Polyphenol-Rich Extract of Apocynum venetum L. Leaves Protects Human Retinal Pigment Epithelial Cells against High Glucose-Induced Damage through Polyol Pathway and Autophagy

Diabetic retinopathy (DR) is a specific microvascular problem of diabetes, which is mainly caused by hyperglycemia and may lead to rapid vision loss. Dietary polyphenols have been reported to decrease the risk of DR. Apocynum venetum L. leaves are rich in polyphenolic compounds and are popular worldwide for their health benefits as a national tea drink. Building on previous findings of antioxidant activity and aldose reductase inhibition of A. venetum, this study investigated the chemical composition of polyphenol-rich extract of A. venetum leaves (AVL) and its protective mechanism on ARPE-19 cells in hyperglycemia. Ninety-three compounds were identified from AVL by LC-MS/MS, including sixty-eight flavonoids, twenty-one organic acids, and four coumarins. AVL regulated the polyol pathway by decreasing the expression of aldose reductase and the content of sorbitol, enhancing the Na+K+-ATPase activity, and weakening intracellular oxidative stress effectively; it also could regulate the expression of autophagy-related proteins via the AMPK/mTOR/ULK1 signaling pathway to maintain intracellular homeostasis. AVL could restore the polyol pathway, inhibit oxidative stress, and maintain intracellular autophagy to protect cellular morphology and improve DR. The study reveals the phytochemical composition and protective mechanisms of AVL against DR, which could be developed as a functional food and/or candidate pharmaceutical, aiming for retina protection in diabetic retinopathy.


Introduction
Dietary polyphenols are natural phytochemical compounds, mainly from herbs, tea, and other beverages, which exhibit multiple biological properties, including antioxidant, antimicrobial, antiviral, anticancer, antiinflammatory, and antidiabetic activities [1].Recently, the epidemiological survey between dietary polyphenol intake and type 2 diabetes shows that the polyphenol intake in diabetic people is lower than in non-diabetic people [2].Researchers have also realized that dietary polyphenols can prevent and cure diabetes mellitus and its complications [3].Diabetic retinopathy (DR) is a specific microvascular problem of diabetes and is mainly caused by hyperglycemia [4].It is the leading cause of vision loss in adults of working age, and the population of DR will reach over 50 million by 2045 [5].Prevention and early intervention are becoming increasingly important due to irreversibility in the late stage of DR.Polyphenol-rich products could treat type 2 diabetes mellitus and its complications by regulating metabolic disorders and alleviating stress pathways [6,7].Hence, the utilization of dietary polyphenols for the prevention and treatment of DR holds significant value.Adsorber Technology Co., Cangzhou, China).The optimum conditions of purification were as follows: sample concentration was 6.0 mg/mL, flow rate was 2 BV/h, and maximum solution treatment capacity was 8 BV; The eluting agent was 5 BV purified water, with the total polyphenols being desorbed by 3 BV of 50% alcohol at the flow rate 2 BV/h [18,20].

Total Phenols Content (TPC)
The total phenol content test was performed according to the Folin-Ciocalteu colorimetric method with slight modification.Folin-Ciocalteu reagent was diluted 10 times before use.Briefly, sample solution (10 µL) and Folin-Ciocalteu reagent diluent (30 µL) were added to each well of a 96-well plate to incubate together for 2 min.Subsequently, 7.5% Na 2 CO 3 (30 µL) and ddH 2 O (180 µL) were added to each well.The absorbance was measured at the wavelength λ = 765 nm.Additionally, the sample was replaced 50% aqueous methanol in the blank group.The total phenol content was calculated from a standard curve of gallic acid at concentrations.

Aldose Reductase Activity Assay
The inhibition of aldose reductase and the half inhibition concentration (IC 50 ) were calculated according to the reference [21].Briefly, Sample solution (4 µL), PBS buffer (146 µL), and aldose reductase solvent (10 µL) were successively added to 96-well plate.Then, nicotinamide adenine dinucleotide phosphate (NADPH) and DL-glyceraldehyde were mixed as a substrate, which was added to incubate for 30 min.The absorption was read at 340 nm.As for blank, 2 µL samples were replaced by DMSO.Quercetin was used as the positive control.

Cell Culture and High Glucose Damage Model
ARPE-19 cells were cultured in the complete growth medium containing DMEM/F-12 with 10% fetal bovine serum, 100 µg/mL streptomycin, and 100 U/mL penicillin.Cells were used between the fifth and fifteenth passages.For the high glucose damage model, ARPE-19 cells were exposed to 30 mM D-glucose and incubated at 37 • C in a humidified chamber of 5% CO 2 for 48 h [22].Untreated cells were cultured with 4.5 mM D-glucose as the normal group.
For the treatment groups, after ARPE-19 cells (5 × 10 5 cells) were cultured in medium with 30 mM glucose for 24 h, then they were treated with AVL (6.25, 12.5, and 25 µg/mL) under the high-glucose condition (HG, 30 mM D-glucose) for another 24 h in a 6-well plate.

Cell Viability Assay
MTT assay was carried out according to the previous description [26].After ARPE-19 cells were cultured in medium with 30 mM glucose for 24 h, and then they were treated with AVL (6.25, 12.5, 25, 50, and 100 µg/mL) under the high-glucose condition (HG, 30 mM D-glucose) for another 24 h.The absorbance value was measured at a wavelength of 490 nm.

Na + K + -ATPase Activity Measurements
The production of Na + K + -ATPase activity was measured using the Na + K + -ATPase Activity Detection Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) following the manufacturer's instructions.

The Content of Sorbitol Assay
The ARPE-19 Cells were boiled in tri-distilled water for 10 min, and the supernatants were obtained by centrifugation for 10 min, 8000× g, room temperature.Sorbitol content detection with supernatants was performed using a D-Sorbitol/Xylitol Assay Kit (Megazyme International Ireland Co., Ltd., Wicklow, Ireland) and protocol.

The Content of the Reduced Glutathione (GSH)
The level of reduced glutathione was measured using Reduced Glutathione Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the direction [17].

Examination of Intracellular ROS Generation
The intracellular ROS level in ARPE-19 cells with AVL was measured by fluorescence microscope with 2, 7-dichlorofluorescein diacetate (DCFH-DA) (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).The treated cells were evaluated by fluorescence microscopy and microplate spectrophotometer according to the direction [22].

Cellular Morphology Electron Microscopy
The cellular morphology was detected by electron microscopy.Cells were fixed with 2.5% glutaraldehyde for 1 h at room temperature.Samples were fixed with 1% osmium tetroxide, dehydrated by successive acetone washes (50%, 70%, 90%, 100%) 4 and embedded with epoxy resin.After polymerization, ultrathin sections were cut using an ultramicrotome and stained with 2% uranyl acetate and Reynold's lead citrate.Sections were assessed using a JEOL 1011 transmission electron microscope (JEOL, Tokyo, Japan) equipped with a GATAN Erlangshen CCD camera (Gatan, Pleasanton, CA, USA).

Statistical Analysis
The experiment results are presented as the mean ± standard deviation (SD).All statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software Inc., Boston, MA, USA).p-values were analyzed by one-way analysis of variance, which is a method of testing differences between more than two groups or treatments.The statistically significant result was represented as # p < 0.05, ## p < 0.01 versus NG, * p < 0.05, ** p < 0.01 versus HG.

The Chemical Composition
In this study, the total polyphenol content of AVL was 48.47 ± 0.41%, the IC 50 value of AVL on AR was 17.38 ± 1.10 µg/mL, and the IC 50 value of quercetin was 5.42 ± 0.40 µg/mL.Based on retention time (t R ), molecular mass, fragment ions, standards, and previous references, 93 compounds were identified from AVL.The results are shown in Table 1, and the base peak chromatograms on positive and negative ion modes are shown in Figure 1.The polyphenolic composition of AVL included sixty-eight flavonoids, twenty-one organic acids, and four coumarins.Based on previous studies, the extract was rich in flavonoid-type phenolic compounds, which had a common C6-C3-C6 backbone structure.The flavonoids of AVL were the main flavonol and flavane derivatives, including glycosides of quercetin, kaempferol, and myricetin, as well as catechin compounds.The hydroxyl groups on the A-ring and B-ring of flavonoids can enhance the inhibition of AR, and the hydroxyl groups on C-5 and C-7 of the A-ring and C-3 ′ and C-4 ′ of the B-ring can significantly increase the inhibition on AR, while the hydrogenation of C2=C3 and the glycosylation on 7-OH and 4 ′ -OH of flavonoids could reduce this inhibition [27].The hydroxyl group of coumarin at C-6 and C-7 had an inhibitory effect on AR [28].AR is the first rate-limiting enzyme involved in the polyol pathway; AR and the polyol pathway were considered as significant mechanisms to explain how hyperglycemia initiates diabetic retinopathy [29].The dietary polyphenols are effective and safe components in functional foods for the treatment of chronic eye diseases [30].It was reported that anthocyanins (such as cyanidin 3-O-galactosides), flavonols (such as quercetin and myricetin), and organic acids (such as neochlorogenic acid) play an improving role in the treatment of DR [31].Hence, AVL had an inhibitory effect on AR, which can be considered as a potential efficacious alternative for intervening in DR.

The Effect of AVL on Cell Viability
The viability of ARPE-19 cells treated with AVL (6.25-100 µg/mL) in high-glucose condition (HG, 30 mM) was shown in Figure S1A.Notably, AVL did not exhibit significant cytotoxicity, but the cell viability slightly decreased with the increase in its concentration.

The Effect in the Polyol Pathway
The polyol pathway is a glucose metabolism pathway, which is considered to be an important factor in the pathogenesis of diabetic eye refractive changes, cataract formation, and diabetic retinopathy [32].The concentration and action time of AVL for treating ARPE-19 cells induced by high-glucose condition were established according to the pre-experiment in the supplementary material (Figure S2A-D).

The Effect of AVL on AR
Aldose reductase, the first rate-limiting enzyme involved in the polyol pathway, catalyzes the conversion of glucose to sorbitol.The results showed that HG treatment significantly up-regulated AR mRNA (Figure 2A) and protein transcriptional levels (Figure 2B) in ARPE-19 cells, while 25 µg/mL AVL had no effect on AR mRNA level, but it downregulated AR protein expression.This result was consistent with the positive drugs that AVL blocks AR expression at the protein level and not at the transcriptional level [33].

The Effect of AVL on Sorbitol Content
Sorbitol is a molecule that poorly penetrates cell membranes, and its accumulation creates hyperosmotic stress.As shown in Figure 2C, the sorbitol content in HG group was significantly increased to 0.39 mg/mg protein (p < 0.01), it was reduced by treatment with AVL in a dose-dependent manner when the AVL concentration was 12.5 µg/mL (0.26 mg/mg protein, p < 0.01), the intracellular sorbitol level is very close to it in positive groups, and when curative concentration arrived to 25 µg/mL (0.24 mg/mg protein, p < 0.01), the content of sorbitol was lower than positive groups (Difrarel, Epalrestat; 0.25 mg/mg protein, 0.26 mg/mg protein; p < 0.01, p < 0.01).

The Effect of AVL on Na + K + -ATPase Activity
Na + K + -ATPase is an ion transporting enzyme that exchanges Na + for K + by hydrolyzing ATP.The effect of AVL and positive groups on Na + K + -ATPase in HG-injured ARPE-19 cells is shown in Figure 2D.The activity of Na + K + -ATPase was significantly decreased in ARPE-19 cells treated in the HG group (NG vs. HG, 0.49 vs. 0.16 U/mg protein), and AVL dose-dependently enhanced Na + K + -ATPase activity.At a concentration of 12.5 µg/mL (0.24 U/mg protein, p < 0.01), the Na + K + -ATPase activity was close to the effect of positive drug of Difrarel (0.26 U/mg protein, p < 0.01), and at a concentration of 25 µg/mL (0.30 U/mg protein, p < 0.01), it was similar to the effect of epalrestat (0.31 U/mg protein, p < 0.01).
Under hyperglycemia, glucose flux, which enters the polyol pathway, is increased due to the saturation of hexokinase with glucose.Aberrant activation of aldose reductase drives the massive production of sorbitol, which is beyond the capacity of sorbitol dehydrogenase [9,32].The accumulation of sorbitol creates osmotic swelling and cell dysfunction and decreases Na + K + -ATPase.In previous studies, the structure-activity relationship

The Effect of AVL on Sorbitol Content
Sorbitol is a molecule that poorly penetrates cell membranes, and its accumulation creates hyperosmotic stress.As shown in Figure 2C, the sorbitol content in HG group was significantly increased to 0.39 mg/mg protein (p < 0.01), it was reduced by treatment with AVL in a dose-dependent manner when the AVL concentration was 12.5 µg/mL (0.26 mg/mg protein, p < 0.01), the intracellular sorbitol level is very close to it in positive groups, and when curative concentration arrived to 25 µg/mL (0.24 mg/mg protein, p < 0.01), the content of sorbitol was lower than positive groups (Difrarel, Epalrestat; 0.25 mg/mg protein, 0.26 mg/mg protein; p < 0.01, p < 0.01).

The Effect of AVL on Na + K + -ATPase Activity
Na + K + -ATPase is an ion transporting enzyme that exchanges Na + for K + by hydrolyzing ATP.The effect of AVL and positive groups on Na + K + -ATPase in HG-injured ARPE-19 cells is shown in Figure 2D.The activity of Na + K + -ATPase was significantly decreased in ARPE-19 cells treated in the HG group (NG vs. HG, 0.49 vs. 0.16 U/mg protein), and AVL dose-dependently enhanced Na + K + -ATPase activity.At a concentration of 12.5 µg/mL (0.24 U/mg protein, p < 0.01), the Na + K + -ATPase activity was close to the effect of positive drug of Difrarel (0.26 U/mg protein, p < 0.01), and at a concentration of 25 µg/mL (0.30 U/mg protein, p < 0.01), it was similar to the effect of epalrestat (0.31 U/mg protein, p < 0.01).
Under hyperglycemia, glucose flux, which enters the polyol pathway, is increased due to the saturation of hexokinase with glucose.Aberrant activation of aldose reductase drives the massive production of sorbitol, which is beyond the capacity of sorbitol dehydrogenase [9,32].The accumulation of sorbitol creates osmotic swelling and cell dysfunction and decreases Na + K + -ATPase.In previous studies, the structure-activity relationship of dietary polyphenol in inhibiting aldose reductase (AR) was reported [34]; Vitamin E could prevent the loss of Na + K + -ATPase, thus significantly reducing the incidence of cataract [35]; Eparastat was also a common AR inhibitor and reduced aldose reductase in ARPE-19 cells under high-glucose condition [25]; the main bio-ingredients of Difrarel were anthocyanin extract of Vaccinium myrtillus L., belonging to the flavonoid class of compounds, which were used to prevent and improve visual fatigue and early stage of diabetes retinopathy [36].We also found that AVL was useful for improving human retinal pigment epithelium cell damage induced by high glucose via decreasing the content of sorbitol, improving the activity of Na + K + -ATPase, and down-regulating AR protein expression to regulate the polyol pathway.

The Effect of AVL on Glutathione (GSH) and Reactive Oxygen Species (ROS)
Excessive levels of glucose stimulate oxidative stress by multiple mechanisms, and the polyol pathway is an important mechanism.Due to the overactivity of the polyol pathway, the excessive consumption of NADPH reduces the content of glutathione (GSH) during the conversion of glucose to sorbitol, and production of NADH increases reactive oxygen species (ROS) during the conversion of sorbitol to fructose [9].

AVL Reduces the Expression of Autophagic Proteins in High-Glucose-Treated ARPE-19 Cells
Autophagy is an important intracellular homeostasis process; abnormal activation of the polyol pathway, oxidative stress, cellular accumulation of ROS, and other stress responses play an important role in the triggering of autophagy [39].
The GSH was decreased, and ROS were increased obviously in APRE-19 cells damaged with the high-glucose condition, which resulted in oxidative stress and accelerated DR process [38].Dietary polyphenols are important natural antioxidants with a wide range of sources.The extract of green tea protected the retina against diabetic retinopathy via an antioxidant mechanism and was rich in polyphenol components [37].The protective effects of AVL were also evaluated in ARPE-19 cells under the high-glucose condition, and these results suggested that AVL increased intracellular GSH levels and decreased ROS levels to protect the retina against oxidative stress caused by the polyol pathway.

AVL Reduces the Expression of Autophagic Proteins in High-Glucose-Treated ARPE-19 Cells
Autophagy is an important intracellular homeostasis process; abnormal activation of the polyol pathway, oxidative stress, cellular accumulation of ROS, and other stress responses play an important role in the triggering of autophagy [39].
The biogenesis of autophagosomes is orchestrated by autophagy-related (ATG) proteins, which first produce phagosomes in hierarchical order and then expand them into autophagosomes.In the initial stage of autophagy, induction of autophagy leads to the formation of autophagosomes mediated by the Vps-Beclin-1 complex, which initiates cell membrane vesicle nucleation.Autophagosome extension requires the involvement of ATG12 and LC3.ATG12 is activated by ATG7 and transported to ATG5 to form the ATG5-ATG12 complex, which further forms ATG5-ATG12-ATG16 to participate in the expansion of autophagosome phagocytic vesicles.In addition, LC3B-I is activated and transferred to ATG3 to form LC3-II-PE with phosphatidylethanolamine (PE) to participate in membrane extension; Phagocytosis and lysosomes fuse to form mature autophagosomes and degrade their contents [40,41].Hence, the expression of key autophagic proteins in high-glucosetreated ARPE-19 cells is analyzed to evaluate the effect of AVL on autophagy levels.
To expound the mechanism of AVL inhibits increased autophagy in the HG group, the expression of significant autophagy proteins Beclin-1, ATG3, ATG5, ATG7, ATG12, ATG16L1, LC3, and P62 were examined by Western Blot in Figure 4A-I.The expression of autophagy-related proteins was significantly increased in ARPE-19 cells treated with high glucose (HG group), indicating that high glucose treatment increased the level of autophagy.However, the protein expressions of Beclin-1 (12.5 µg/mL, 25 µg/mL; p < 0.05, p < 0.01), ATG3 (12.5 µg/mL and 25 µg/mL; p < 0.05, p < 0.01), ATG5 (25 µg/mL; p < 0.05), ATG7 (12.5 µg/mL and 25 µg/mL; p < 0.05, p < 0.01), ATG12 (12.5 µg/mL and 25 µg/mL; p < 0.01, p < 0.01), ATG16L1 (12.5 µg/mL and 25 µg/mL; p < 0.05, p < 0.01), and LC3-II/LC3-I (12.5 µg/mL and 25 µg/mL; p < 0.05, p < 0.05) were diminished and P62 (6.25 µg/mL, 12.5 µg/mL and 25 µg/mL; p < 0.01, p < 0.01, p < 0.01) was increased significantly after treated by AVL.The mechanism of AVL was consistent with that of epalrestat, but the Difrarel group only significantly improved the protein expression of Atg7, ATG16L1, LC3-II/LC3-I, and P62.This indicated that autophagy stages of elongation and maturation were influenced by Difrarel, which was not fully consistent with AVL.The accumulation of autophagy proteins was due to the increase of autophagy initiation or the disorder of fusion between autophagosome and lysosome under high glucose conditions.The level of the P62 protein, negatively correlated with autophagy activity, was combined with the ratio of LC3-II/LC3-I to evaluate the level of autophagy.Autophagy plays a protective and repair role under normal conditions, while dysregulation of autophagy could mediate the damage of high glucose to ARPE-19 cells directly [39].This result is consistent with Gao et al.; they found that the level of intracellular autophagy was increased after high glucose injury in ARPE-19 cells, and Lycium barbarum polysaccharide can decrease the autophagy levels of ARPE-19 cells [39,42].In addition, it has been reported that A. venetum leaf extract has a positive effect on injured neurons by regulating the levels of autophagy and apoptosis in PC12 cells [16].Our result demonstrates that AVL reduced autophagy caused by the high-glucose condition via down-regulation of the over-activated autophagic proteins to maintain autophagy homeostasis in ARPE-19 cells.The AMPK/mTOR/ULK1 signaling pathway is the switch of intracellular anabolic and catabolic processes and is also an important regulatory pathway upstream of autophagy.
AMP-activated protein kinase (AMPK) is an energy-sensitive kinase that monitors the ratio of AMP/ATP; the mammalian target protein of rapamycin (mTOR) regulates cellular processes and unc-51-like kinase 1 (ULK1) is the initial switch of autophagy.The level of AMPK, mTOR, and ULK1 proteins in ARPE-19 cells is clarified in Figure 5A-C.The expression level of P-AMPK(Thr172)/AMPK and p-ULK1(ser555)/ULK increased, and

Measurement of Autophagic Flux
LC3-I is converted into LC3-II, which directly participates in autophagosome formation and elongation and is considered an important autophagy marker.Autophagy is a highly dynamic pathway; chloroquine (CQ) is an autophagy inhibitor that blocks the last stage of autophago-lysosomal fusion and inhibits the degradation of LC3-II from lysosomes in the cytoplasm [43].When autophagy is activated, a CQ is added, LC3-II degradation is inhibited, and the expression of LC3-II is higher than without the inhibitor.Hence, the autophagic fluxn is evaluated by LC3-II/LC3-I ratio in the presence or absence of the autophagy inhibitor CQ.
The previous study has shown that inhibition of the autophago-lysosomal fusion by CQ significantly increased the LC3-II/LC3-I ratio and, thus, autophagic flux in H 2 O 2treated ARPE-19 and hRPE cells [41].ARPE-19 Cells in the CQ + group were treated with 10 µM CQ for 1 h before glucose treatment.There was a significant difference between the HG group and HG+CQ group on the level of LC3-II/LC3-I (p < 0.01) in Figure 4J.When autophagy activity was enhanced, the expression of LC3-II/LC3-I was higher than before the addition of the CQ, indicating that high-glucose stimulated the activation of autophagic flux.Additionally, there was no significant difference between the LC3-II/LC3-I in cells treated with AVL alone or combined with CQ (AVL group vs. AVL + CQ group), indicating that AVL can inhibit the initial stage of autophagy to protect ARPE-19cells from high glucose injury.

AVL Inhibit Upstream Autophagy Induced by High Glucose in ARPE-19 Cells via the AMPK/mTOR/ULK1 Signaling Pathway
The AMPK/mTOR/ULK1 signaling pathway is the switch of intracellular anabolic and catabolic processes and is also an important regulatory pathway upstream of autophagy.
AMP-activated protein kinase (AMPK) is an energy-sensitive kinase that monitors the ratio of AMP/ATP; the mammalian target protein of rapamycin (mTOR) regulates cellular processes and unc-51-like kinase 1 (ULK1) is the initial switch of autophagy.The level of AMPK, mTOR, and ULK1 proteins in ARPE-19 cells is clarified in Figure 5A-C.The expression level of P-AMPK(Thr172)/AMPK and p-ULK1(ser555)/ULK increased, and the level of P-mTOR/mTOR decreased in ARPE-19 cells with the high-glucose condition (HG group) compared with the NG group, markedly.However, AVL significantly reduced the expressions of P-AMPK/AMPK (6.25 µg/mL, 12.5 µg/mL and 25 µg/mL; p < 0.05, p < 0.01, p < 0.01) and p-ULK1/ULK1 (6.25 µg/mL, 12.5 µg/mL and 25 µg/mL; p < 0.01, p < 0.01, p < 0.01) compared with the HG group.The level of P-mTOR/mTOR was greatly increased in the AVL group (12.5 µg/mL, 25 µg/mL; p < 0.05, p < 0.01) compared with the HG group, which is consistent with the positive group of epalrestat.However, this finding was different from the result of the Difrarel group in that only the protein level of P-ULK1/ULK1 was inhibited compared with the HG group, indicating that Difrarel may act through additional signaling pathways to improve DR, such as the inflammatory/antioxidative signaling [44].In addition, it is also possible that the dosage of Difrarel is insufficient.Therefore, the mechanism of Difrarel in the treatment of DR can be further explored.
The AMPK activated by phosphorylation of Thr172 promoted phosphorylation of the rapamycin target site on mTORC1 to inhibit the activity of mTORC1, thereby activating the Ser555 site of ULK1 and finally inducing autophagy; in addition, AMPK can also directly phosphorylate ULK1 to initiate autophagy [45].Autophagy was up-regulated in vascular smooth muscle cells (VSMCs) of DM via activation of the AMPK/mTOR pathway, and epigallocatechin-3-gallate from green tea can inhibit autophagy against cardiomyopathy in diabetic Goto-Kakizaki rats [46,47].And when ARPE-19 cells were treated with a high glucose condition (30 mM) for 48 h, the p-AMPK/AMPK ratio was higher than the normal glucose condition, LC3 II expression was significantly increased, and autophagy was activated [22].Additionally, the phosphorylation level of mTOR was down-regulated in autophagy-activated ARPE-19 cells under high glucose conditions [39].Thus, AVL decreased the phosphorylation of AMPK, increased mTOR activity, and decreased ULK1 activity, regulating the level of autophagy finally.
was activated [22].Additionally, the phosphorylation level of mTOR was down-regulated in autophagy-activated ARPE-19 cells under high glucose conditions [39].Thus, AVL decreased the phosphorylation of AMPK, increased mTOR activity, and decreased ULK1 activity, regulating the level of autophagy finally.

The Effect of AVL on Cellular Morphology
The cellular morphology of ARPE-19 cells was observed by transmission electron microscopy (TEM).The ultrastructural features are shown in Figure 6A; lipid droplets, necrosis, edema, and a blurred mitochondrial outline were observed when cells were exposed to high glucose treatment for 48 h, which suggests that cell damage was clear in the HG group [40,48].After AVL (25 µg/mL) and positive group treatment, these damage conditions were relieved with significantly reduced lipid droplets, necrosis, and edema.This observation indicated that AVL and positive groups could attenuate the damage of retina cells caused by high glucose and have a protective effect.Furtherly, the autophagy state of ARPE-19 cells was observed in Figure 6B.Autophagosomes are mainly composed of double membranes, and a few of them are closed circular structures formed by multilayer or monolayer membranes, which are surrounded by damaged organelles or proteins; autophagic lysosomes are formed by the fusion of autophagosome and lysosome, with monolayer membrane structure, which is surrounded by degraded cytoplasmic components [22].Autophagosomes and autophagic lysosomes were observed in the HG group but decreased in the AVL group and positive treatment group.These results suggested that AVL could protect the cellular morphology and restore the autophagy level of high glucose-injured ARPE-19 cells.
tophagic lysosomes are formed by the fusion of autophagosome and lysosome, with monolayer membrane structure, which is surrounded by degraded cytoplasmic components [22].Autophagosomes and autophagic lysosomes were observed in the HG group but decreased in the AVL group and positive treatment group.These results suggested that AVL could protect the cellular morphology and restore the autophagy level of high glucoseinjured ARPE-19 cells.ARPE-19 cells with long-term exposure to high glucose conditions trigger abnormal polyol pathways with the accumulation of sorbitol and decreased Na + K + -ATPase, oxidative stress with the decrease of GSH, and the generation of ROS, resulting in activating autophagy eventually.AVL can restore the polyol pathway via inhibiting AR, protecting ARPE-19 cells from oxidative stress.The AMPK/mTOR/ULK1 signaling pathway, the switch of anabolic and catabolic processes, is an important regulatory pathway upstream of autophagy.Previously reported that AR regulated ROS/SIRT1/AMPK/mTOR pathway to protect human umbilical vascular endothelial cells (HUVECs) from dysfunction [49], Fidarestat, as an AR inhibitor, regulated the expression of AMPK, mTOR, and P53 to prevent colorectal cancer cell (CRC) growth [50].The AR correlated with AMPK/mTOR pathway and disrupted autophagy, and AR inhibitor epalrestat is neuroprotective against brachial plexus root avulsion injury through the regulation of autophagy to alleviate neuroinflammation and rescue massive motoneurons death in mice [51].Therefore, AVL could regulate AR and the AMPK/mTOR/ULK1 pathway to maintain intracellular autophagy and protect ARPE-19 cells from high glucose conditions.

Conclusions
AVL enriched polyphenolic compounds, including sixty-eight flavonoids, twentyone organic acids, and four coumarins, which can effectively inhibit aldose reductase in vitro.AVL could decrease the protein level of AR and sorbitol content and enhance the

Figure 2 .
Figure 2. The effect of AVL on the polyol pathway in high glucose-induced ARPE-19 cells: (A) The relative expression of the AR gene by quantitative real-time PCR.(B) The expression of protein on AR by Western blo ing.(C) The effect of AVL on sorbitol content.(D) The effect of AVL on Na + K + -ATPase activity.n = 3 independent experiments.A p-value < 0.05 was considered as a significant difference between each group (## p < 0.01 versus NG; ** p < 0.01 versus HG).

Figure 2 .
Figure 2. The effect of AVL on the polyol pathway in high glucose-induced ARPE-19 cells: (A) The relative expression of the AR gene by quantitative real-time PCR.(B) The expression of protein on AR by Western blotting.(C) The effect of AVL on sorbitol content.(D) The effect of AVL on Na + K + -ATPase activity.n = 3 independent experiments.A p-value < 0.05 was considered as a significant difference between each group (## p < 0.01 versus NG; ** p < 0.01 versus HG).

18 Figure 3 .
Figure 3.The effect of AVL on the GSH and ROS in high glucose-induced ARPE-19 cells: (A) The effect of AVL on intracellular glutathione (GSH) levels.(B) The effect of AVL on intracellular reactive oxygen species (ROS) content.(C) The effect of AVL on DCFH-DA staining for ROS.n = 3 independent experiments.A p-value < 0.05 was considered as a significant difference between each group (## p < 0.01 versus NG; ** p < 0.01 versus HG).

Figure 3 .
Figure 3.The effect of AVL on the GSH and ROS in high glucose-induced ARPE-19 cells: (A) The effect of AVL on intracellular glutathione (GSH) levels.(B) The effect of AVL on intracellular reactive oxygen species (ROS) content.(C) The effect of AVL on DCFH-DA staining for ROS.n = 3 independent experiments.A p-value < 0.05 was considered as a significant difference between each group (## p < 0.01 versus NG; ** p < 0.01 versus HG).