Effects of Lycium barbarum polysaccharide on the photoinduced autophagy of retinal pigment epithelium cells

INTRODUCTION

The morphology of autophagosomes in the ARPE-19 cells was observed through TEM (Figure 1). Typical dual-membraniform autophagosomes and autophagy characteristics were observed.Autophagosomes and autolysosomes were hardly observed in the control group. These results indicated that illumination can induce autophagy in ARPE-19 cells.

(LBP) is a major bioactive component of Chinese wolfberry fruit and is a protein polysaccharide composed of monosaccharides, such as arabinose, rhamnose, xylose, mannose, galactose, and glucose. Modern pharmacological studies have demonstrated that LBP has antioxidative, anti-aging, immunoregulatory, and antifatigue functions, provides protection against radiation, and protects the reproductive system. In ocular regions, LBP can protect ganglion cells from retinal ischemic and reperfusion injuries, exerts neuroprotective effects in acute and chronic intraocular hypertension rat models, plays an important role in the antioxidative stress pathway, and protects photoinduced retina models.

In previous studies, we have found that the fruit of Chinese wolfberry can protect RPE cells to some extent possibly through antioxidative stress and by regulating Bcl2/Bax protein expression ratio and inhibiting mitochondrial and endoplasmic reticulum apoptosis signal transduction pathway.The principles underlying the roles of Chinese wolfberry fruit in “tonifying kidney and improving eyesight” have been elaborated. To elucidate how LBP protects human RPE cells from photoinduced injuries during the photoinduced autophagy of RPE cells, we discuss the relationship between autophagy and apoptosis on the basis of cellular morphology and proteomics. This study is expected to provide new insights into AMD prevention and methods decreasing the risk of AMD and facilitate the development of natural drugs for AMD.

MATERIALS AND METHODS

ARPE-19 was bought from China Beena Biological Company.

The main reagents included fetal calf serum (Bioind Company, Ⅰsrael), Dulbecco’s modified Eagle medium (DMEM)/F12 (Bioind Company, Israel),cell counting kit-8 (CCK8) detection kit (Shanghai Beibo Biotechnology Co., Ltd., China), annexinV-FITC apoptosis detection kit (Shanghai Bebo Biotechnology Co., Ltd.,China), BC protein quantification kit, whole protein extraction kit (Jiangsu Kaiji Biotech Co., Ltd., China), LC3Ⅰ‐specific antibody, LC3Ⅱ‐specific antibody, and P62/SQSTM1 antibody(all bought from Wuhan Sanying Biotech Co., Ltd., China);rabbit anti-beclin 1 antibody, rabbit anti-PI3 kinase p110 beta antibody, Akt antibody, Akt-phospho-S473 antibody, rabbit anti-phospho-mTOR antibody (all puchased from Proteintech,China-US joint venture), 3-methyladenine (3MA; Apexbio Technology LLC, USA). The major instruments included Multiskan ELⅠSA (Thermo Company, USA), FACSAriaⅡflow cytometer (BD Company, USA), and TES‐1332A digital illuminometer (TaiBei TASS Electronics Industries, China),transmission electron microscopy (H7800, Hitachi, Japan).

ARPE-19 cells with a good growth state were used for experiments. The original culture solution was discarded after illumination, then washed and stored at 4℃ for 20min in a refrigerator. The cells were then transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm/min in a horizontal rotor centrifuge for 10min. The cells were allowed to settle, mixed with 1 mL of glutaraldehyde,and left to stand at 4℃ for 30min in the refrigerator. The cells were centrifuged again, allowed to settle, mixed with 2 mL of buffer, and left to stand at 4℃ for 30min in the refrigerator(repeated three times). The cells were soaked in 1% sour for 1h, and the the buffer was rinsed twice for 15min each.Ultra-thin slices were prepared by dehydrating the cells with increasing concentrations of alcohol , gradually penetrated with epoxy resin, and fixed with acetate. The sections were stained with citrate, and cell ultrastructure was observed through transmission electron microscopy (TEM).

All cells were incubated in a humidified incubator at 37℃ with 5% CO. ARPE-19 cells were incubated in a humidified incubator at 37℃ with 5% CO, and the liquid was changed every other day. When cell density increased to 80%-90%, the cells were digested and passed on in a ratio of 1:3. The third to sixth generations of the cells in the logarithmic phase were selected. Cell suspension was incubated into a 96-well culture plate at a concentration of 1×10cells/well (100 μL per well). The plate was placed in an incubator at 37℃ and 5% COfor 12h, and the experiment was started after cell adherence. The experimental samples were divided into five groups: ARPE-19 cell group (control group), ARPE-19 cell +illumination group (model group), ARPE-19 cells + illumination for the first 2h + autophagy inhibitor 3MA group (3 mmol/mL),ARPE‐19 cells + illumination for the first 2h + LBP low‐dose group (10 mg/L), ARPE-19 cells + illumination for the first 2h + LBP middle-dose group (50 mg/L), and ARPE-19 cells +illumination for the first 2h + LBP high‐dose group (100 mg/L).Indexes were tested after illumination for 24h.

The third to sixth generations of ARPE-19 cells in the logarithmic phase were selected and cultured in a 6-well plate in a closed incubator under illumination without natural light interference. A three-primary-color light-emitting diode cold light lamp was used as the light source. It was hung at the top of the incubator for straight illumination. According to previous experimental results, the illuminometer was used in monitoring illumination strength. The illumination strength of the cells was controlled at 16500±500 lx, and the illumination time was 24h. During illumination, temperature fluctuations in the cell plane were controlled between 36.5℃ and 37.5℃.This procedure eliminated the possibility of photoinduced injuries at increasing temperature.

据笔者统计，唐圭璋于《全宋词》中收录《卜算子》共计236首，其中《卜算子·和惜惜》与《卜算子·答幼谦》两首皆宋元小说话本中人物词，本文不做分析。另有3首闺情，皆为元明小说话本中紫竹、玉娇娘、无名氏依托宋人词，亦不做分析。故而真正作于两宋时期的《卜算子》只有231首，另外，《全宋词》中有脱文8首，本文亦不做研究。综上，本文研究的完整词作共有223首。

The second to fourth generations of ARPE-19 cells in the logarithmic phase were inoculated into a 96-well plate after digestion at a density of approximately 5×10cells/well. The cells were starved in media without fetal calf serum overnight in an incubator. All groups were terminated from culture at the end of illumination, and the old media were eliminated through absorption. Then, 100 µL of preheated new media were added to each well (containing 10 µL of CCK8 solution)and incubated under 37℃ for 2.5h. The 96‐well plate had three replicates of holes for each group, and a control group was set.The optical density (OD) of each hole at 450 nm was detected through ELISA. The experiment was repeated three times.According to mean±standard deviation, data were calculated,and a cell survival rate diagram was drawn. Cell survival rate was calculated as follows: [(OD of cells with drugs–OD of blank control group)/(OD of control cells–OD of blank cells)]×100%.

ARPE-19 cells grew well, and cells in the logarithmic phase were selected as experimental samples. After illumination treatment, all groups were processed, and the old culture solution was collected and then rinsed with warm phosphate buffer saline (PBS) two or three times. The cells were digested with 0.25% pancreatin without ethylene diamine tetraacetic acid (EDTA) and phenol red. The cells were mixed evenly for the preparation of a single-cell suspension. The suspension was transferred to a centrifuge tube, and then centrifuged for 5min at a rotation rate of 1200 rpm/min. The supernate was eliminated, and residues were rinsed with 2 mL of PBS once, mixed evenly, and finally used in preparing a singlecell suspension. The suspension was centrifuged again for 5min at a rotation rate of 1200 rpm/min, and the supernate was eliminated. The process was repeated twice. The cells were suspended in 400 µL of annexin-V binding buffer, and 5 µL of FITC was added under the dark. The mixture was left to stand for 15min under 4℃. Subsequently, 10 µL of proteinase inhibitor was added in the dark for 5min before the loading,and flow cytometry was accomplished in 1h.

[2] James E. Dougherty, Robert L. Pfaltzgraff, Contending Theories of International Relations: a Comprehensive Survey(5th Edition), 北京：北京大学出版社, 2004年, pp. 165-166.

LC3 and P62 proteins were located in the cytoplasm and appear as claybank particles after staining. The immunofluorescence method was used to detect the staining degree of LC3Ⅱ,LC3Ⅰ, P62 before and after photoinduced authophagy and after intervention with different concentrations of LBP. These results indicated that LBP inhibits autophagy and increases cell survival rate (Figure 4).

SPSS 21.0 or Graphpad Prism 6.0 was used in statistical analysis. Data were expressed as mean±standard deviation(s). The normal distribution of data was tested.One-factor ANOVA was used for data comparison, which conformed to normal distribution and homogeneity of variance. Multiple comparisons of several sample means were tested with the SNK‐Q method, and the significance level was determined at 0.05 (=0.05).

RESULTS

Age-related macular degeneration (AMD) is a disease characterized by degenerative changes and angiogenesis in photoreceptors in the retinal macular zone and retinal pigment epithelium (RPE). It is a major chronic disease that causes blindness in aged people and greatly reduces not only their quality of life but also that of their families. Stimulated under certain conditions and influenced by autolysosomes or autophagy vesica, autophagy provides substances essential to basic cell survival by degrading and eliminating subcellular fragments. The role of autophagy in the optical injuries of the retina is controversial. Autophagy disorder can induce the apoptosis of RPE cells, and inhibiting autophagy can prevent light-induced damage to photoreceptors. Therefore,elaborating the role and action mechanism of autophagy in photoinduced retinal injuries and searching an effective therapeutic strategy for AMD are necessary.

Figure 2 shows that the cell survival rates of the model group after 24h of photodamage (16500±500 lx)decreased significantly compared with that of the control group (<0.01). The cell survival rates of the 3MA groups and the LBP-L, LBP-M, and LBP-H groups with photodamage increased compared with the cell survival rate of the model group (<0.01). A certain intensity of photodamage can considerably decreased cell survival rate, and modeling was successful. LBP increased the survival rate of RPE cells in a dosedependent manner and protected RPE cells from photodamage.

Figure 3 shows that the apoptosis level of the model group increased dramatically compared with that of the control group(<0.01) and the apoptosis level of the 3MA group decreased compared with that of the model group (<0.01). The apoptosis levels of the ARPE-19 cells in the LBP-L, LBP-M,and LBP-H groups were lower than the apoptosis level of the model group (<0.01). These results indicated that LBP can inhibit the photoinduced apoptosis of ARPE-19 cells and its ability to inhibit apoptosis is positively related to its concentration.

Proteins in the cells of each group were extracted using protein lysates, and protein quantification was performed with a BCA quantitative determination kit. Specifically,50 µg of each sample was loaded through degeneration after computer adjustment. The samples were transferred to a PVDF transmembrane after SDS-PAGE electrophoresis, and the primary antibody was added. The mixture was mixed with 5% skim milk powder for 2h, sealed overnight at 4℃, rinsed with tris buffered saline tween (TBST) three times (10min),mixed with the second antibody, and incubated in a shaker at 37℃ for 2h. A luminescent solution was added in a dark room for exposure. After X-ray development and photographic fixing, the gray values of the protein stripes were analyzed with Ⅰmage J. The gray values of different protein stripes were quantitatively analyzed, and β‐actin was used as the internal reference. A histogram was drawn, and data were expressed as mean±standard deviation (=3).

As shown in Figure 5, the expression levels of beclin1 and LC3 in the model group increased compared with those of the control group, LC3Ⅱ/LC3Ⅰ level increased and P62 expression level was low. These results indicated that autophagy was activated to a higher degree in the model group than that in the control group. In other words, illumination induced autophagy and increased autophagy flux. After intervention with different concentrations of LBP, the expression levels of beclin1 and LC3Ⅱ, which were proteins related to photoinduced autophagy,and LC3Ⅱ/LC3Ⅰ decrease, whereas the expression level of P62 increased. The expression levels of beclin1, LC3Ⅱ and LC3Ⅱ/LC3Ⅰ were negatively related to LBP concentration,whereas the expression level of P62 was positively related to LBP concentration. These results showed that LBP can inhibit photoinduced autophagy in ARPE-19 cells. The LBP-H group(100 mg/L) shows a higher degree of autophagy inhibition in ARPE-19 cells than the LBP-L group (10 mg/L).

Figure 6 shows that the expression levels of PI3K, P-mTOR,and P-Akt, which were proteins related to the proliferation pathway in the model group, decrease after 24h of illumination compared with those in the control group (<0.01). Similarly,P-mTOR/mTOR and P-Akt/Akt (<0.01) were reduced. The expression levels of PI3K, P-mTOR, and P-Akt in the LBP-L,LBP-M, and LBP-H groups increased compared with those in the model group (<0.01). P-mTOR/mTOR and P-Akt/Akt levels are also increased (<0.01). We concluded that LBP may promotes the phosphorylation of mTOR and Akt, induces the activation of the PI3K/Akt/mTOR signal pathway, and inhibits the expression of beclin1 and LC3Ⅱ.

Autophagy is conducive to the maintenance of steady balance and metabolic adaptation of normal tissues and is an indispensable biological process. However, excessive autophagy has serious consequences. Autophagy eliminates lipid peroxides in RPE cellsand is extremely active in RPE and photoreceptor cells. Moreover, impaired autophagy might lead to the early degradation of RPE cells. The physiological functions of RPE can be associated with autophagy in age-related retinal diseases and photoinduced retinal degenerative diseases, such as AMD. Degenerative changes in RPE cells are the major characteristics of AMD, and excessive illumination can accelerate the death of RPE cells and intensify the development of AMD. Furthermore, autophagy can damage RPE cells under photoinduction, and inhibiting autophagy has a protective effect on RPE cells damaged by photodamage-induced. This result conforms to the research conclusions of Song,that might be a potential therapeutic strategy for early AMD.Beclin 1 is an important factor in the formation of autophagosomes and is formed for positively regulated autophagy. LC3 is the most classical autophagic molecular markerand can realize target location in autophagosomes. When autophagy is activated, LC3-I in the cytoplasm is transformed into LC3-II in membrane bonding form. In general, increasing LC3-II or LC3II/I level indicates the formation of autophagyand reflects strengthened autophagy in cells. P62 is an ubiquitination substrate associated with LC3 and integrated into autophagosomes. P62 binds with lysosomes to form autolysosomes. When autophagy is activated, organelles, such as P62, in autophagy vesicles are degraded, and the expression level of P62 decreases; otherwise, the expression level of P62 increases when autophagy is inhibited.In this experiment, ARPE-19 cells were exposed to 16500±500 lx of illumination. According to TEM observation, classical dualmembrane autophagosome was present in the ARPE-19 cells of the model group, indicating that illumination at this intensity can induce autophagy in ARPE-19 cells. The expression level of beclin1, which was an autophagy marker, in the model group increases significantly (<0.01), and LC3II/I level is increased (<0.01), whereas the expression level of P62 was low (<0.01). After 24h of intervention was performed on photoinduced RPE cells with different concentrations of LBP, the survival rate was higher than that of the model group(<0.01). According to flow cytometry, the LBP-L, LBP-M,and LBP-H groups showed lower levels of apoptosis than the model group (<0.01). Moreover, compared with the model group, the dependence of LC3II/I and expression level of beclin1 on LBP dosage in the LBP-L, LBP-M, and LBP-H group decrease (<0.05), and the expression level of P62 increased (<0.05). These results showed that LBP can inhibit the apoptosis of the ARPE-19 in the model group and dosage dependence decreased the autophagy levels of ARPE-19 cells.The detection of LC3II/I and expression level of P62 with the cell immunofluorescence method further proved that LBP can inhibit autophagy in ARPE-19 cells and protect ARPE-19 cells from photodamage.

DISCUSSION

According to Figure 7, the 3MA group had a lower expression level of beclin1 in the ARPE-19 cells after 24h of illumination than the model group (<0.01), and the LC3Ⅱ/LC3Ⅰ decreases (<0.01),but the expression level of P62 was increased (<0.05). The expression level of beclin1 was higher than that in the control group (<0.01), and LC3Ⅱ/LC3Ⅰ level increased slightly(<0.05). The P-mTOR/mTOR level of the 3MA group was higher than that of the model (<0.05) but lower than that of the control group (<0.01). These result indicateed that autophagy activated by illumination in the 3MA group was inhibited after the upregulation of P-mTOR.

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The solution containing the third to fourth generations of ARPE-19 cells in the logarithmic phase were collected for hepatocytes growing on glass coverslips at a density of 6×10cells/piece. The cells were mixed evenly and cultured for 48h. Drugs were added according to different groups, and 24h of illumination was provided after 2h. Cells were collected and rinsed with PBS three times, fixed with 4% paraformaldehyde for 30min,and stored at 4℃. The samples were then rinsed three times(3min each) and then incubated for 15min in 3% endogenous blocker hydrogen peroxide under 37℃. The samples were subsequently washed with PBS three times for 3min each. A working solution of goat serum was added, and the mixture was incubated for 30min at 37℃. The primary antibody was added, and the solution was left to stand at 4℃ overnight.Subsequently, the cells were warmed for 1h at 37℃ and rinsed with PBS three times for 10min each. The second antibody was added dropwise, and the solution was incubated for 30min at 37℃ before rinsing with PBS three times for 5min each.The samples were treated with DAB for 2-3min, and excessive deep colors were prevented. Then, the samples were re-stained with hematoxylin for 3min. The gradient dehydration of alcohol and dripping of neutral vegetable glue were performed.Finally, glass coverslips were sealed.

The PI3K/Akt/mTOR signal pathway plays an important role in the survival, proliferation, apoptosis, and autophagy of cells. The PI3K/Akt/mTOR signal pathway can weaken autophagy, and inhibit cell apoptosis. Thus, the effects of LBP on autophagy and apoptosis in ARPE-19 cells were discussed preliminarily. Moreover, how LBP protects ARPE-19 cells through the PI3K/Akt/mTOR signaling pathway in the photoinducedARPE-19 cell model was explored. The expression level of PI3K in the model group decreased compared with that of the control group (<0.01), and P-mTOR/mTOR and P-Akt/Akt levels were low (<0.01). Moreover, PI3K,P-mTOR/mTOR, and P-Akt/Akt levels in the LBP-L, LBP-M,and LBP-H groups increased relative to those in the model group (<0.01). These data showed that LBP can protect RPE cells from photodamage, and such protective effect was related to the activation of the PI3K/Akt/mTOR signaling pathway.

冬天也想保持水嫩光泽的皮肤，除了挑对护肤品外，也要会挑食物。有些食物是天然的“护肤品”，吃对了也能让你的皮肤年轻滋润。

As a PI3K inhibitor, 3MA is extensively used in studies on autophagy. In the present study, 3MA was used in processing cells. The expression levels of LC3II/I and beclin1 in the 3MA group increased slightly, whereas the expression level of P62 decreased relative to those in the control group. Moreover, the expression levels of LC3II/I and beclin1 in the 3MA group are decreased, but the expression level of P62 is increased compared with those of the model group. These results further demonstrated that 3MA inhibits photoinduced excessive autophagy in ARPE-19 cells.According to our previous studies, LBP is involved in the PI3K/Akt/mTOR signaling pathway and can inhibit the formation of reactive oxygen species and malonaldehyde(MDA) and oxidative stress to protect cells from photodamage.Oxidative stress can activate the autophagy of nerve cells and is thus conducive to the decomposition of macromolecules and collection of their components. This feature retains cell energy and eliminates damaged proteins and mitochondria.However, long-term autophagy can cause cell apoptosis.This study further discussed. LBP could protect cells by inhibiting excessive autophagy. LBP might protect ARPE-19 from photodamage by inhibiting cell apoptosis and autophagy.To protect RPE cells from photodamage, LBP promotes the phosphorylation of mTOR and Akt, thus activating the PI3K/AKt/mTOR signaling pathway and inhibiting autophagy.However, LBP may also protect RPE cells from photodamage through other pathways, and this possibility deserves further study.

和谐是一种完美，是自然界、人类社会、人类思维存在的最理想状态。“凡是美的都是和谐的和比例合度的，凡是比例和谐的和比例合度的就是真的，凡是既美又真的也就在结果上是愉快的和善的。”这段话揭示出和谐与真、善、美的关系。教学的最终目的是使学生的基本素质和个性品质得到全面、充分、和谐的发展。

Gao YY performed the review,analyzed the data, and wrote and edited the manuscript. Yu Y, Li J, Huang J, and Li WJ each contributed to writing and editing of the manuscript.

Supported by National Natural Science Foundation of China (No.82060885); Ningxia Scientific Research Program for Institution of Higher Education(No.NGY2018-99); Ningxia Natural Science Foundation(No.2020AAC03172).

2.2 性别和年龄分布特征 2012-2017年的524例麻疹病例中，男性311例，阳性为177例，阳性率56.91%；女性213例，阳性为108例，阳性率50.70%，男女间阳性率差异无统计学意义（χ2=1.964 6，P＞0.05）。麻疹抗体阳性病例中，年龄最小的14 d，最大的54岁，按年龄的大小，病例分为5个年龄组，0～7月组阳性率最高，其次是8月～6岁组，两个年龄组的阳性例数占比为69.82%（199/285），15～22岁组阳性率最低，阳性例数占比仅为2.46%（7/285）。不同年龄组间阳性率差异有统计学意义（χ2=47.933 3，P＜0.05），见表1。

None;None;None;None;None.