.