The elastin-derived peptide (VGVAPG) activates autophagy in neuroblastoma (SH-SY5Y) cells via peroxisome proliferator-activated receptor gamma (PPAR γ )

Autophagy is a self-degradative process important for balancing the sources of energy and involved in the development of Alzheimer's disease (AD). To date, a number of papers have shown that elastin-derived peptides (EDPs) affect the expression and activation of peroxisome proliferator-activated receptor gamma (PPAR γ ), which is crucial for the development of AD and autophagy initiation. Therefore, the aim of the present study was to determine whether EDPs with a Val – Gly – Val – Ala – Pro – Gly (VGVAPG) amino acid sequence activate the auto-phagic process in undifferentiated SH-SY5Y human neuroblastoma cells. Our study is the first to show that EDPs with the VGVAPG sequence initiate the autophagy process in the undifferentiated SH-SY5Y cell line exhibiting a number of features of normal neuroblasts. In particular, we observed in our study that VGAVPG peptide increased ULK1, AKT, PPAR γ , and LC3B protein expression. Moreover, our experiments with the agonist (rosi-glitazone) and antagonist (GW9662) of PPAR γ confirm that the studied EDP acts through the PPAR γ pathway affecting mTOR and finally autophagy. Some studies have shown that autophagy disturbances are involved in the development of AD. Therefore, we believe that our study will provide new evidence of the possible involvement of EDPs (especially VGVAPG) in the development of AD.


Introduction
Autophagy is a self-degradative process important for the balancing sources of energy at critical times in development and in response to nutrient stress (Glick et al., 2010).This process also plays a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, such as mitochondria, endoplasmic reticulum (ER) and peroxisomes, as well as eliminating intracellular pathogens (Anding and Baehrecke, 2017;Glick et al., 2010).A number of studies have shown that autophagy disturbances may occur in an early stage of Alzheimer's disease (AD) (Li et al., 2017;Uddin et al., 2018).In the development of AD, autophagy plays an important role in the generation and metabolism of β-amyloid (Aβ); tau protein assembling and dysfunction may lead to the progress or aggravation of AD (Li and Sun, 2017).
Elastin is one of the crucial proteins building the extracellular matrix (ECM) of the brain (Li et al., 2021).It is well-documented that, during aging and due to various physiological and pathological processes, elastin is degraded and the EDPs formed bind with high affinity to EBP (Senior et al., 1984).An increased level of EDPs was also observed in a patient with ischemic stroke in cerebrospinal fluid (CSF) (Nicoloff et al., 2008;Tzvetanov et al., 2008).Moreover, Ma et al. (2019) clearly showed that EDPs increased the levels of Aβ, associated with AD, in in vitro and in vivo murine models (Ma et al., 2019).Therefore, now there is a growing amount of evidence indicating that, through different mechanisms, EDPs are the primary cause of the development of neurodegenerative diseases, especially AD [reviewed in (Szychowski et al., 2022;Szychowski and Skóra, 2021)].Previous studies conducted by our team showed that EDPs have an impact on ROS production, PPARγ protein and mRNA expression, and cell proliferation in mouse primary astrocytes and the human neuroblastoma (SH-SY5Y) cell line, which exhibits many features of normal human neuroblasts in an undifferentiated state (Szychowski et al., 2019;Szychowski andGmiński, 2019, 2020).
Therefore, due to involvement of ROS and the PPARγ receptor in autophagy induction and AD development, the aim of the present study was to determine whether EDPs with the VGVAPG amino acid sequence activate the autophagic process in undifferentiated SH-SY5Y human neuroblastoma cells.

SH-SY5Y cell culture and treatment
Cells from the human neuroblastoma (SH-SY5Y) cell line were obtained from the American Type Culture Collection (ATCC distributor: LGC Standards, Łomianki, Poland).The SH-SY5Y cells were maintained in DMEM/F12 medium supplemented with 10 % heat-inactivated FBS at 37 • C in a humidified atmosphere with 5 % CO 2 .The cells were seeded in 96-well culture plates at a density of 5 × 10 3 (for resazurin, caspase-1, and caspase-3 analysis), in 12-well culture plates at a density of 7.5 × 10 4 (for mRNA isolation), and in 6-well culture plates at a density of × 10 5 (for ELISA and Western Blot) per well and initially cultured before the experiment for 24 h.

Resazurin reduction assay
The assay was performed according to Ivanov et al. (2014) with modifications.On the day of analysis, a working solution of 60 μM resazurin was prepared in a medium containing 1 % FBS.After 48-h treatment of the cells with the VGVAPG peptide and in co-treatment with the tool compounds, the medium in the wells was replaced with a working solution of resazurin (100 μL) and the plates were placed at 37 • C. Fluorescence was measured with excitation and emission wavelengths of 530 and 590 nm, respectively, in a FilterMax F5 Multi-Mode microplate reader (Molecular Devices, Corp., Sunnyvale, CA, USA) for h after addition of the dye.

Real-time PCR for genes encoding Ki67, PPARγ, S100B, p62/ SQSTM1, LC3B, and mTOR
After the 24-h exposure of the SH-SY5Y cells to 10 nM of the VGVAPG peptide or to the co-treatment with 1 μM of GW9662, 1 μM of rosiglitazone, or 1 μM of atorvastatin, the samples were collected and total RNA was extracted using the Universal RNA Purification Kit according to the manufacturer's protocol (EURx).The level of the RNA was determined with the use of the Nanodrop device (ND/1000 UV/Vis; Thermo Fisher, USA).The reverse transcription reaction was performed at a final volume of 20 μL with 800 ng of RNA (as a cDNA template) using the cDNA reverse transcription kit according to the manufacturer's protocol (ThermoFisher).Products from the RT reaction were amplified using the Fast Probe qPCR Master Mix (2×) kit with TaqMan probes and primers for specific genes encoding GAPDH, S100B, PPARG, MKI67, MTOR, LC3B (MAP1LC3B), and p62/SQSTM1 in a total volume of 20 μL containing 1 μL of the RT product, which was used as a PCR template.K.A. Szychowski and B. Skóra The standard qPCR procedure was performed as follows: 2 min at 50 • C and 10 min at 95 • C followed by 40 cycles of 15 s at 95 • C and 1 min at 60 • C. The threshold value (C t ) for each sample was set during the exponential phase, and the ΔΔCt method was used for data analysis.GAPDH was used as a reference gene.To study the gene expression levels, three candidate reference genes (ACTB, GAPDH, 18S) were selected and validated.The RefFinder web-based comprehensive tool was used to evaluate the reference gene expression.
2.6.ELISA for Ki67, PPARγ, S100B, p62/SQSTM1, LC3B, and mTOR The levels of Ki67, PPARγ, S100B, LC3B, p62/SQSTM1, and mTOR proteins were determined with the use of ELISA after 48-h treatment with 10 nM of the VGVAPG peptide and in the co-treatment with 1 μM of GW9662, 1 μM of rosiglitazone, or 1 μM of atorvastatin.After the experiment, the samples were collected using RIPA buffer.Specific detections of the studied proteins were obtained using the ELISA method.The assay was conducted according to the manufacturer's instructions from Elabscience Biotechnology and Wuhan Fine Biotech Co., Ltd.(WuHan, China).Briefly, a 96-well plate was pre-coated with monoclonal antibodies specific for Ki67, PPARγ, S100B, LC3B, p62/SQSTM1, and mTOR.Standard references and collected cell extracts were added to the wells and incubated for 90 min at 37 • C.Then, after the supernatant was removed, 100 μL of a biotinylated detection antibody was added for 60 min.After washing three times to remove any unbound substances, horseradish peroxidase-conjugated avidin was added.
Following additional washing, 90 μL of the substrate solution was added to the wells for 15 min.Then, 50 μL of the reaction termination solution was added, and absorbance was measured at 450 nm using a microplate reader (FilterMax F5); this value was proportional to the amount of Ki67, PPARγ, S100B, LC3B, p62/SQSTM1, and mTOR.The protein concentration was measured in each sample and determined in triplicate for each sample using the Thermo Fisher NanoDrop device.

Western blot for PI3K, AKT, PCNA, ULK1, and PPARγ protein expression
The Western Blot method was performed as proposed by Skóra et al. (2023).Briefly, after the 48-h exposure of the SH-SY5Y cells to 10 nM of the VGVAPG peptide or to the co-treatment with 1 μM of GW9662, 1 μM of rosiglitazone, or 1 μM of atorvastatin, the samples were collected by RIPA buffer containing protease inhibitors.Subsequently, the protein concentration was determined in each sample (using the Bradford method), standardized, and denaturated with 5× Laemmli buffer (Bio-Rad) at 95 • C for 5 min (Bradford, 1976).Subsequently, the samples were separated by 7.5 % SDS-polyacrylamide vertical gel electrophoresis (Bio-Rad).Afterwards, the proteins were transferred onto a PVDF membrane with 30 V and kept overnight at 4 • C. Subsequently, nonspecific side blocking was performed using 1 % BSA in TBST buffer for 1 h, followed by overnight incubation of the membranes with anti-PI3K (1:1000), anti-AKT (1:1000), anti-ULK1 (1:1500), and anti-PCNA (1:2000) primary antibodies.After this time, the membranes were washed three times with TBST, and secondary HRP-conjugated antimouse (1:10000) or anti-rabbit (1:10000) antibodies were added for 1 h in RT.Subsequently, the antibodies were discarded, and the membranes were washed four times with TBST.Next, the chemiluminescence method was used to detect the presence of certain proteins by adding the luminol reagent onto the membrane.The signal was measured by a chemiluminescence scanner (LiCor C-DiGit).The density of each blot was assessed, using GelQuant.Net software.After detection, the membranes were stripped with Mild Stripping Buffer (Abcam) and the anti-GAPDH (1:10000) primary antibodies were applied and regarded as a loading control.

Statistical analysis
The data are presented as the means ± standard deviation (SD).The results were used in the statistical analysis performed in the GraphPad Prism 8.0 Statistical Analysis Mode.The data were analyzed via one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison procedure and denoted as ***p < 0.001, **p < 0.01, and *p < 0.05 vs. the control.Moreover, the t-test was used to compare statistical significance between two groups and denoted as # at p < 0.05.

Cell metabolism and caspase activity
The cellular metabolism (resazurin reduction), induction of inflammation process (caspase-1) and/or apoptosis (caspase-3) are essential to determine of the mechanism of many tested substances.Therefore, in the first stage of the study, we examined the effect of the VGVAPG peptide of afore-described parameters.For this studies agonist and antagonist of the PPARγ and HMG-coA inhibitor, have been applied.After the 48-h exposure of the SH-SY5Y cells to 10 nM of VGVAPG and to the co-treatment with GW9662 (PPARγ antagonist), rosiglitazone (PPARγ agonist), or atorvastatin (HMG-CoA inhibitor), a decrease in cell metabolism measured by the resazurin reduction test was observed in the three study groups.The VGVPAG peptide decreased resazurin reduction slightly and not statistically significantly, compared to the control.GW9662 alone decreased resazurin reduction by 16.59 %, compared to the control.However, GW9662 in co-treatment with the VGVAPG peptide did not change the level of resazurin reduction, compared to the control group.In cells treated with atorvastatin, we observed a 57.71 % decrease in cell metabolism, compared to the control.Similarly, we observed a 48.31 % decrease in resazurin reduction in cells co-treated with atorvastatin and the VGVAPG peptide, compared to the control (Fig. 1 A).
After the 48-h exposure of the SH-SY5Y cells to 10 nM of VGVAPG and to the co-treatment with GW9662, rosiglitazone, or atorvastatin, increased caspase-1 activity was observed only in the rosiglitazoneexposed groups.Rosiglitazone caused a 41.17 % increase in caspase-1 activity, compared to the control group.Similarly, in the group cotreated with rosiglitazone and the VGVAPG peptide, a 27.94 % increase in caspase-1 activity was observed, compared to the control group.Atorvastatin alone decreased caspase-1 activity by 59.39 %, compared to the control; similarly, in the group co-treated with atorvastatin and the VGVAPG peptide, a 48.31 % decrease was observed, compared to the control group.No changes in caspase-1 were observed in the groups treated with VGVAPG and GW9662 alone and in the group co-treated with GW9662 and the VGVAPG peptide (Fig. 1 B).
In the experiments, the after 48-h cell exposure to the studied compounds, only rosiglitazone alone and rosiglitazone with the VGVAPG peptide increased caspase-3 activity by 53.84 and 43.58 %, respectively, compared to the control.In the other studied group, we did not observe any changes in caspase-3 activity, compared to the control (Fig. 1 C).

Real-time PCR for Ki67, PPARγ, S100B, p62/SQSTM1, LC3B, and mTOR
In the next part of the study, the genes related to proliferation (KI67), metabolism control (PPARγ, mTOR), autophagy and inflammation (p62, LC3B and S100B) were examined.24 h after the experiments, 10 nM of the VGVAPG peptide did not affect KI67 mRNA expression in the undifferentiated SH-SY5Y cell line.GW9662 increased KI67 mRNA by 39.76 %, compared to the control.In the cell co-treatment with GW9662 and the VGVAPG peptide, we did not observe any changes in KI67 mRNA.Rosiglitazone decreased KI67 mRNA by 29.09 %, compared to the control.However, in the cell co-treatment with rosiglitazone and the VGVAPG peptide, we did not observe any changes in KI67 mRNA.Both in the cells treated with atorvastatin alone and in those exposed to atorvastatin with the VGVAPG peptide, a decrease in KI67 mRNA expression was observed (by 26.94 and 24.58 % respectively, compared to the control) (Fig. 2A).
After 24 h, a decrease in PPARγ mRNA (by 7.87 %, compared to the control) was observed only in the group exposed to rosiglitazone.No changes in PPARγ mRNA were observed in the other experimental groups (Fig. 2B).In the cells treated with GW9662 alone and co-treated with GW9662 and the VGVAPG peptide, S100B mRNA decreased by 24.58 and 19.81 %, respectively, compared to the control.In the rosiglitazone and VGVAPG co-treated cells, a 20.51 % increase in S100B mRNA was observed, compared to the control.Interestingly, the atorvastatin and VGAVPG co-treated cells exhibited a 13.74 % decrease in S100B mRNA, compared to the VGAVPG alone treatment (Fig. 2C).
In our experiments, the VGVAPG peptide increased mTOR mRNA by 24.40 %, compared to the control.A similar increase in mTOR mRNA was observed in the group treated with rosiglitazone, atorvastatin alone, and co-treated with atorvastatin and VGVAPG peptide (increase by 21.76, 42.84, and 28.25 % respectively, compared to the control) (Fig. 2D).
After 24 h of the SH-SY5Y cell exposure to the studied compounds, GW9662 and atorvastatin increased the p62/SQSTM1 mRNA expression by 22.01 and 16.81 %, respectively, compared to the control.A similar increase in the p62/SQSTM1 mRNA expression was observed in the group co-treated with GW9662 and the VGVAPG peptide (increase by 16.00 %, compared to the control).We observed an increase in the p62/ SQSTM1 mRNA expression in the groups co-treated with rosiglitazone and the VGVAPG peptide and co-treated with atorvastatin and the VGVAPG peptide (increase by 12.61 and 13.34 %, compared to the group exposed to the VGVAPG peptide alone) (Fig. 2E).
In the case of LC3B, an increase in mRNA expression was observed in the group treated with atorvastatin alone and co-treated with atorvastatin and VGVAPG (increase by 52.23 and 34.26 %, respectively, compared to the control).However, in the GW9662 and VGVAPG co-treated group, we observed an 8.27 % decrease in LC3B mRNA expression, compared to the VGVAPG alone treated group (Fig. 2F).
In the experiments, the VGVAPG peptide did not affect the KI67 protein expression measured with the ELISA method.Similarly, GW9662 alone and in co-treatment with the VGAVPG peptide did not change the KI67 protein expression, compared to the control.Rosiglitazone alone decreased the KI67 protein expression by 10.45 ng/mL, compared to the control.In turn, no changes were observed in the rosiglitazone and VGVAPG co-treated cells, compared to the control group.Similarly, atorvastatin decreased the KI67 protein expression by 6.82 ng/mL, while no changes were observed in the cells co-treated with atorvastatin and the VGVAPG peptide, compared to the control group (Fig. 3A).
In our experiments, the ELISA method showed that the VGVAPG peptide increased PPARγ protein expression by 158.76 ng/mL, compared to the control.GW9662 increased the PPARγ protein expression by 64.35 ng/mL, compared to the control, while the GW9662 and VGVAPG co-treated cells exhibited a decrease in the PPARγ protein expression by 42.25 ng/mL, compared to the control.Rosiglitazone alone increased the PPARγ protein expression by 150.91 ng/mL; however, only a slight increase in the protein level (by 49.48 ng/mL) was observed in the cells co-treated with rosiglitazone and the VGVAPG peptide, compared to the control.Atorvastatin alone increased the PPARγ protein expression by 151.09 ng/mL, compared to the control.In turn, no changes were observed in the group co-treated with atorvastatin and the VGVAPG peptide, compared to the control (Fig. 3B).
After the 48-h exposure of the SH-SY5Y cells to the VGAVPG peptide, no significant changes in the S100B protein level were observed.In the cells treated with GW9662, rosiglitazone, and atorvastatin alone, a decrease by 1.02, 0.82 and 1.01 ng/mL, respectively, compared to the control, was observed.However, in the cells co-treated with GW9662, rosiglitazone, or atorvastatin and the VGVAPG peptide, there were no changes in the S100B protein level (Fig. 3C).
In our experiments, the VGVAPG peptide did not affect the mTOR protein level in SH-SY5Y after the 48-h exposure.GW9662 increased the mTOR protein expression by 1.85 ng/mL, compared to the control.In turn, no changes in the mTOR protein level were observed in the GW9662 and VGVAPG co-treated cells.In the groups treated with rosiglitazone alone and co-treated with rosiglitazone and the VGVAPG peptide, no changes in the mTOR protein expression were observed.In the group treated with atorvastatin alone and co-treated with atorvastatin and the VGVAPG peptide, the mTOR protein level increased by 1.14 and 1.22 ng/mL, respectively, compared to the control (Fig. 3D).
In the case of p62/SQSTM1, the VGVAPG peptide, GW9662 alone, and the co-treatment did not change the expression of the studied protein.Similarly, rosiglitazone alone did not change the p62/SQSTM1 protein expression.In turn, the cell co-treatment with rosiglitazone and the VGVAPG peptide increased the p62/SQSTM1 protein expression by 2.10 ng/mL, compared to the control.The cell treatment with atorvastatin decreased the p62/SQSTM1 protein expression by 1.29 ng/mL, compared to the control.In contrast, the cell co-treatment with atorvastatin and the VGVAPG peptide did not change the p62/SQSTM1 protein expression (Fig. 3E).
The experiments revealed that the VGVAPG peptide increased the LC3B protein expression by 6.99 ng/mL.Both in the cells treated with GW9662 and co-treated with VGVAPG, a decrease in the LC3B protein expression was observed (by 3.11 and 2.83 ng/mL, respectively, compared to the control).On the other hand, in the cells treated with rosiglitazone and co-treated with rosiglitazone and the VGVAPG peptide, an increase in the LC3B protein expression was observed (by 16.81 and 24.45 ng/mL, respectively, compared to the control).Atorvastatin alone decreased the LC3B protein expression by 2.78 ng/mL, compared to the control.In turn, the cells co-treated with atorvastatin and the VGVAPG peptide exhibited no change in the LC3B protein level, compared to the control (Fig. 3F).

Western blot protein expression
Finally, to confirm the effect of the VGVAPG peptide on protein expression related to the proliferation (PCNA) and activation of the autophagy process (ULK1), control protein (PPARγ) and control kinases (AKT, PI3K) were examined.For this purpose PPARγ antagonist (GW9662), PPARγ agonist) (rosiglitazone), and HMG-CoA inhibitor (atorvastatin) have been applied.
After the 48-h exposure to the VGVAPG peptide, we observed a 55.39 % increase in the PPARγ protein expression, compared to the control, measured with the Western Blot method.Similarly, GW9662 also increased the PPARγ protein expression by 41.19 %, compared to the control.However, the cell co-treatment with GW9662 and the VGVAPG peptide increased the PPARγ protein expression only by 9.49 %, compared to the control.Rosiglitazone increased the PPARγ protein expression to a similar level as the VGVPAG peptide alone (by 57.10 %, compared to the control).In turn, the cell co-treatment with rosiglitazone and the VGVAPG peptide increased the PPARγ protein expression by 40.57%, compared to the control.Atorvastatin alone increased the PPARγ protein expression by 12.01 %, compared to the control, while the cells co-treated with atorvastatin and the VGVAPG peptide exhibited a 57.35 % decrease in the PPARγ protein expression, compared to the control (Fig. 4 A).
In our experiments, we did not observe significant changes in the PCAN protein level the after SH-SY5Y cell exposure to both the VGVAPG peptide and GW9662 alone.However, in the cell co-treatment with GW9662 and the VGVAPG peptide, a slight but statistically significant decrease (by 2.24 %, compared to the control) in the PCNA protein was observed.Rosiglitazone alone decreased the PCNA protein level by 10.84 %, compared to the control, while a 7.22 % decrease in PCNA was observed in the cell co-treatment with rosiglitazone and the VGVAPG peptide, compared to the control.Atorvastatin alone decreased the PCNA protein level by 33.49 %, compared to the control; the cell cotreatment with atorvastatin and VGVAPG potentiated this effect and decreased the PCNA protein level by 47.79 %, compared to the control (Fig. 4 B).
In the SH-SY5Y cells after the 48-h exposure, the VGVAPG peptide increased the ULK1 protein expression by 66.74 %, compared to the control.A similar increase in the ULK1 protein expression was observed in the groups treated with GW9662, rosiglitazone, and atorvastatin (an increase in the protein expression by 111.11, 46.93, and 135.59 %, respectively, compared to the control).Similarly, the groups co-treated with GW9662 and the VGVAPG peptide, rosiglitazone and the VGVAPG peptide, and lastly atorvastatin and the VGVAPG peptide, exhibited an increase in the ULK1 protein expression (by 124.23, 49.10, and 156.67 % respectively, compared to the control (Fig. 4C).
Our experiments showed that the VGVAPG peptide increased the AKT kinase expression by 65.74 %, compared to the control.Similarly, the SH-SY5Y cell treatment with GW9662, rosiglitazone, and atorvastatin increased the AKT kinase expression by 88.89, 67.77, and 84.62 % respectively, compared to the control.The cell co-treatment with GW9662 and VGVAPG increased the AKT kinase expression by 16.56 %, compared to the VGVAPG peptide alone variant.Interestingly, in the cells co-treated with rosiglitazone and VGVAPG as well as atorvastatin with the VGVAPG peptide, the AKT kinase expression decreased by 29.46 and 36.72 %, respectively, compared to the VGVAPG peptide alone treatment (Fig. 4 D).
In the experiments, the VGVAPG peptide decreased the PI3K protein expression by 14.80 %, compared to the control.A similar decrease in the PI3K protein expression (by 41.78 %) was observed in the group treated with GW9662, compared to the control.Interestingly, in the cell co-treatment with the VGVAPG peptide and GW9662, an increase in the PI3K protein expression was observed (by 27.41 %, compared to the control).In the rosiglitazone-treated cells, a 267.01 % increase in the PI3K protein expression was observed, compared to the control cells.In the cells co-treated with rosiglitazone and the VGVAPG peptide, a 318.39 % increase in the PI3K protein expression was observed, compared to the control cells.Atorvastatin increased the PI3K protein expression by 212.24 %, compared to the control cells.In turn, in the cells co-treated with atorvastatin and the VGVAPG peptide, only a 55.00 % increase in the PI3K protein expression was observed, compared to the control cells (Fig. 4 E).

Discussion
In the first stage of our study, we observed a strong decrease in the cell metabolism measured with the resazurin reduction test in the atorvastatin-treated groups.Moreover, an increase in caspase-1 activity was observed in the rosiglitazone-treated groups, while a decrease in caspase-1 was observed in the groups treated with atorvastatin.Finally, caspase-3 activity increased only in the rosiglitazone-treated groups.We did not observe a significant effect of the VGVAPG peptide alone or in co-treatment with the tool compounds on the measured parameters.The observed effects of the action of the tool compounds are consistent with the current state of knowledge.To date, it has been well described that, through inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthesis, statins (especially atorvastatin) decrease cell metabolism and inflammation markers, such as caspase-1 expression and activity, interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α) (Gong et al., 2017;Peng et al., 2018).On the other hand, rosiglitazone increased the activity of both studied caspases (− 1 and − 3), as mentioned above.The VGVAPG peptide slightly but not statistically significantly reduced the effect of rosiglitazone, which was probably related to the interaction with its main rosiglitazone target, i.e.PPARγ.To date, it has been reported that caspase-1 and -3 are involved in autophagy; moreover, as a PPARγ agonist, rosiglitazone itself also activates this process (Ge et al., 2022;Tsapras and Nezis, 2017).Therefore, based on our preliminary data, we decided to study autophagy markers with particular reference to the PPARγ-dependent activation pathway.
In our experiments, the VGVAPG peptide did not significantly change the mRNA and protein expression of KI67, i.e. a well accepted marker of proliferation, and the expression of the proliferating cell nuclear antigen (PCNA) protein.The PPARγ receptor agonist (rosiglitazone) alone decreased the expression of both KI67 and PCNA, while the VGVAPG peptide inhibited the rosiglitazone-induced changes.On the other hand, the data on the atorvastatin-stimulated groups are inconclusive.Similar to rosiglitazone, atorvastatin decreased the KI67 and PCNA protein expression, while the VGVPAG peptide in the atorvastatin-stimulated group prevented the decrease in KI67 but potentiated the decrease in the PCNA protein level.This phenomenon may be correlated with the different functions of the KI67 and PCNA during the cell cycle, i.e.KI67 is correlated with the G1, S, G2, and M phases, while PCNA is mainly expressed in the G1/S phase, which shows that KI67 is a better marker of the overall proliferation properties (Bologna-Molina et al., 2013).However, the rest of the data are consistent with previous discoveries, in which EDP VGVAPG did not change or slightly decreased cell proliferation measured by KI67 in the SH-SY5Y cell line, which exhibited a number of features of normal human neuroblasts in an undifferentiated state (Szychowski et al., 2019).However, the effect of EDP VGVAPG depends on the cell type, exposure time, and concentrations.In mouse primary astrocytes and in different human gliomas, EDPs (especially the VGVAPG peptide) were found to increase cell proliferation (Jung et al., 1998;Szychowski and Gmiński, 2020).Previous studies suggest that the EDP mechanism of action, including the effect on proliferation and cell functioning, is dependent on the interaction with the PPARγ pathway (Szychowski et al., 2019;Szychowski andGmiński, 2020, 2019).
Our present experiments showed that EDP VGVAPG increased the PPARγ protein expression in the SH-SY5Y cell line.Similar expression of the PPARγ protein was observed in all the tool compound-treated groups.Interestingly, all groups co-treated with the tool compounds and the VGVAPG peptide, exhibited a decrease in the PPARγ protein expression, compared to the VGVAPG peptide alone treatment.Our data support previous discoveries, in which the VGVAPG peptide interfered and/or acted through the PPARγ-dependent pathway in SH-SY5Y cells, mouse primary astrocytes, and mouse fibroblasts (3T3-L1) (Hocine et al., 2020;Szychowski et al., 2020Szychowski et al., , 2019;;Szychowski and Gmiński, 2019).Due to the important role of the PPARγ pathway in autophagy regulation, we decided to investigate the impact of the tested VGVAPG on the key kinase involved in this process, namely the mammalian target of rapamycin kinase (mTOR).mTOR is a key regulatory point in autophagy induction (Zhu et al., 2019).Many studies showed that direct inhibition of mTOR increased the autophagy process inter alia in human hepatoma cancer cells (HepG2) or in male Sprague-Dawley rats (Song et al., 2017;Yang et al., 2021).Since PPARγ agonists cause inhibition of mTOR and in this way promote autophagy, mTOR seems to be an interesting protein for studies (Soto-Avellaneda and Morrison, 2020).Our results showed that the VGVAPG peptide did not affect the level of the mTOR protein, whereas GW9662 (PPARγ antagonist) increased the mTOR protein expression.The VGVAPG peptide inhibited the increase in the mTOR protein expression stimulated by GW9662.Rosiglitazone alone and in co-treatment with the VGVAPG peptide did not significantly change the mTOR protein expression, while atorvastatin alone and in the co-treatment with the VGVAPG peptide increased the mRNA and protein expression of mTOR, compared to the control.
However, many literature data show that indirect mTOR inhibition, i.e. via the PPARγ-related pathway (agonists), causes suppression of the mTOR and in this way promotes autophagy (Soto-Avellaneda and Morrison, 2020), which is consistent with our results.Interestingly, Zhao et al. showed that the PPARγ antagonist used (GW9662) caused a decrease in the mTOR protein expression in the murine microglial cell line (BV-2) (Zhao et al., 2020).These data suggest that inhibition of basal PPARγ activity also affects mTOR.Finally, it was shown that atorvastatin induced autophagy through the AKT/mTOR-dependent pathway in hypertensive Wistar-Kyoto rats (Wang et al., 2015).
Therefore, given the impact of atorvastatin and both PPARγ agonists and antagonists on autophagy markers, in the last stage of our study, we decided to analyze the main proteins involved in autophagy pathways, such as p62/SQSTM1, microtubule-associated protein 1 light chain 3 isoform B (MAP1LC3B, hereafter referred to as LC3B), and S100 calcium binding protein B (S100B).Our data showed that the VGVAPG peptide increased the p62/SQSTM1 mRNA expression only in the co-treatment with GW9662, in comparison to the control.On the other hand, an increase in the p62/SQSTM1 protein expression was observed in the group co-treated with rosiglitazone and the VGVAPG peptide.Moreover, we observed that the VGVAPG peptide increase the LC3B protein level.GW9662 alone and in the co-treatment with the VGVAPG peptide caused a decrease in the LC3B protein level.In turn, a strong increase in the LC3B protein expression was observed in the rosiglitazone-treated group.An even stronger increase in the LC3B protein expression was observed in the rosiglitazone and VGVAPG co-treated group than in the rosiglitazone or VGVAPG alone treatments.In both groups treated with atorvastatin, an increase in the LC3B mRNA expression was observed, but a decrease in the LC3B protein level was observed at the same time.The observed decrease in the amount of protein with a simultaneous increase in its mRNA expression suggests protein degradation after activation.Moreover, the present data revealed that VGVAPG activated autophagy at least partly through the PPARγ pathway.As cited above, autophagy is regulated at the receptor-dependent level with a vital role of p62/SQSTM1.As reviewed by Kumar et al., p62/SQSTM1 is activated as a result of mTORC1 pathway induction through pro-autophagy signals (Kumar et al., 2022).Subsequently, the p62/SQSTM1 complex, the Keap1 protein, and Nrf2 act as transcriptional factors for stress-related genes or induce the activity of the LC3 protein, which is a major point in selective autophagy induction (Kageyama et al., 2021).Moreover, atorvastatin treatment was found to upregulate the expression of autophagy-related LC3B and downregulate the expression of p62/ SQSTM1, which suggested that autophagy was activated in vulnerable plaques (Peng et al., 2018).Therefore, we can assume that our data are consistent with the current state of knowledge.
To date, an increase in the S100B protein level and an increase in the LC3B protein level during the autophagy process have been described (Du et al., 2018).Moreover, the S100B protein is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is associated with senile plaques (Cristóvão et al., 2018).In our experiments, the VGVAPG peptide alone did not significantly change the S100B mRNA and protein level.Interestingly, the PPARγ antagonist decreased the S100B protein level, while the PPARγ agonist did not change it.Moreover, the cell co-treatment with the PPARγ agonist (rosiglitazone) and VGVAPG contributed to a significant increase in the S100B protein level.To date, a decrease in the S100B protein has been described in rosiglitazone-treated ischemic rat brains, compared to a non-treated group (Sayan-Ozacmak et al., 2011).Moreover, it has also been reported that the S100B protein in the SH-SY5Y cell line can act as suppressor of Aβ42 aggregation and toxicity (Cristóvão et al., 2018).Therefore, our data are consistent with the current state of knowledge of the role of the S100B protein and in line with previous discoveries, in which EDPs caused an increase in Aβ (Ma et al., 2019).
Finally, to confirm the induction of autophagy, we examined three kinases involved in the regulation of the autophagy process, namely Unc-51 like autophagy activating kinase 1 (ULK1), phosphatidylinositol 3-kinase (PI3K), and serine-threonine protein kinase (AKT).In the experiments, the VGVAPG peptide alone increased ULK1 and AKT kinases and slightly decreased the PI3K protein level.The PPARγ antagonist and atorvastatin increased the ULK1 protein level more potently than the VGVAPG peptide.In turn, rosiglitazone increased the ULK1 level likewise the VGVAPG peptide.In the case of AKT kinase, the VGVAPG peptide and the tool compounds increased the AKT kinase expression to a similar level.In contrast, the cell co-treatment with rosiglitazone and the VGVAPG peptide as well as atorvastatin and the VGVAPG peptide decreased the AKT kinase level, compared to the VGVAPG-treated group.The PPARγ antagonist, similar to VGVAPG alone, decreased the PI3K expression.Interestingly, an increase in the PI3K expression was observed in the cells co-treated with the PPARγ antagonist and the VGVAPG peptide.The PPARγ agonist increased the PI3K protein expression in the group co-treated with the PPARγ agonist and the VGVAPG peptide, and the increase was even greater than that in the group exposed to rosiglitazone alone.Atorvastatin alone increased the PI3K protein expression but, compared to the group treated with atorvastatin alone, the VGVPAG peptide decreased the PI3K expression.Therefore, it can be assumed that the VGVAPG peptide, similar to the PPARγ agonist and atorvastatin, activates the autophagy process in SH-Y5Y cells and interferes with the PPARγ signaling pathway.Previous studies described that the VGVAPG peptide acted through PI3Kγ in bone marrow cells derived from C57Bl/6J mice (Gayral et al., 2014).The authors described that the absence of PI3Kγ prevented EDP-induced atherosclerosis development (Gayral et al., 2014).Moreover, atorvastatin was found to induce autophagy through the AKT/mTORdependent pathway in Wistar-Kyoto rats and cortical neurons isolated from Sprague-Dawley rats (Jin et al., 2012;Wang et al., 2015).
Unfortunately, the impact of EDPs on the ULK1 protein level or markers of autophagy has never been studied.However, it has been shown that atorvastatin induces autophagy in human prostate cancer cell line PC3 (Toepfer et al., 2011).Toepfer et al. described that atorvastatin increased the LC3B, p62, and ULK1 mRNA expression in PC3 cells (Toepfer et al., 2011), which is consistent with our data.Recent data have shown that walnut-derived peptides promote autophagy via the activation of the AMPK/mTOR/ULK1 pathway to ameliorate hyperglycemia in type 2 diabetic mice, which is also connected with the PPARγ pathway (Hou et al., 2023).Therefore, our data are mainly consistent with the current state of knowledge.

Conclusion
Our data have confirmed previous studies that the VGVAPG peptide in SH-SY5Y cells does not initiate cell proliferation.Our study is the first to show that EDPs with the VGVAPG sequence initiate the autophagy process in the undifferentiated SH-SY5Y cell line, which exhibits a number of features of normal neuroblasts.Moreover, the present data confirm that the studied EDP acts through the PPARγ pathway.As reported in a number of studies, autophagy disturbances are involved in the development of AD.Therefore, we believe that our study will provide evidence for involvement of EDPs (especially VGVPAG) in the development of AD.Moreover, by disrupting the normal PPARγ signaling, the VGVAPG peptide dysregulates normal brain metabolism.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Effect of 10 nM of the VGVAPG peptide and the VGVAPG peptide in co-treatment with 10 μM of GW9662 (PPARγ antagonist), 10 μM of rosiglitazone (PPARγ agonist), or 10 μM of atorvastatin (HMG-CoA inhibitor) on the metabolic activity (A), caspase-1 activity (B), and caspase-3 activity (C) in the SH-SY5Y cell line after the 48-h exposure.In each group, data are expressed as a mean (n = 18) with standard deviation.Statistically significant values determined by Tukey's test for each study group * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control cells.#p < 0.5; ##p < 0.01; ###p < 0.001 the group treated with the VGVAPG peptide alone vs. the group co-treated with the VGVAPG peptide and the respective tool compound.

Fig. 2 .
Fig. 2. Effect of 10 nM of the VGVAPG peptide and the VGVAPG peptide in co-treatment with 10 μM of GW9662 (PPARγ antagonist), 10 μM of rosiglitazone (PPARγ agonist), or 10 of μM atorvastatin (HMG-CoA inhibitor) on the Ki67, PPARγ, S100B, LC3B (MAP1LC3B), p62/SQSTM1, and mTOR on mRNA expression in the SH-SY5Y cell line after the 24-h exposure.In each group, data are expressed as a mean (n = 9) with standard deviation.The mRNA expression was normalized to GAPDH mRNA.Statistically significant values determined by Tukey's test for each study group * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control cells.#p < 0.5; ##p < 0.01; ###p < 0.001 the group treated with the VGVAPG peptide alone vs. the group co-treated with the VGVAPG peptide and the respective tool compound.

Fig. 3 .
Fig. 3. Effect of 10 nM of the VGVAPG peptide and the VGVAPG peptide in co-treatment with 10 μM of GW9662 (PPARγ antagonist), 10 μM of rosiglitazone (PPARγ agonist), or 10 μM of atorvastatin (HMG-CoA inhibitor) on the Ki67, PPARγ, S100B, LC3B (MAP1LC3B), p62/SQSTM1, and mTOR protein expression measured with the ELISA method in the SH-SY5Y cell line after the 48-h exposure.The protein expression was normalized to total protein in the sample.In each group, data are expressed as a mean (n = 6) with standard deviation.Statistically significant values determined by Tukey's test for each study group * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control cells.#p < 0.5; ##p < 0.01; ###p < 0.001 the group treated with the VGVAPG peptide alone vs. the group co-treated with the VGVAPG peptide and the respective tool compound.

Fig. 4 .
Fig. 4. Effect of 10 nM of the VGVAPG peptide and the VGVAPG peptide in co-treatment with 10 μM of GW9662 (PPARγ antagonist), 10 μM of rosiglitazone (PPARγ agonist), or 10 μM of atorvastatin (HMG-CoA inhibitor) on the PPARγ (A), PCNA (B), ULK1 (C), AKT (D), and PI3K (E) protein expression measured with the Western Blot method in the SH-SY5Y cell line after the 48-h exposure.In each group, data are expressed as a mean (n = 3) with standard deviation.The protein expression was normalized to GAPDH protein expression.The representative blots are given in the panels below.Statistically significant values determined by Tukey's test for each study group * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control cells.#p < 0.5; ##p < 0.01; ###p < 0.001 the group treated with the VGVAPG peptide alone vs. the group co-treated with the VGVAPG peptide and the respective tool compound.