Lycium barbarum Polysaccharides Regulating miR-181/Bcl-2 Decreased Autophagy of Retinal Pigment Epithelium with Oxidative Stress

Disturbed structure and dysfunction of the retinal pigment epithelium (RPE) lead to degenerative diseases of the retina. Excessive accumulation of reactive oxygen species (ROS) in the RPE is thought to play an important role in RPE dysfunction and degeneration. Autophagy is a generally low-activity degradation process of cellular components that increases significantly when high levels of oxidative stress are present. Agents with antioxidant properties may decrease autophagy and provide protection against RPE dysfunction and damage caused by ROS. Lycium barbarum polysaccharide (LBP) has been widely studied as an antioxidant and cell-protective agent. Therefore, we designed this study to investigate the effects of LBP, which inhibits miR-181, on autophagy in retinal pigment epithelium (RPE) with oxidative stress in vitro and in vivo. In the current study, we found that the highly expressed miR-181 downregulated the expression of Bcl-2 in hydrogen peroxide- (H2O2-) induced ARPE-19 cells, resulting in an increase in ROS, apoptosis, and autophagy flux. LBP inhibited the expression of miR-181, decreased the levels of ROS, apoptosis, and autophagy flux, and increased cell viability in H2O2-induced ARPE-19 cells, suggesting that LBP provides protection against oxidative damage in ARPE-19 cells. We also found that LBP decreased RPE atrophy and autophagy flux in rd10 mice. Taken together, the results showed that LBP has a protective effect for RPE under oxidative stress by inhibiting miR-181 and affecting the Bcl-2/Beclin1 autophagy signaling pathway.


Introduction
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells arranged in the outermost layer of the retina [1]. The RPE sheet is an important structure that connects the outer Bruch's membrane and choroid to the inner photoreceptor cells [2]. There are microvilli structures extending between the outer photoreceptor segments inside the RPE cells, which are involved in phagocytosis of the RPE [2,3]. The single-layered RPE forms a choroid-blood-retinal barrier with Bruch's membrane and the choroid outside the retina, which controls the transport of material by retinal neurons. The RPE contains melanin, which can reduce ultraviolet light damage to the retina and inner nerves. In addition, the RPE is critical for visual function and photoreceptor homeostasis, because RPE phagocytosis recycles photoreceptor outer segments and the complex metabolic system of the RPE reduces excessive accumulation of reactive oxygen species (ROS) [3][4][5].
Disrupted structure and dysfunction of the RPE can lead to retinal degenerative diseases, such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD), in which RPE dysfunction and damage are involved in the development [6]. Therefore, understanding the role of RPE in retinal degeneration and delaying the dysfunction and damage of RPE cells could effectively alleviate retinal degeneration (RD). There is increasing evidence that oxidative stress plays a critical role in RPE dysfunction and damage [7,8]. Exposure of the retina to ROS results in altered expression of microRNAs (miRNAs) as well as altered expression of genes that may be involved in the pathogenesis and progression of RD [9]. Previous studies have shown that microRNA-181 (miR-181), as a posttranscriptional regulatory molecule, has taken part in the injury of RPE cells by oxidative stress [10].
Autophagy is a degradation process mediated by lysosomes for damaged cellular components, including those damaged by ROS [11]. In this way, the metabolic needs of the cell itself and the renewal of certain organelles are satisfied. Autophagy is a defense and stress regulatory mechanism and acts in the development of immunity, infections, inflammation, tumors, cardiovascular diseases, and neurodegenerative diseases of the body [12][13][14]. In the state of oxidative stress, mitochondrial ROS homeostasis is disturbed, and its excess accumulation can contribute to membrane lipid peroxidation and structural damage to organelles and also stimulate autophagy to eliminate the damaged sites [15]. Studies have found that autophagy plays an important role in the alleviation of oxidative stress by exogenous antioxidants [16,17]. Most antioxidants alleviate oxidative stress and reduce apoptosis by regulating the autophagy signaling pathway. Beclin1 has been reported to be a key factor in autophagy formation by regulating the lipid kinase activity of VPS34 (vacuolar protein-sorting 34); Beclin1 is also a multifunctional protein with a BH3 domain that can interact with antiapoptotic protein B-cell lymphoma/leukemia-2 (Bcl-2) and plays a dual role in regulating autophagy and apoptosis [18].
Lycium barbarum polysaccharide (LBP) is the active ingredient extracted from the ripe fruit of Lycium barbarum and performs a vital function in the antiaging and antioxidant function of wolfberry [19]. The effects of LBP on antioxidants and cell protection have been extensively researched. LBP can effectively resist free radical peroxidation and maintain mitochondrial function [20,21]. LBP has exhibited high efficacy against oxidative damage and cell apoptosis in hydrogen peroxide-(H 2 O 2 -) induced ARPE-19 cells [22]. In this study, we investigated the effects of LBP, which inhibits miR-181, on autophagy in the oxidative stress response of RPE in vitro and vivo.

Preparation of LBP Solution.
The impurity of LBP powder mainly contains betaine, zeaxanthine, physalein, carotene, riboflavin, niacin, vitamin C, and so on. The powder was dissolved in distilled water at a concentration of 160 mg/L. The dissolved LBP was filtered using a mixed cellulose ester membrane with a pore size of 0.22 μm (ROJB91514, MF-Millipore). After dilution of the solution, the effects of different concentrations of solubilized LBP on the survival rate of ARPE-19 cells were detected by CCK8.
The ARPE-19 cell line was purchased from Jennio Biotech Co., Ltd. (Guangzhou, China) and was cultured in a 24-well plate (702011, NEST) at a concentration of 1 × 10 5 cells/mL for 24 h. When cell confluence reached about 80%, small RNAs (miR-181a mimic and NC) and plasmids (wt pBcl-2 and mut pBcl-2) were transfected, and luciferase   Digest the adherent ARPE-19 cells with trypsin (HY-P71773, MedChemExpress), take 5 × 10 5 cells/mL after cell counting, centrifuge at 1000 rpm at 4°C for 10 minutes, and discard the supernatant. Add 1 mL of phosphatebuffered saline (PBS, P1010, Solarbio), shake gently to suspend the cells, then centrifuge at 1000 rpm for 10 min at    4°C, and discard the supernatant. Suspend the cells in 200 μL of binding buffer, add 10 μL of Annexin V-APC, mix gently, and allow to react at room temperature in the dark for 15 minutes. Add 300 μL binding buffer (total reaction volume of 500 μL) and 5 μL of propidium iodide stain and test the apoptosis rate within 1 hour by flow cytometry.
Treated cells were collected by digestion, washed once with PBS buffer, and then resuspended in 1 mL of PBS buffer. The fluorescent probe CM-H2DCFDA was added at a final concentration of 1 M/mL and incubated at 37°C for 30 minutes, and the accumulation of ROS in live cells was analyzed by flow cytometry.
2.7. Immunofluorescence. After treatment of ARPE-19 monolayer cells, cells were fixed in 4% PFA for 30 minutes at room temperature, washed twice with PBS for 3 minutes, and treated for 2 minutes on 0.1% TX-100 (X100, Supelco) at room temperature to permeabilize the cell membrane. Add 30 μL of the diluted primary antibody in PBS with 1% BSA/TBS to the parafilm, incubate the cells for 30 minutes at room temperature, and then wash three times with PBS for 3 minutes. The procedure for incubating the secondary antibody is the same as that for the primary antibody. Finally, add 10 μL of mounting medium (03989, Sigma-Aldrich), let it stand at 37°C for 30 minutes, and then you can observe it with the Leica DM4 B fluorescence microscope.

Protein Extraction and Western Blot Assay In Vitro.
Wash the treated ARPE-19 cells at 4°C with PBS and then add RIPA lysis buffer and isolate the protein. Follow the instructions of the BCA protein quantification kit to measure the protein concentration. Depending on the protein quantification result, transfer the sample to the SDS-PAGE gel (P1200, Solarbio) and start electrophoresis at 75 V until the bromophenol blue reaches the bottom. The transferred membranes were saturated with 5% skim milk in PBS for 2 hours. The primary antibodies (Table 3) were incubated for 12 hours at 4°C with a shaker, and the secondary antibodies (Table 4) were incubated for 90 minutes at room temperature with a shaker. The membrane was slid onto ECL chemiluminescence reagent and incubated for 1 minute. The film was exposed and developed in the darkroom. Quantity One software was used to analyze the grayscale of the images.
2.9. Animals. Retinal degeneration in rd10 mice begins 16 days after birth, has a late onset, and progresses slowly, so it is often used as a model for RD research [23]. The experimental protocols were approved by the Animal Ethics   The model group and the control group were administered 0.9% saline orally. The LBP-LD group and the LBP-HD group received oral doses of 3.6 g/kg and 7.2 g/kg per day, respectively. The miR-181 mimic group and the miR-181 inhibitor group received a local injection into the vitreous at a dose of 0.5 μg per eye every four days using a microsyringe (Hamilton, 700 syringe and 33 G needle). Mice were fed at a temperature of approximately 22°C, and oral administration lasted 28 days (a course of treatment with the Chinese medicine Lycium barbarum).

Histopathological Examination of Retinal Tissues.
After the ERG examination was completed, the mice were killed and the eyeballs were harvested. The eyeballs were fixed with eyeball fixed liquid (B0006, Powerful Biology). The fixed tissue was sequentially dehydrated, dipped in wax, embedded, and sectioned. HE staining was performed according to the instructions of the staining kit for histopathological examination (HE) (C0105S, Beyotime Biotechnology). Images were captured using a Leica DCM8 microscope.
2.13. Western Blot Assay In Vivo. The retina was collected from the mice to perform a Western blot test. The process, procedures, and reagents are described in Section 2.5.
2.14. Statistical Analysis. Statistical analyses were performed using SPSS version 25.0. All data was expressed as the mean ± standard deviation (SD). For comparison of quantitative data between two groups, the normality test was performed first. If all groups accorded with normality and the variance between the two groups was equal, t-test was used for comparison between groups. Otherwise, take into account the nonparametric Wilcoxon rank-sum test. For comparison between multiple groups, one-way analysis of variance (one-way ANOVA) was used for comparison between groups while the continuous data followed the normal distribution and homogeneity of variance, and the Bonferroni method was further used for the pairwise comparison as the difference between groups was statistically significant. If there was no normal distribution or unequal variance, the Kruskal-Wallis rank-sum test was used for comparison among multiple groups. When there are overall statistical differences between groups, DSCF method is further used for multiple comparisons. p < 0:05 was considered statistically significant.

Results
3.1. The Cell Viability of LBP Detected by CCK8. We first examined the effect of LBP on the viability of ARPE-19 cells, which are commonly used in RD cell experiments. The results of CCK8 assay showed that the concentration of LBP in dissolved form at 40 mg/L had the best effect on the survival rate of ARPE-19 cells (Figure 1).
Western blot assay was performed to determine the regulatory effect of hsa-miR-181a-5p on human Bcl-2 gene. The results showed that hsa-miR-181a-5p has a significant regulatory effect on the Bcl-2 gene (Figure 4).  Oxidative Medicine and Cellular Longevity 3.3. LBP Decreased the Generation of Excessive ROS in ARPE-19. The generation of excessive ROS is the main factor leading to oxidative stress in the RPE, and oxidative stress is considered to be one of the main causes of RPE cell damage and apoptosis. Flow cytometry results found that hydrogen peroxide led to a remarkable increase in cellular ROS in ARPE-19 cells ( Figure 5). Inhibition of miR-181 contributes to a decrease in cellular ROS levels, while LBP treatments cause the same change in ARPE-19 cells. Comparison between LBP-LD and LBP-HD groups showed that with the increase of LBP dose, the amount of ROS in cells decreased significantly. Possibly, LBP decreases the formation of excessive ROS in ARPE-19 cells in a dosedependent manner.

LBP Protected ARPE-19 from Apoptosis.
To evaluate the protective effect of LBP on hydrogen peroxide-induced ARPE-19 cells, apoptosis detection was carried out by flow cytometry. The results of the Annexin V-FITC assay showed that the apoptosis rate of ARPE-19 cells induced by hydrogen peroxide increased significantly (see Figure 6). Inhibition of miR-181 leads to a decrease in the apoptotic rate of ARPE-19 cells with oxidative stress, and LBP treatments induce the same change in ARPE-19 cells with excessive ROS accumulation. Comparison between the LBP-LD and LBP-MD groups showed that the apoptotic rate of cells decreased significantly with increasing LBP dose. 7 Oxidative Medicine and Cellular Longevity cytometry in hydrogen peroxide-pretreated ARPE-19 cells, we further employed immunofluorescence assay to evaluate the protein expression of Bcl-2 in oxidative stress ARPE-19 cells, which implies the ARPE-19 cell viability with oxidative damage. Images were taken by Leica fluorescence microscope (scale bars, 25 μm, Figure 8). The integrated optical density (IOD) displayed that hydrogen peroxide decreased expression in ARPE-19 cells; inhibition miR-181 increased expression in ARPE-19 cells. With sufficient dose of LBP (LBP-HD and LBP-MD), the expression of Bcl-2 has a tendency to increase, and there is no difference in the expression between LBP-HD and miR-181 inhibitor.

Beclin1
Signaling Was Involved in LBP Regulating miR-181/Bcl-2-Protected Oxidative Stress ARPE-19. In order to explore whether autophagy is involved in LBP-mediated protection against hydrogen peroxide-induced oxidative stress ARPE-19 cells, Western blot was employed to investigate the protein expression of Bcl-2, Beclin1, LC3BII/I, and Atg5 in ARPE-19 groups (Figure 9). Compared with the normal group, the relative level of Bcl-2 protein was significantly decreased in the model group. The relative level of Bcl-2 protein in all LBP group was significantly increased compared with the model group. The results suggest that the protein expression of Bcl-2 decreased and autophagy protein including Beclin1, LC3B, and Atg5 increased expression in ARPE-19 cell with oxidative stress; inhibition of miR-181 increased the protein expression of Bcl-2 and decreased protein expression of Beclin1, LC3B, and Atg5, and LBP-MD and LBP-HD can play the same effect as miR-181 inhibitor.

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Oxidative Medicine and Cellular Longevity ( Figure 10). The figures displayed that the amplitude of the a-wave and b-wave was recorded in all the groups. The amplitude of a-wave and b-wave in the mice model group both was at low levels, and inhibition miR-181 markedly increased the amplitude of a-wave and b-wave. LBP-HD works the same as inhibitor miR-181. The results suggested LBP and inhibition miR-181 has a beneficial impact on the visual function of rd10 mice.
3.9. Effect of LBP on RPE Changes in rd10 Mice. HE staining was carried out to observe the RPE changes in rd10 mice retinal tissue, C57 mice as the normal group. As shown in Figure 11, the structure of retinal tissue in the model group (rd10 mice) had a rupture or disappearance of the RPE layer (green arrow) and loss of a large number of photoreceptor cells (red arrow). The miR-181 mimic group has the identical changes of morphology to the model group. In comparison to the model group, the overall retina in the LBP group was well structured. There may be a dose-dependent manner of LBP in the survival of RPE cells and the retinal structural arrangement. The miR-181 inhibitor group has the same outcome as the LBP-HD group. In addition, LBP and inhibition miR-181 could effectively protect photoreceptor from death.

Discussion
The understanding of RPE demonstrates that oxidative damage is one of the main causes of retinal degeneration [3,24]. The retina is a tissue with high oxygen consumption and high oxidation products. Hydrogen peroxide is produced by the RPE as part of cellular oxidative metabolism, which provides several mechanisms to support normal photoreceptor function [25]. Excessive accumulation of ROS in the retina and diminished antioxidant capacity in the cells leads to RPE cell dysfunction and damage [24,26]. Besides, exposure of the RPE to ROS-altered expression of miRNA may be involved in the pathogenesis and progression of RD [27]. The ARPE-19 cell line used in this study was isolated from primary human RPE cells and is commonly used as an in vitro model of RD [28]; rd10 mice have a missense mutation in phosphodiesterase 6B, and these mice have retinal degeneration that complete at 60 days of age [29]. Our study showed that miR-181 significantly increased in the oxidative stress response of ARPE-19 cells and was associated with an increase in oxidative stress and autophagy, whereas antioxidants and inhibition of miR-181 decreased oxidative stress and autophagy flux. Moreover, injection of antioxidants and miR-181 inhibitors ameliorated RPE injury in rd10 mice. Agents with antioxidant properties could rebalance miRNA expression and provide protection against RPE dysfunction and damage induced by ROS. Previously, miR-181 has been reported to be closely related to the degeneration of visual impairment. miR-181 was highly expressed in the lens tissue of age-related cataract  14 Oxidative Medicine and Cellular Longevity and negatively regulates Bcl-2, thereby inhibiting the proliferation of lens epithelial cells [30]. Recently, a study found that miR-181a is involved in the process of neurodegenerative complaint, and overexpression of miR-181a can activate autophagy in cells [31]. Moreover, one study found that miR-181 is involved in the oxidative stress response of RPE cells as a posttranscriptional regulatory molecule [10]. The Bcl-2 gene is regulated by miR-181, which is also known as the most important regulatory gene in apoptosis and can mediate the release of Beclin1 and other substances by mitochondrial oxidative stress [32,33]. Our findings hint that oxidative stress-induced miR-181 inhibits Bcl-2 and affects Beclin1 autophagy signaling in the RPE oxidative stress response. Antioxidant regulation of miR-181/Bcl-2 attenuated Beclin1 autophagy signaling and resulted in a protective effect on oxidative stress in the RPE. Autophagy in the RPE is responsible for retinal homeostasis, and any defect in autophagy disrupts RPE function and triggers retinal degeneration diseases [34]. The Beclin1 gene, an autophagy-related gene, is in antagonism to the Bcl-2 gene to regulate autophagy [35]. Studies have demonstrated that oxidative stress-induced damage to proteins and organelles can cause Beclin1 to separate from Bcl-2 and form a Beclin1-VPS34 complex, initiating autophagy to eliminate the damaged site [15]. Oxidative stress that decreased the expression of Bcl-2 also abrogates mitochondrial function and exacerbates the accumulation of ROS [36]. Prolonged or severe oxidative stress leads to damage of organelles such as mitochondria, resulting in expansion of autophagy flux which in turn leads to abnormal mitochondrial function [37]. Researchers found that suppressing the expression of Atg5 and Beclin1 can prevent cell death because blocking autophagy significantly decreases caspase-3 [38]. At low stress levels, autophagy is already activated and may serve as an important protec-tion against oxidative injuries [39]. Our results suggest that protein expression of Beclin1, LC3B, and Atg5 is at a low level in C57 mice and maintains this justification. When oxidative stress is high, autophagy is overactivated and contributes to cell injury [40,41]. The protein expression of Beclin1, LC3B, and Atg5 in the model group in vitro and in vivo is consistent with previous studies.
LBP is the active ingredient extracted from the ripe fruit of Lycium barbarum and has been widely studied as an antioxidant and cell-protective agent [19]. One study found that LBP exhibited protective effects against neurotoxicity by reversing H 2 O 2 -induced elevated ROS levels, decreased cell viability, and decreased mitochondrial membrane potential, indicating the amelioration of mitochondrial apoptosis [42]. LBP attenuates chemotherapy-induced oxidative stress by enhancing the efficacy of antioxidant enzymes and attenuating the increased concentrations of oxidation products after cyclophosphamide injection [43]. In recent years, some studies have also demonstrated that the cytoprotective effect of LBP is mediated by autophagy. In an optic nerve injury microglia model induced by bipolar pulsed current, LBP attenuated the inhibition of autophagy by electrical stimulation and attenuated apoptosis and oxidative stress [44]. Treatment of hippocampal neurons with LBP during oxygen-glucose deprivation and reperfusion decreased intracellular expression of LC3B and Beclin1, resulting in a decrease in autophagosomes and an increase in p62 protein levels [45]. Moreover, LBP exerts an excellent function in antioxidant activity, increasing cell viability and against apoptosis effects by regulating miRNAs.
In conclusion, this study shows that oxidative stress upregulates miR-181 and autophagy flux in ARPE-19 cells and rd10 mice. The cytoprotective effects of LBP in oxidatively damaged ARPE-19 cells and rd10 mice were associated with antioxidant activity and a reduction in autophagy  The protein expression relative level of Bcl-2, Beclin1, LC3B, and Atg5 in rd10 mice retina; the data appeared as the mean ± SD, * p < 0:05 and * * p < 0:01.