ETM Experimental and Therapeutic Medicine 1792-0981 1792-1015 D.A. Spandidos ETM-0-0-09843 10.3892/etm.2021.9843 Articles Chlorogenic acid enhances autophagy by upregulating lysosomal function to protect against SH-SY5Y cell injury induced by H2O2 GaoLi-Juan 1 2 * DaiYuan 2 * LiXiao-Qiong 1 2 MengShi 1 2 ZhongZhan-Qiong 1 3 XuShi-Jun 1 2 Institute of Meterial Medica Integration and Transformation for Brain Disorders, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan 611137, P.R. China School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan 611137, P.R. China School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan 611137, P.R. China Correspondence to: Professor Shi-Jun Xu, Institute of Meterial Medica Integration and Transformation for Brain Disorders, Chengdu University of Traditional Chinese Medicine, 1166 Liutai Avenue, Chengdu, Sichuan 611137, P.R. China xushijun@cdutcm.edu.cn

Miss Zhan-Qiong Zhong, School of Basic Medical Sciences, Chengdu University of Traditional Chinese Medicine, 1166 Liutai Avenue, Chengdu, Sichuan 611137, P.R. China zhongzhanqiong@cdutcm.edu.cn

*Contributed equally

 05 2021 26 02 2021 21 5 426 04 11 2019 11 08 2020 Copyright: © Gao et al. 2020 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Autophagy serves an important role in amyloid-β (Aβ) metabolism and τ processing and clearance in Alzheimer's disease. The progression of Aβ plaque accumulation and hyperphosphorylation of τ proteins are enhanced by oxidative stress. A hydrogen peroxide (H2O2) injury cell model was established using SH-SY5Y cells. Cells were randomly divided into normal, H2O2 and chlorogenic acid (5-caffeoylquinic acid; CGA) groups. The influence of CGA on cell viability was evaluated using a Cell Counting Kit-8 assay and cell death was assessed using Hoechst 33342 nuclear staining. Autophagy induction and fusion of autophagic vacuoles assays were performed using monodansylcadaverine staining. Additionally, SH-SY5Y cells expressing Ad-mCherry-green fluorescent protein-LC3B were established to detect autophagic flow. LysoTracker Red staining was used to evaluate lysosome function and LysoSensor™ Green staining assays were used to assess lysosomal acidification. The results demonstrated that CGA decreased the apoptosis rate, increased cell viability and improved cell morphology in H2O2-treated SH-SY5Y cells. Furthermore, CGA alleviated the accumulation of autophagic vacuoles, reduced the LC3BII/I ratio and decreased P62 levels, resulting in increased autophagic flux. Additionally, CGA upregulated lysosome acidity and increased the expression levels of cathepsin D. Importantly, these effects of CGA on H2O2-treated SH-SY5Y cells were mediated via the mTOR-transcription factor EB signaling pathway. These results indicated that CGA protected cells against H2O2-induced oxidative damage via the upregulation of autophagosomes, which promoted autophagocytic degradation and increased autophagic flux.

 Alzheimer's disease chlorogenic acid autophagy transcription factor EB lysosomal Funding: The present study was supported by the e National Natural Science Foundation of China (No. 82074150), Key Research and Development Project of Sichuan Province (Major Science and Technology Projects; grant no. 19ZDYF0600) and the Chengdu Technological Innovation R & D project (grant no. 2019-YF05-01332-SN). Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss, cognitive dysfunction, personality changes and decreased mobility (1). Furthermore, AD is pathologically characterized by senile plaques (SP) and neurofibrillary tangles (1). According to a 2019 report by the Alzheimer's Association, the risk of developing AD has increased to 1:10 for individuals older than 65 years and is as high as 1:2 among individuals older than 85 years in USA (2). Therefore, effective treatments and controlling the pathological processes underlying AD are of great concern in the medical field.

The pathogenesis of AD is complex and there are numerous hypotheses regarding its occurrence, including neuroinflammation, cholinergic dysfunction, metabolic alterations, amyloid-β (Aβ) peptide accumulation and abnormalities in τ protein metabolism (3-5). Although these hypotheses are controversial, the accumulation of Aβ is one of the major theories recognized by researchers (6). According to the Aβ cascade hypothesis, mutations in the amyloid precursor protein (APP), presenilin-1 (PS-1) and PS-2 upregulate Aβ secretion, leading to the accumulation of Aβ, which forms small molecule oligomers that are further aggregated into fibers and deposited to form extracellular SPs (7). Aβ plaques create an environment that allows for the rapid induction of pathological τ aggregates from endogenous τ and may enhance or sustain τ pathologies through additional mechanisms, including intracellular protein degradation impairment (8). Furthermore, Aβ plaques induce oxidative stress and mediate damage to numerous biological macromolecules, including proteins, nucleic acids and lipids, resulting in changes of neuronal structure and function, and potential neuronal loss (9). A previous study has indicated an association between oxidative stress and protein aggregation processes, which are involved in the development of proteinopathies (10). Therefore, oxidative stress can be considered either causative or consecutive in regard to protein aggregation (11). Furthermore, recent studies have demonstrated that medications used to successfully treat AD-like symptoms act by modulating oxidative stress (12,13).

Autophagy is involved in numerous diseases, including cancer, diabetes and neurodegenerative disorders (14). Autophagy consists of a dynamic pathway, including the formation of surrounding membranes and the degradation of diverse cellular components, such as organelles and protein aggregates (15). Additionally, autophagy serves an important role in Aβ metabolism and τ processing and clearance (16). Furthermore, autophagy contributes to endoplasmic reticulum stress and mitophagy in relation to AD pathology (17,18).

Chlorogenic acid (5-caffeoylquinic acid; CGA) is a polyphenolic substance that is widely found in the human diet (19). CGA has various pharmacologic actions, including antioxidant, anti-inflammatory and anticarcinogenic activities, that exert hypoglycemic and hypolipidemic effects (20). A number of studies have indicated that dietary consumption of CGA reduces the risk of developing neurodegenerative diseases (21,22). An in vitro biological study revealed that CGA has an enhanced inhibitory effect on Aβ40 aggregation and protects PC12 cells from Aβ-aggregation-induced cell death (23). Additionally, a previous study demonstrated the neuroprotective effects of CGA and its major metabolites in primary cultures of rat cerebellar granule neurons (24). Furthermore, numerous studies have reported that CGA reduces the risk of neurodegenerative diseases (21,22); however, the mechanism by which CGA confers this protective effect remains unclear. To investigate the underlying mechanism, the present study employed a hydrogen peroxide (H2O2)-induced SH-SY5Y cell model and investigated whether CGA regulated autophagic flow and lysosomal function via the mTOR/transcription factor EB (TFEB) pathway to protect against H2O2-induced cell damage.

 Materials and methods <sec> <title>Cell model and CGA treatment

SH-SY5Y human neuroblastoma cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (cat. no. 20161116-02) and were authenticated by a short tandem repeat profiling service provided by American Type Culture Collection. CGA (purity, ≥98%; assessed by high performance liquid chromatography) was purchased from Baoji Herbest Bio-Tech Co., Ltd. The details were as follows: Instrument: Spd-16 High performance liquid chromatography (Shimadzu Corporation); chromatographic column, Sepax Bio-C18 [Yuexu Technology (Shanghai) Co., Ltd.; 4.6x250 mm; 5 µl]; temperature, 30˚C; injection volume, 20 µl; mobile phase, acetonitrile-0.4% acetic acid aqueous solution; velocity of flow: 1.0 ml/min; and detection wavelength, 350 nm. H2O2 (34.01 g/mol) was purchased from Sigma-Aldrich; Merck KGaA.

The cell model was established as previously described (25). Briefly, SH-SY5Y cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Zhejiang Tianhang Bio Polytron Technologies Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and incubated at 37˚C in a humidified atmosphere containing 5% CO2 (3111 CO2 incubator; Thermo Fisher Scientific, Inc.).

Cells in the logarithmic growth phase were selected and digested with 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.). Subsequently, cells were seeded into 96-well plates (seeding density, 3x104 cells/ml) for 16 h. To determine the optimal concentration and duration of H2O2 treatment to elicit toxic effects on SH-SY5Y cells, cells were treated with 50, 100, 200, 400, 500, 600, 700 or 800 µM H2O2 for 4, 8, 12 or 48 h. After 12 h of plating, the medium was replaced with CGA (100, 50, 25, 12.5, 6.25 and 3.125 µM) in the CGA groups or fresh DMEM in the normal and model groups. These cells were incubated for an additional 24 h prior to analyses.

 Cell viability assessment using a Cell Counting Kit-8 (CCK-8) assay

Cell viability was measured using CCK-8 assays according to the manufacturer's protocol. Briefly, cells were treated with different concentrations of H2O2 and viability was measured at 12, 24 and 36 h. Cells were treated with 10 µl CCK-8 reagent (cat. no. JE914; Dojindo Molecular Technologies, Inc.) for 2 h at 37˚C. Following this, optical density (OD) values were measured at a wavelength of 450 nm using an automatic microplate reader (Thermo Fisher Scientific, Inc.). The SH-SY5Y cells were treated with different concentrations of CGA (0, 100, 200, 300, 400, 500, 600, 700 and 800 µM) for 12, 24 and 36 h to determine the optimal concentration, and cell viability was detected using the CCK-8 assay. Cell viability (%) was calculated as follows: ODvalue of the administration well - ODvalue of the zero well/ODvalue of the normal well - ODvalue of the zero well. Morphologic changes were observed using phase-contrast microscopy (x400), and analysis of the length of cell processes and numbers of SH-SY5Y cells were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.).

 Monodansylcadaverine (MDC) and Hoechst fluorescence staining

MDC (Sigma-Aldrich; Merck KGaA) is a lysosomotropic compound used for the identification of autophagic vesicles via fluorescence microscopy and for the assessment of autophagy induction via the accumulation of MDC-labeled vacuoles (26). Hoechst staining was used to test for cell apoptosis. CGA (50, 25, 12.5 and 6.25 µM) and/or H2O2 (500 µM) was added to each well at 37˚C. After 24 h, cells (1x106/well) were washed twice with PBS and treated with 500 µl MDC solution (25 µM) or Hoechst solution (50 µM) and incubated for 30 min at 37˚C. Fluorescence images were captured using an inverted fluorescence microscope (DMI3000; Leica Microsystems GmbH) and analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.).

 Autophagic flow test

mCherry-green fluorescent protein (GFP)-LC3 adenoviruses (cat. no. 030217170302; Beyotime Institute of Biotechnology) were infected into SH-SY5Y cells, according to the manufacturer's protocol. SH-SY5Y cells were inoculated into 24-well plates (2.5x105/well) and cultured for 24 h at 37˚C. Subsequently, the culture medium was removed and a virus solution at MOI=5 was added. Cells that were not exposed to the virus served as the control group. Cells were cultured for 12 h at 37˚C with 5% CO2. After 24 h of treatment with CGA (50 µM) and/or H2O2 (500 µM), mean fluorescence intensities were measured using Image-Pro Plus software.

 Western blotting

The primary antibodies used in the present study were as follows: Anti-rabbit-sequestosome 1 (SQSTM1)/p62 (1:1,000; Cell Signaling Technology, Inc.), anti-rabbit-Lamin A/C (1:1,000; Cell Signaling Technology, Inc.), anti-rabbit-phosphorylated-mTOR Ser 2448 (P-mTOR; 1:1,000; Cell Signaling Technology, Inc.), anti-rabbit-TFEB (1:1,000; Cell Signaling Technology, Inc.), anti-rabbit-mTOR (1:1,000; Cell Signaling Technology, Inc.), anti-rabbit-LC3B (1:500; Cell Signaling Technology, Inc.), anti-rabbit-Beclin 1 (1:500; BIOSS), anti-rabbit-autophagy protein 5 (Atg5)/autophagy related 5 (1:1,000; BIOSS), anti-rabbit GAPDH (1:5,000; Wuhan Servicebio Technology Co., Ltd.), anti-rabbit-cathepsin D (CTSD; 1:500; Beyotime Institute of Biotechnology), anti-rabbit-Bcl-2 (1:500; Wuhan Sanying Biotechnology), anti-rabbit-Bax (1:500; Wuhan Sanying Biotechnology Co., Ltd.). Cells were washed with PBS and subjected to lysis at 4˚C in RIPA lysis buffer (Servicebio Technology Co., Ltd.) supplemented with phenyl methylsulfonyl fluoride [Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.], phosphatase inhibitors (Boster Biological Technology) at a ratio of 100:1:1 and a cocktail of protease inhibitors [Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.]. Cell suspensions were centrifuged at 800 x g for 10 min at 4˚C and protein concentrations were quantified using a BCA kit (Sichuan Mike Biological Technology Co., Ltd.). Protein extracts (30 µg/lane) were separated and fractionated via 8-15% SDS-PAGE and transferred to a PVDF membrane (Merck KGaA). The membranes were blocked with 5% BSA (Guangzhou Saiguo Biotech Co., Ltd.) for 90 min at room temperature. Cells were incubated with primary antibodies overnight at 4˚C. After washing with TBS with 0.1% Tween-20, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000; BIOSS) for 2 h at room temperature. The best blocking reagent was 5% BSA at 90 min. Following washing, bands were visualized using an ECL kit (Beijing 4A Biotech Co., Ltd.). Quantity One software (v4.6.6; Bio-Rad Laboratories, Inc.) was used for quantification.

 LysoTracker Red and LysoSensor™ Green staining

Lysosomal staining was performed using LysoTracker Red and LysoSensor Green staining assays in order to assess lysosomal acidification. Following 16 h of treatment with CGA (50 µM) at 37˚C, Earle's Balanced Salt Solution (EBSS; cat. no. 354655A; incubation, 30 min; Beyotime Institute of Biotechnology), chloroquine (CQ; 25 µM; incubation, 2 h; cat. no. J0102A; Dalian Meilun Biology Technology Co., Ltd.) and H2O2 (500 µM; incubation, 12 h) were added. Cells (2.5x105/well) were subsequently incubated with LysoTracker Red (5 µl) and LysoSensor Green (10 µl) for 30 min at 37˚C, washed with PBS and examined using fluorescence microscopy (Leica Microsystems GmbH). The mean fluorescence intensity was measured using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.).

 Statistical analysis

Statistical analysis was performed using SPSS software (version 21.0; IBM Corp.). Quantitative data are presented as the mean ± SD. All experiments were repeated at least three times Non-parametric tests were used for data that were not normally distributed. Comparisons among different groups were performed using one-way ANOVA. If the datasets contained more than three groups, Tukey's multiple comparison analysis was used. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>Effect of CGA on SH-SY5Y cells

SH-SY5Y cells were treated with different concentrations of H2O2 to determine the optimal concentration for subsequent experiments, as assessed by CCK-8 assays. Different concentrations of H2O2 (100-800 µM) affected cells differently at 12 and 24 h compared with the normal group (Fig. 1A). Treatment with H2O2 decreased cell viability in a dose- and time-dependent manner, and the IC50 of 500 µM H2O2 regarding cell injury at 24 h was 973.90 µM. The cell viability was 69.25±3.17% following treatment with 500 µM H2O2 for 24 h. Based on the results of this experiment and previous studies, 500 µM H2O2 and 24 h were selected as the optimal damage conditions for subsequent experiments (27,28).

According to previous study, CGA has a protective effect induced by H2O2 (29). However, CGA treatment decreased cell viability and exhibited drug toxicity at concentrations of 50-400 µM at 12-36 h (Fig. 1B). Therefore, 6.25-50 µM was regarded as the optimum concentration range for CGA treatment in the present study.

 Neuroprotective effect of CGA on SH-SY5Y cells injured by H<sup>2</sup>O<sup>2</sup>

To investigate the effects of CGA on SH-SY5Y cells injured by H2O2, four concentrations of CGA (50, 25, 12.5 and 6.25 µM) were used. The morphology of the cells was altered following 24 h of incubation with CGA (Fig. 1C). Data revealed that the length of cell processes significantly reduced in the H2O2 group, luckily, it could be rescue by CGA, CGA concentrations of 50 and 12.5 µM ameliorated the morphological alterations (length of cell processes) of the cell model (Fig. 1D). Similarly, the numbers and viability of the SH-SY5Y cells were rescued by all concentrations of CGA (Fig. 1E and G).

Cell apoptosis was assessed with Hoechst staining and the average cell apoptosis rate was significantly decreased in each CGA treatment group compared with the H2O2 treatment group (Fig. 1F and H). The changes of Bax and Bcl-2 protein expression may play an important role in cell apoptosis (30). Therefore, the results demonstrated that CGA upregulated Bcl-2 expression and downregulated Bax expression to inhibit cell apoptosis (Fig. 1I). It has been suggested that the Bax/Bcl-2 ratio may be more important than either protein alone in determining apoptosis (31). These results indicated that CGA alleviated the morphological damage and effectively reduced apoptosis in H2O2-treated SH-SY5Y cells.

 Effect of CGA on autophagic flux in SH-SY5Y cells treated with H<sup>2</sup>O<sup>2</sup>

The effects of CGA on the accumulation of autophagic vacuoles in SH-SY5Y cells injured by H2O2 were assessed. H2O2 treatment increased the average fluorescence intensity of MDC in a time-dependent manner when compared with the control group (Fig. 2A and B). Furthermore, the different concentrations of CGA significantly reduced the average fluorescence intensity of MDC in the cell model at 12 and 24 h (Fig. 2A and B).

Autophagic flux is a dynamic process and includes the formation of autophagosomes, fusion of autophagosomes with lysosomes and cargo degradation. Atg5 and Beclin-1 are involved in the formation of autophagosomes (32) and the results indicated that H2O2 increased Atg5 and Beclin-1 levels, which likely promoted an enhancement of autophagosome formation. To further characterize the effects of CGA on autophagic flux in the SH-SY5Y cell model, autophagy-associated proteins were assessed in each group. Western blotting demonstrated that the expression levels of Beclin-1 in the 50 and 25 µM CGA groups and Atg5 in the 25, 12.5 and 6.25 µM CGA groups were significantly downregulated (Fig. 2C). These data indicated that CGA inhibition of autophagosome production may occur via the downregulation of Beclin-1 and Atg5 expression.

To determine whether CGA had any effect on H2O2-mediated autophagy activation, western blotting was performed. The results indicated that the intensity of LC3B-II/I bands in the SH-SY5Y cell model was decreased in the 25, 12.5 and 6.25 µM CGA groups compared with the H2O2 treatment group. P62/SQSTM1 is an autophagy-selective substrate complex and LC3B-II is localized on the inner membrane to form the complex (33). The expression levels of P62/SQSTM1 were decreased in each CGA treatment group (Fig. 2D). Therefore, these results suggested that CGA increased autophagic flux by reducing the levels of LC3-II/I and P62/SQSTM1.

SH-SY5Y cells successfully transfected with mCherry-GFP-LC3 were used as vectors to investigate the effect of CGA on autophagic flow. When autophagy is activated, the fluorescent GFP signal enters the lysosome. However, during autophagy flux, lysosomes fuse with autophagosomes, and the green (GFP) fluorescence is quenched by the acidic microenvironment whereas the mCherry signal remains stable (34). After entering the lysosome, the mCherry signal can still be observed (34). The co-localization of GFP and mCherry (observed as yellow fluorescence) represents the blockage of autophagic flow. Compared with the H2O2 treatment group, treatment with CGA reduced the occurrence of mCherry+ GFP+ (yellow) spots induced by H2O2 and increased the occurrence of mCherry+ GFP- (red) spots (Fig. 2E).

 Effect of CGA on lysosome function in SH-SY5Y cells injured by H<sup>2</sup>O<sup>2</sup>

To investigate how CGA activity may affect autophagy, lysosome function in the SH-SY5Y cell model was assessed. CQ is a lysosomal blocker that blocks the autophagy-lysosomal pathway (ALP) by alkalizing the acidic environment of lysosome and inhibiting the degradation of lysosomes (35). Following 25 µM CQ pretreatment for 2 h to inhibit lysosome activity, the results indicated that CQ successfully reversed the decreased cell viability conferred by H2O2 treatment (Fig. 3A). These results indicated that H2O2 reduced cell viability, and CGA significantly relieved the damage caused by H2O2 in SH-SY5Y cells. However, CQ had an antagonistic effect on the anti-H2O2 effects of CGA in regard to cytotoxicity (Fig. 3B).

Following this, the effects of CGA on the lysosomal acidity of H2O2-treated SH-SY5Y cells were investigated. LysoTracker Red specifically labels intracellular lysosomes and these are presented as red fluorescent spots under a fluorescence microscope. LysoSensor Green exhibits a pH-dependent increase in fluorescence intensity following acidification, which is used to detect the effect of CGA on the lysosomes. EBSS is a balanced salt solution that induces autophagosome formation via starvation while rapidly upregulating the acidic environment of the lysosomes and, therefore, promoting lysosomal degradation and enhancing the ALP (36). H2O2 treatment significantly decreased the average fluorescence intensity of LysoTracker Red (Fig. 3C). Compared with the H2O2 treatment group, the 50, 25 and 12.5 µM CGA groups exhibited significant upregulation of the mean fluorescence intensity of LysoTracker Red induced by H2O2. The acidic intracellular compartments (lysosomes) in SH-SY5Y cells were measured using LysoSensor Green. H2O2 (500 µM) exposure significantly reduced the fluorescence signal compared with the control group (Fig. 3D), indicating a H2O2-mediated reduction of intracellular acidic components. Furthermore, treatment with CGA significantly increased LysoSensor Green fluorescence compared with the H2O2 treatment group. Finally, the expression levels of CTSD in the different CGA groups were determined. These data revealed that the levels of CTSD were significantly enhanced in the 50, 25 and 12.5 µM CGA + H2O2 groups compared with the H2O2 treatment group (Fig. 3E).

 Effect of CGA on the mTOR-TFEB signaling pathway in SH-SY5Y cells treated with H<sup>2</sup>O<sup>2</sup>

Western blotting was used to determine total protein and TFEB levels in cell nuclei. The results indicated that H2O2 had no significant effect on the overall levels of TFEB in cells (Fig. 4). However, H2O2 significantly decreased the levels of nuclear TFEB and increased the ratio of P-mTOR/mTOR compared with the normal group. Furthermore, CGA treatment (50, 25 and 6.25 µM) significantly increased the levels of nuclear TFEB and decreased the ratio of P-mTOR/mTOR compared with the H2O2 treatment group.

 Discussion

A previous study has reported that natural compounds reduce the risk of developing neurodegenerative conditions, including vitamins A, C, and E and β-carotene (37). The present study provided additional evidence for the pivotal role of CGA in AD pathogenesis. CGA attenuated cell injury and apoptosis, reduced autophagy overactivation, increased autophagic flux and enhanced lysosomal function in SH-SY5Y cells injured by H2O2. Additionally, the results demonstrated that CGA may inhibit excessive autophagy by regulating the mTOR-TFEB signaling pathway and decreasing TFEB nuclear translocation.

AD is a neurodegenerative disorder with no known cure (1). The progression of Aβ plaque accumulation and hyperphosphorylation of τ proteins are enhanced by oxidative stress (38). The risk of AD increases significantly with age and, based on the hypotheses proposed to explain the causes of aging and AD, it has been reported that oxidative stress serves a central role in this occurrence (37). A previous study has indicated that brain tissues of patients with AD are exposed to oxidative stress during the development of the disease (39). Over time, oxidative stress contributes to a range of aging-related degenerative diseases, including cancer, diabetes, macular degeneration and AD (37). Furthermore, oxidative stress is one of the main causes of neuronal degeneration and loss-of-function in patients with AD and it can interact with β-amyloid peptides and activate signaling pathways to promote the occurrence and progression of AD (37). Neuroblastoma cell lines, including SH-SY5Y, are the most frequently utilized models in neurodegenerative studies and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades (40).

CGA is a phenylpropanoid compound produced in plants via the shikimic acid pathway during aerobic respiration (22). Polyphenols are secondary plant metabolites, comprising several antioxidant compounds and are generally considered to be involved in the defense against numerous chronic human diseases, including prostate cancer among others, cardiovascular diseases, diabetes mellitus and neurodegenerative diseases, such as Alzheimer's and Parkinson's disease (41). CGA is a polyphenolic substance widely found in the human diet (22). CGA has various pharmacologic actions, including antioxidant, anti-inflammatory and anticarcinogenic functions, and exerts hypoglycemic and hypolipidemic effects (20). Furthermore, CGA is mainly found in Eucommia ulmoides, sunflowers, coffee, burdock, honeysuckle and cocoa, and contains a plurality of phenolic hydroxyl groups that form hydrogen radicals and possesses strong antioxidant properties (19). Previous studies have demonstrated that CGA reduces the risk of developing neurodegenerative conditions, such as Alzheimer's and Parkinson's disease, via its inhibitory effect on Aβ40 aggregation (21,23). Additionally, a clinical study has demonstrated that patients who exhibit subjective memory loss of composite and verbal memory show improved complex attention, cognitive flexibility, executive function, motor speed domains of the central nervous system following a 6-month intake of a test beverage containing CGAs prior to bedtime (42). The present study observed that H2O2 decreased cell viability and affected the morphology of SH-SY5Y cells. Importantly, CGA ameliorated these morphological alterations and reduced the occurrence of cell apoptosis in a dose- and time-dependent manner following damage by H2O2.

To further clarify the mechanism of CGA action in this neuroprotective role, the ALP in H2O2-treated SH-SY5Y cells was assessed. The data indicated that CGA treatment reduced the average fluorescence intensity of MDC within the cells and inhibited autophagosome production by downregulating the levels of Beclin-1 and Atg5. Additionally, CGA enhanced lysosomal function in SH-SY5Y cells. Defects of the ALP are strongly associated with AD and the ALP has been reported to regulate APP turnover and Aβ metabolism (43). The accumulation of proinflammatory cytokines and dysfunction of the ALP may damage hippocampal neuronal cells, which is associated with the pathogenesis of AD (44). Autophagy influences the secretion of Aβ to the extracellular space, thereby directly affecting Aβ plaque formation, a pathological hallmark of AD (45). During autophagy, LC3-I is conjugated with phosphatidylethanolamine to form LC3-II, recruited to the autophagosomal membranes and degraded by lysosomal hydrolases following the fusion of autophagosomes with lysosomes (46). Therefore, the LC3-II/I ratio is directly proportional to the number of autophagosomes and is considered to be an important measure of autophagosome levels (46). Cytoplasmic components are selected and tagged prior to being sequestered into an autophagosome by p62/SQSTM1 during selective autophagy and the transcription of p62 is markedly increased during conditions in which selective autophagy substrates have accumulated (47). Therefore, H2O2 treatment induced the levels of LC3-II/I and P62/SQSTM1, while CGA prevented this, preventing the accumulation of autophagic vacuoles and the increase of autophagy-related proteins. Furthermore, defects in lysosomal function appear at the earliest stages of AD development and progress to a widespread failure of intraneuronal waste clearance, neuritic dystrophy and neuronal cell death, as well as inhibition of autophagic flow and decreased levels of autophagosome degradation (48). The results of the present study indicated that CGA enhanced lysosomal function by increasing the levels of CTSD. CTSD upregulation may be an adaptive response to AD-related processes, leading to neurofibrillary degeneration (49). Additionally, the activity of CTSD has been associated with the metabolism of cholesterol and glycosaminoglycans, which accounts for its involvement in neuronal plasticity (50). Therefore, CGA likely serves a vital neuroprotective role during the progression of AD.

The present study aimed to identify the signaling pathway by which CGA affected the progression of AD. TFEB is a transcription factor that upregulates lysosomal function when translocated into the nucleus. mTOR is a key protein involved in regulating factors involved upstream of TFEB nuclear transcription and these factors mainly function to promote TFEB entry into the nucleus by inhibiting phosphorylation of mTOR at the serine 2448 phosphorylation site (51). The results of the present study demonstrated that CGA treatment enhanced the levels of nuclear TFEB and decreased the ratio of P-mTOR/mTOR. It was hypothesized that CGA serves an indispensable neuroprotective role in the process of AD, likely via the mTOR-TFEB signaling pathway. Prior research has revealed that alterations of mTOR signaling and autophagy occur at the early stages of AD and are associated with autophagy impairment (52). Additionally, autophagy is strictly regulated by the mTOR signaling pathway, and regulation of the functional status of autophagy via the mTOR signaling pathway during physical activity is used for the prevention and treatment of AD (53).

In summary, the present study demonstrated that CGA inhibited the activation of the mTOR-TFEB signaling pathway by upregulating lysosomal function and promoting autophagic flux to exert neuroprotective effects.

 Acknowledgements

Not applicable.

 Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

 Authors' contributions

SJX conceived, designed the experiments and critically revised the manuscript. LJG devised the methodology, performed the experiments and wrote the original draft. XQL performed the data analyses and participated in the revision of the manuscript. YD and ZQZ contributed the sample assays and revised the manuscript. SM performed data analysis and assisted in preparing the figures. All authors read and approved the final manuscript.

 Ethics approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

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Neuroprotective effects of CGA on SH-SY5Y cells injured by H2O2. (A) Cell viability of SH-SY5Y cells damaged by different concentrations of H2O2 at 4, 8, 12 and 24 h. (B) Cell viability of SH-SY5Y cells following treatment with different concentrations of CGA for 12, 24 and 36 h. (C) Morphology of SH-SY5Y cells in the different groups. Scale bar, 20 µm. (D) Concentrations of 50 and 12.5 µM CGA restored the morphology of SH-SY5Y cells damaged by H2O2 for 24 h. (E) Numbers of SH-SY5Y cells were rescued by different concentrations of CGA. (F) Hoechst staining of SH-SY5Y cells in each group. Scale bar, 10 µm. (G) Cell viability of SH-SY5Y cells was significantly increased by different concentrations of CGA compared with control group. (H) Average cell apoptosis rate was significantly decreased in each CGA group. (I) Representative western blotting images of Bcl-2 and Bax expression in SH-SY5Y cells (left panel). GAPDH was used as the loading control. Relative optical density values of Bcl-2/Bax (right panel). *P<0.05, **P<0.01 and ***P<0.001 vs. control group. #P<0.05, ##P<0.01 and ###P<0.001 vs. H2O2 treatment group. CGA, chlorogenic acid; H2O2, hydrogen peroxide; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2 associated X.

Effects of CGA on autophagic flux in SH-SY5Y cells injured by H2O2. (A) Effects of CGA on autophagic vacuole accumulation in SH-SY5Y cells injured by H2O2. Scale bar, 10 µm. (B) CGA treatments significantly reduced the average fluorescence intensity of monodansylcadaverine at 6 (25 and 50 µM), 12 and 24 h compared with the H2O2 group. (C) Intensity of Beclin-1 bands in SH-SY5Y cells was decreased following incubation with 50 or 25 µM CGA compared with the H2O2 group. Additionally, the intensity of Atg5 bands was decreased in the 25, 12.5 and 6.25 µM CGA groups. Beclin-1 and Atg5 levels were normalized to GAPHD. (D) Intensity of LC3B-II/I bands in the SH-SY5Y cells was decreased following incubation with 25, 12.5 and 6.25 µM CGA compared with the H2O2 group. Additionally, the intensity of the SQSTM/p62 bands was decreased in each CGA group. LC3-II/I and p62 levels were normalized to GAPHD. (E) mCherry-GFP-LC3 adenovirus transfection was used to detect the effect of CGA on SH-SY5Y cells. mCherry-GFP-LC3 specifically labels autophagosomes. Scale bar, 15 µm. **P<0.01 and ***P<0.001 vs. control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. H2O2 treatment group. CGA, chlorogenic acid; H2O2, hydrogen peroxide; Atg5, autophagy protein 5; SQSTM, sequestosome 1; GFP, green fluorescent protein.

Effect of CGA on lysosome function in SH-SY5Y cells injured by H2O2. (A) CQ pretreatment prevented damage to SH-SY5Y cells as the cell viability of SH-SY5Y cells was significantly decreased in the H2O2 + CQ group. ***P<0.001 vs. H2O2 group. (B) Cell viability of SH-SY5Y cells was reduced by H2O2 treatment; however, cell viability was rescued by CGA treatment. However, cell viability was lower in the H2O2 + CQ + CGA group compared with the H2O2 + CGA group. ***P<0.001, ##P<0.05 and ###P<0.01, as indicated. (C) LysoTracker Red was used to specifically label intracellular lysosomes. Scale bar, 5 µm. ***P<0.001 vs. control group; ##P<0.01 and ###P<0.001 vs. H2O2 treatment group. (D) Lysosomes were labeled with LysoSensor Green and observed by fluorescence microscopy. Scale bar, 25 µm. ***P<0.001 vs. control group; ##P<0.01 and ###P<0.001 vs. H2O2 treatment group. (E) Representative western blotting images of CTSD in SH-SY5Y cells (left panel). ***P<0.001 vs. control group; #P<0.05 and ##P<0.01 vs. H2O2 treatment group. GAPDH was used as the internal control. Relative optical density values of CTSD in the experimental groups (right panel). CGA, chlorogenic acid; H2O2, hydrogen peroxide; CQ, chloroquine; CTSD, cathepsin D; EBSS, Earle's Balanced Salt Solution.

Effect of CGA on the mTOR-TFEB signaling pathway in H2O2-treated SH-SY5Y cells. Nuclear TFEB expression was increased in the 50, 25 and 6.25 µM CGA groups. However, total TFEB levels were not significantly different among groups. The ratio of P-mTOR/mTOR TFEB was significantly reduced in all CGA groups. TFEB and P-mTOR/mTOR were normalized to GAPHD. ***P<0.001 vs. control group. #P<0.05, ##P<0.01 and ###P<0.001 vs. H2O2 treatment group. CGA, chlorogenic acid; H2O2, hydrogen peroxide; TFEB, transcription factor EB; P-, phosphorylated; ser, serine.