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The Adeno-associated virus (AAV) was provided by Shanghai Jima Pharmaceutical Technology Co., LTD (Qingdao, China). The brain stereoscopic locator was purchased from Anhui Zhenghua Biological Instrument Equipment Co., LTD (Anhui, China). The Morris Maze was purchased from Shanghai Xin Soft Information Technology Co., LTD (Shanghai, China). Commercial assay kits for total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-PX) were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). Detailed information on the primary antibodies used is presented in Table 1. Horseradish-peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG were purchased from Proteintech (Wuhan, China).
Table 1. Detailed information of primary antibodies used
Antibody Producers Catalogue
numberSource Dilution APP Cell Signaling Technology 15126S Rabbit 1:1,000 Aβ40 Cell Signaling Technology 12990S Rabbit 1:1,000 Aβ42 Cell Signaling Technology 14974s Rabbit 1:1,000 p-Tau Cell Signaling Technology 23214S Rabbit 1:1,000 Tau Cell Signaling Technology 46687S Rabbit 1:1,000 Ox-DJ1 Emd Millipore Corporation MABN1773 Rat 1:10,000 DJ1 Abcam ab18257 Rabbit 1:1,000 Nrf2 Cell Signaling Technology 12721S Rabbit 1:1,000 Keap1 Cell Signaling Technology 8047S Rabbit 1:1,000 HO-1 Cell Signaling Technology 86806S Rabbit 1:1,000 p62/SQSTM1 Cell Signaling Technology 23214S Rabbit 1:1,000 LC3 Proteintech 14600-1-AP Rabbit 1:5,000 Beclin1 Servicebio GB11228 Rabbit 1:4,000 p-AMPK Abclonal AP883 Rabbit 1:2,000 AMPK Proteintech 18167-1-AP Rabbit 1:2,000 P-mTOR Abcam ab109268 Rabbit 1:10,000 mTOR Abcam ab32028 Rabbit 1:5,000 Caspase3 Proteintech 19677-1-AP Rabbit 1:2,000 Bax Proteintech 50599-2-Ig Rabbit 1:10,000 Bcl-2 Proteintech 26593-1-AP Rabbit 1:2,000 PCNA Cell Signaling Technology 13110S Rabbit 1:1,000 β-actin Proteintech 20536-1-AP Rabbit 1:5,000 GAPDH Abcam ab181602 Rabbit 1:10,000 -
Forty male 7-month-old SPF APP/PS1 mice were selected and purchased from Kavenberg Model Animal Research Co., LTD (Suzhou, China). The animals were raised in the barrier environment of the Experimental Animal Center, School of Public Health, Zhengzhou University. The experimental mice were housed in cages with free access to water and food, and had light/dark cycling of 12 h. The ambient temperature was 20–25 °C and the humidity was 50%–60%. After one week of adaptive feeding, the 40 APP/PS1 mice were randomly divided into four equal groups. Overexpression or knockdown of AAV was injected into the hippocampus of two groups of mice through brain localization surgery to construct AD model mice with DJ1 overexpression or knockdown. The four groups were: AD model control group (MC), AAV vector control group (NC), DJ1 up-regulation group (DJ1+), and DJ1 knockdown group (DJ1−). After 21 days of feeding, behavioral tests were performed, followed by anesthesia and euthanasia, and samples were collected. The protocol was reviewed and approved by the Life Science Ethics Committee of Zhengzhou University (ethical approval number zzuGZR2018-03).
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AAV is a single-stranded linear DNA virus that is a safe, durable, efficient, and highly specific gene-manipulation tool. To date, AAV-based gene vectors have been used in a large number of clinical trials for gene therapy[26]. AAV has multiple serotypes (1-9, Rh10), among which AAV-9 is capable of vectorial translocation and long-term expression in the hippocampus[27]. We used the brain localization injection technique to inject AAV9-DJ1 into the bilateral hippocampal regions of the mice. During localization, a locating needle was used to locate and drill holes 2 mm behind the fontanelle and approximately 2 mm beside the sagittal suture, and then 2 µL of AAV-DJ1 was injected into each hippocampus.
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The Morris water maze (MWM) behavioral study was performed on APP/PS1 mice in each group three weeks after brain localization injection. MWM was performed according to previous reports with some modifications[28]. Before the formal water maze experiment began, the APP/PS1 mice were acclimatized for two days. In the hidden platform training, the mice were trained for four days and were given a limit of 90 s to find the platform that was submerged 1 cm below the water surface. The mice performed 4 trials per day at 15 min intervals. On the last day of the space exploration test, the platform was removed, and the number of times the mice crossed the platform region for 1 min was recorded. All data were recorded using a computer program.
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All APP/PS1 mice were fasted overnight, weighed, and euthanized using sodium pentobarbital (40 mg/kg). Blood samples collected by cardiac aspiration were centrifuged at 3,000 rpm at 4 °C for 15 min to separate the upper serum. Samples were stored at –80 °C[29] before further biochemical analysis. For histological studies, the brains of mice were removed and immediately divided into two halves on ice. One half was fixed in 4% paraformaldehyde for morphological identification, and hippocampal tissue from the other half was collected and stored at –80 °C for subsequent experiments.
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AD is a progressive neurological dysfunction disease and studies have found that as the disease progresses, the brains of patients with AD experience brain cell death and atrophy compared to the brains of healthy individuals[30]. The brain index is a simple and effective index for evaluating brain atrophy and was calculated as: Brain index = brain weight (g)/rat total body weight (g).
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Mouse brains were fixed in 4% paraformaldehyde for 48 h, followed by graded dehydration in ethanol, xylene removal, and paraffin-embedding. Paraffin sections (10 μm thick) were soaked in hematoxylin stain for 5 min, washed with water, and then fractioned and liquefied. Slides were washed with water and then with performing anti-blue. The sections were dehydrated with gradient alcohol and soaked in an eosin staining solution for 5 min. The stained sections were soaked in anhydrous ethanol and xylene and then sealed with neutral resin. The stained images were acquired and analyzed using an inverted microscope.
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Paraffin sections were soaked in Congo Red A solution overnight, washed with water for 2 min, and fractioned with Congo Red B solution. The differentiated sections were placed in Congo Red C solution for 1 min, washed with water, and stained with anti-blue. The treated sections were dehydrated with anhydrous ethanol and xylene and finally sealed with neutral gum. The stained images were acquired and analyzed using an inverted microscope. The number of age spots was quantified using ImageJ software.
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Paraffin sections were fixed in 4% paraformaldehyde for 30 min and washed 3 times with perbenzoic acid (PBA) for 5 min each. After antigen repair, the sections were washed three times with PBA for 5 min. The sections were blocked with BSA in a circle around the tissue for 30 min and incubated overnight at 4 °C with the primary antibody. The corresponding secondary antibody was added to cover the tissue and incubated for 50 min at room temperature in the dark. Sections were double-stained with DAPI for cell nuclei and sealed with a fluorescent quencher. The stained images were acquired and analyzed using a fluorescent microscope. The intensity of the positive product fluorescence images was quantified using ImageJ software.
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T-SOD, MDA, T-AOC, and GSH-PX are important indicators of the level of oxidative damage in the body. T-SOD plays a crucial role in the balance between oxidation and antioxidant activity in the body, and can scavenge superoxide anion free radicals, thus protecting the body from damage. MDA level often reflects the degree of lipid peroxidation in the body and indirectly reflects the degree of tissue and cell damage. GSH-PX is an important peroxidase enzyme widely present in the body, and its level can reflect the strength of the body’s antioxidant capacity. T-AOC can reflect the strength of the antioxidant capacity of the body’s defense system. These four indicators were measured in the hippocampus and serum of APP/PS1 mice, and the assays were performed strictly according to the manufacturer’s instructions.
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First, the ipsilateral hippocampal tissue of each group of APP/PS1 mice was weighed using an electronic analytical balance, and protein extract was added at a ratio of 1:10. The protein extract solution was prepared with RIPA lysate, phenyl methane sulfonyl fluoride (PMSF), and phosphatase inhibitor at a ratio of 98:1:1. The tissues were completely ground in an ice and water bath. The supernatant was extracted after centrifugation, and tissue protein content was quantified using the BCA protein assay. Intranuclear Nrf2 protein was extracted using an Intranuclear Protein Extraction Kit (Cell Signaling Technology). The tissue protein solution was mixed with 5 × sodium dodecyl sulfate (SDS) loading buffer at 95 °C and boiled for 5 min to denature the protein. Equal amounts of protein samples (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes. Subsequently, the membranes were blocked in 5% skim milk for 2 h at room temperature, after which they were incubated at room temperature for 1.5 h with the following primary antibodies: APP (1:10,000), p-Tau (1:50,000), DJ-1 (1:1,000), Nrf2 (1:2,000), AMPK (1:10,000), mTOR (1:10,000), Bax (1:10,000), Caspase3 (1:2,000), B cell lymphoma-2 (Bcl-2) (1:2,000), and β-actin (1:5,000) (details of primary antibodies are listed in Table 1). The membranes were incubated with an HRP-coupled secondary antibody (1:10,000) for 1.5 h at room temperature. Finally, the proteins were visualized using an enhanced chemiluminescence kit (Biosharp), and the density values of the protein bands were quantified using ImageJ software. Most of the proteins were normalized to β-actin as a control, whereas their phosphorylated and oxidized proteins were normalized to the corresponding total proteins.
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All experiments were conducted in triplicate, and the experimental data were analyzed using SPSS 25.0 software (SPSS, Chicago, IL, USA). Measures that conform to a normal distribution are expressed as mean ± standard deviation (M ± SD). One-way ANOVA was used for comparisons between groups, and significance was calculated using the LSD test. If the data did not conform to a normal distribution or the variance was not uniform, the Kruskal–Wallis rank-sum test was used. The data from each group of the MWM training period were processed with repeated ANOVA measures and simple effects analysis and are expressed as mean ± standard error (M ± SE). All test levels were bilateral (α = 0.05).
doi: 10.3967/bes2023.133
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Abstract:
Objective To explore whether the protein Deglycase protein 1 (DJ1) can ameliorate Alzheimer’s disease (AD)-like pathology in Amyloid Precursor Protein/Presenilin 1 (APP/PS1) double transgenic mice and its possible mechanism to provide a theoretical basis for exploring the pathogenesis of AD. Methods Adeno-associated viral vectors (AAV) of DJ1-overexpression or DJ1-knockdown were injected into the hippocampus of 7-month-old APP/PS1 mice to construct models of overexpression or knockdown. Mice were divided into the AD model control group (MC), AAV vector control group (NC), DJ1-overexpression group (DJ1+), and DJ1-knockdown group (DJ1−). After 21 days, the Morris water maze test, immunohistochemistry, immunofluorescence, and western blotting were used to evaluate the effects of DJ1 on mice. Results DJ1+ overexpression decreased the latency and increased the number of platform traversals in the water maze test. DJ1− cells were cured and atrophied, and the intercellular structure was relaxed; the number of age spots and the expression of AD-related proteins were significantly increased. DJ1+ increased the protein expression of Nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase-1 (HO-1), light chain 3 (LC3), phosphorylated AMPK (p-AMPK), and B cell lymphoma-2 (BCL-2), as well as the antioxidant levels of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), and Glutathione peroxidase (GSH-PX), while decreasing the levels of Kelch-like hydrates-associated protein 1 (Keap1), mammalian target of rapamycin (mTOR), p62/sequestosome1 (p62/SQSTM1), Caspase3, and malondialdehyde (MDA). Conclusion DJ1-overexpression can ameliorate learning, memory, and AD-like pathology in APP/PS1 mice, which may be related to the activation of the NRF2/HO-1 and AMPK/mTOR pathways by DJ1. -
Key words:
- Alzheimer’s disease /
- DJ1 /
- NRF2/HO-1 /
- Oxidative stress /
- AMPK/mTOR /
- Autophagy /
- Apoptosis.
The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study.
&These authors contributed equally to this work.
注释:1) AUTHOR CONTRIBUTIONS: 2) CONFLICT OF INTEREST: -
Figure 1. DJ1 expression effects in the up-regulated and down-regulated models in APP/PS1 mice hippocampus. (A) Western blot for DJ1 and β-actin. (B) Densitometry analyses of DJ1 normalized on β-actin. (C) Mean fluorescence intensity of DJ1. (D) Immunofluorescence results of DJ1 protein (red) and DAPI (blue) in the same field of view; MC, AD model control group; NC, AAV vector control group; DJ1+, DJ1 up-regulation group; DJ1−, DJ1 knockdown group. *Р < 0.05, **Р < 0.001. Data are expressed as mean ± SD (n = 3). Scale bar in main image 50 µm and merged inset 25 µm.
Figure 2. AD characteristic index in APP/PS1 mice hippocampus. (A) HE and Congo red staining. Scale bar for HE and Congo red inset 50 µm and main Congo red image 250 µm. (B) Number of senile plaques. (C) Cerebral index. (D) Western blot for APP, Tau, p-Tau, and β-actin. Densitometry analyses of (E) APP, (G) Tau, and (F) p-Tau normalized on β-actin, and (H) p-Tau/Tau ratio. (I) Immunofluorescence results of Aβ40 (red), Aβ42 (green), and DAPI (blue) and colocalization in hippocampal CA1 region. Scale bar 25 µm. Mean fluorescence intensity of (J) Aβ40 and (K) Aβ42. (L) Colocalization coefficient of Aβ40 and Aβ42. *Р < 0.05, **Р < 0.001. Data are expressed as mean ± SD (n = 3).
Figure 3. Morris water maze evaluation of the effects of DJ1 expression up-regulation or knockdown on cognitive performance in APP/PS1 mice. (A) Representative movement trajectories of one mouse from each group during the training period. The red dot is the starting position of the mouse, blue dot is the ending position, and red circle is the target platform position. (B) Escape latency of mice in each group during the training period. (C) Escape latency comparison between the DJ1+ and DJ1− groups on day 1 and day 4 during the training period. (D) Representative movement trajectories of one mouse from each group during the test period. (E) Escape latency of mice in each group during the test period. (F) Target quadrant entry times of mice in each group during the test period. (G) Swimming speed of mice in each group during the test period; MC, AD model control group; NC, AAV vector control group; DJ1+, DJ1 up-regulation group; DJ1−, DJ1 knockdown group. *Р < 0.05 and **Р < 0.001 for groups compared with the MC group; #Р < 0.05 and ##Р < 0.001 for DJ1+ group compared with the DJ1− group; $Р < 0.05 for DJ1+ group Day 1 compared with Day 4. Data are expressed as mean ± SEM (n = 6).
Figure 4. DJ1 up-regulation or knockdown effects on oxidative stress index and Nrf2/HO-1 signaling pathway related proteins in APP/PS1 mice. The levels of oxidative stress in the (A) serum and (B) hippocampus. (C) Western blot analysis of the seven proteins of interest (β-actin used for normalization). Densitometry analyses of (D) DJ1, (E) total Nrf2, (F) cytoplasmic Nrf2, (G) nuclear Nrf2, (H) Keap1, and (I) HO-1. Mean fluorescence intensity of (J) DJ1 and (L) Nrf2. (K) Immunofluorescence co-location of DJ1 (red) and Nrf2 (green), with DAPI nuclear staining (blue). (M) Colocalization coefficient of DJ1 and Nrf2; MC, AD model control group; NC, AAV vector control group; DJ1+, DJ1 up-regulation group; DJ1−, DJ1 knockdown group. *Р < 0.05, **Р < 0.001. Data are expressed as mean ± SD (n = 3). Scale bar 25 µm.
Figure 5. DJ1 up-regulation or knockdown effects on autophagy and apoptosis in the hippocampus of APP/PS1 mice. (A) Western blot analysis of the six proteins of interest in the AMPK/mTOR pathway (β-actin used for normalization). Densitometry analyses of (B) LC3, (C) p62/SQSTM1, (D) AMPK, (E) p-AMPK, (F) AMPK/p-AMPK, (G) mTOR, (H) p-mTOR, and (I) p-mTOR/mTOR. (K) Immunofluorescence results of Beclin1 (red) protein, with DAPI nuclear staining (blue). (J) Mean fluorescence intensity of Beclin1. (L) Western blot analysis of the three proteins of interest involved with apoptosis (GAPDH used for normalization). Densitometry analyses of (M) Caspase3, (N) Bax, (O) Bcl-2, and (P) Bax/Bcl-2. Mean fluorescence intensity of (Q) Caspase3 and (R) TUNEL assay. (S) Immunofluorescence results of Caspase3 (red), with DAPI nuclear staining (blue). (T) Results of TUNEL fluorescence assay; MC, AD model control group; NC, AAV vector control group; DJ1+, DJ1 up-regulation group; DJ1−, DJ1 knockdown group. *Р < 0.05, **Р < 0.001. Data are expressed as mean ± SD (n = 3). Scale bar 25 µm.
Table 1. Detailed information of primary antibodies used
Antibody Producers Catalogue
numberSource Dilution APP Cell Signaling Technology 15126S Rabbit 1:1,000 Aβ40 Cell Signaling Technology 12990S Rabbit 1:1,000 Aβ42 Cell Signaling Technology 14974s Rabbit 1:1,000 p-Tau Cell Signaling Technology 23214S Rabbit 1:1,000 Tau Cell Signaling Technology 46687S Rabbit 1:1,000 Ox-DJ1 Emd Millipore Corporation MABN1773 Rat 1:10,000 DJ1 Abcam ab18257 Rabbit 1:1,000 Nrf2 Cell Signaling Technology 12721S Rabbit 1:1,000 Keap1 Cell Signaling Technology 8047S Rabbit 1:1,000 HO-1 Cell Signaling Technology 86806S Rabbit 1:1,000 p62/SQSTM1 Cell Signaling Technology 23214S Rabbit 1:1,000 LC3 Proteintech 14600-1-AP Rabbit 1:5,000 Beclin1 Servicebio GB11228 Rabbit 1:4,000 p-AMPK Abclonal AP883 Rabbit 1:2,000 AMPK Proteintech 18167-1-AP Rabbit 1:2,000 P-mTOR Abcam ab109268 Rabbit 1:10,000 mTOR Abcam ab32028 Rabbit 1:5,000 Caspase3 Proteintech 19677-1-AP Rabbit 1:2,000 Bax Proteintech 50599-2-Ig Rabbit 1:10,000 Bcl-2 Proteintech 26593-1-AP Rabbit 1:2,000 PCNA Cell Signaling Technology 13110S Rabbit 1:1,000 β-actin Proteintech 20536-1-AP Rabbit 1:5,000 GAPDH Abcam ab181602 Rabbit 1:10,000 -
[1] GBD 2019 Dementia Forecasting Collaborators. Estimation of the global prevalence of dementia in 2019 and forecasted prevalence in 2050: an analysis for the Global Burden of Disease Study 2019. Lancet Public Health, 2022; 7, e105-25. [2] 2020 Alzheimer’s disease facts and figures. Alzheimers Dement, 2020; 16, 391−460. [3] Liu CG, Meng S, Li Y, et al. MicroRNA-135a in ABCA1-labeled exosome is a serum biomarker candidate for Alzheimer’s disease. Biomed Environ Sci, 2021; 34, 19−28. [4] Li WL, Li YY, Li YX, et al. Gene-environment interactions between environmental noise and ApoE4 causes AD-like neuropathology in the hippocampus in male rats. Biomed Environ Sci, 2022; 35, 270−5. [5] Chen ZC, Zhong CJ. Oxidative stress in Alzheimer’s disease. Neurosci Bull, 2014; 30, 271−81. doi: 10.1007/s12264-013-1423-y [6] Ali T, Kim T, Rehman SU, et al. Natural dietary supplementation of anthocyanins via PI3K/Akt/Nrf2/HO-1 pathways mitigate oxidative stress, neurodegeneration, and memory impairment in a mouse model of Alzheimer’s disease. Mol Neurobiol, 2018; 55, 6076−93. doi: 10.1007/s12035-017-0798-6 [7] Fão L, Mota SI, Rego AC. Shaping the Nrf2-ARE-related pathways in Alzheimer’s and Parkinson’s diseases. Ageing Res Rev, 2019; 54, 100942. doi: 10.1016/j.arr.2019.100942 [8] Zhou YY, Xie N, Li LB, et al. Puerarin alleviates cognitive impairment and oxidative stress in APP/PS1 transgenic mice. Int J Neuropsychopharmacol, 2014; 17, 635−44. doi: 10.1017/S146114571300148X [9] Kanninen K, Heikkinen R, Malm T, et al. Intrahippocampal injection of a lentiviral vector expressing Nrf2 improves spatial learning in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA, 2009; 106, 16505−10. doi: 10.1073/pnas.0908397106 [10] Frias DP, Gomes RLN, Yoshizaki K, et al. Nrf2 positively regulates autophagy antioxidant response in human bronchial epithelial cells exposed to diesel exhaust particles. Sci Rep, 2020; 10, 3704. doi: 10.1038/s41598-020-59930-3 [11] Jo C, Gundemir S, Pritchard S, et al. Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52. Nat Commun, 2014; 5, 3496. doi: 10.1038/ncomms4496 [12] Pajares M, Rojo AI, Arias E, et al. Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A. Autophagy, 2018; 14, 1310−22. doi: 10.1080/15548627.2018.1474992 [13] Abelaira HM, Réus GZ, Neotti MV, et al. The role of mTOR in depression and antidepressant responses. Life Sci, 2014; 101, 10−4. doi: 10.1016/j.lfs.2014.02.014 [14] Fan XD, Wang J, Hou JC, et al. Berberine alleviates ox-LDL induced inflammatory factors by up-regulation of autophagy via AMPK/mTOR signaling pathway. J Transl Med, 2015; 13, 92. doi: 10.1186/s12967-015-0450-z [15] Liu SX, Sun YQ, Li ZM. Resveratrol protects Leydig cells from nicotine-induced oxidative damage through enhanced autophagy. Clin Exp Pharmacol Physiol, 2018; 45, 573−80. doi: 10.1111/1440-1681.12895 [16] Li GH, Lin XL, Zhang H, et al. Ox-Lp(a) transiently induces HUVEC autophagy via an ROS-dependent PAPR-1-LKB1-AMPK-mTOR pathway. Atherosclerosis, 2015; 243, 223−35. doi: 10.1016/j.atherosclerosis.2015.09.020 [17] Ou ZR, Kong XJ, Sun XD, et al. Metformin treatment prevents amyloid plaque deposition and memory impairment in APP/PS1 mice. Brain Behav Immun, 2018; 69, 351−63. doi: 10.1016/j.bbi.2017.12.009 [18] Hijioka M, Inden M, Yanagisawa D, et al. DJ-1/PARK7: a new therapeutic target for neurodegenerative disorders. Biol Pharm Bull, 2017; 40, 548−52. doi: 10.1248/bpb.b16-01006 [19] Jang J, Jeong S, Lee SI, et al. Oxidized DJ-1 levels in urine samples as a putative biomarker for Parkinson’s disease. Parkinsons Dis, 2018; 2018, 1241757. [20] Yan YF, Yang WJ, Xu Q, et al. DJ-1 upregulates anti-oxidant enzymes and attenuates hypoxia/re-oxygenation-induced oxidative stress by activation of the nuclear factor erythroid 2-like 2 signaling pathway. Mol Med Rep, 2015; 12, 4734−42. doi: 10.3892/mmr.2015.3947 [21] Clements CM, McNally RS, Conti BJ, et al. DJ-1, a cancer- and Parkinson’s disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2. Proc Natl Acad Sci USA, 2006; 103, 15091−6. doi: 10.1073/pnas.0607260103 [22] Kitamura Y, Inden M, Kimoto Y, et al. Effects of a DJ-1-binding compound on spatial learning and memory impairment in a mouse model of Alzheimer’s disease. J Alzheimers Dis, 2017; 55, 67−72. [23] Hardy J. The discovery of Alzheimer-causing mutations in the APP gene and the formulation of the “amyloid cascade hypothesis”. FEBS J, 2017; 284, 1040−4. doi: 10.1111/febs.14004 [24] Zhang W, Bai M, Xi Y, et al. Early memory deficits precede plaque deposition in APPswe/PS1dE9 mice: involvement of oxidative stress and cholinergic dysfunction. Free Radic Biol Med, 2012; 52, 1443−52. doi: 10.1016/j.freeradbiomed.2012.01.023 [25] Ruan LF, Kang ZJ, Pei G, et al. Amyloid deposition and inflammation in APPswe/PS1dE9 mouse model of Alzheimers disease. Curr Alzheimer Res, 2009; 6, 531−40. doi: 10.2174/156720509790147070 [26] Santiago-Ortiz JL, Schaffer DV. Adeno-associated virus (AAV) vectors in cancer gene therapy. J Control Release, 2016; 240, 287−301. doi: 10.1016/j.jconrel.2016.01.001 [27] Cearley CN, Wolfe JH. Transduction characteristics of adeno-associated virus vectors expressing cap serotypes 7, 8, 9, and Rh10 in the mouse brain. Mol Ther, 2006; 13, 528−37. doi: 10.1016/j.ymthe.2005.11.015 [28] Fan YG, Guo T, Han XR, et al. Paricalcitol accelerates BACE1 lysosomal degradation and inhibits calpain-1 dependent neuronal loss in APP/PS1 transgenic mice. eBioMedicine, 2019; 45, 393−407. doi: 10.1016/j.ebiom.2019.07.014 [29] Aleissa MS, Alkahtani S, Eldaim MAA, et al. Fucoidan Ameliorates oxidative stress, inflammation, DNA damage, and hepatorenal injuries in diabetic rats intoxicated with Aflatoxin B. Oxid Med Cell Longev, 2020; 2020, 9316751. [30] Bi WY, Cai SL, Hang ZC, et al. Transplantation of feces from mice with Alzheimer’s disease promoted lung cancer growth. Biochem Biophys Res Commun, 2022; 600, 67−74. doi: 10.1016/j.bbrc.2022.01.078 [31] Torromino G, Maggi A, De Leonibus E. Estrogen-dependent hippocampal wiring as a risk factor for age-related dementia in women. Prog Neurobiol, 2021; 197, 101895. doi: 10.1016/j.pneurobio.2020.101895 [32] Nunomura A, Zhu XW, Perry G. Modulation of Parkinson’s disease associated protein rescues Alzheimer’s disease degeneration. J Alzheimers Dis, 2017; 55, 73−5. [33] Prasad KN. Oxidative stress and pro-inflammatory cytokines may act as one of the signals for regulating microRNAs expression in Alzheimer’s disease. Mech Ageing Dev, 2017; 162, 63−71. doi: 10.1016/j.mad.2016.12.003 [34] Guo JP, Cheng J, North BJ, et al. Functional analyses of major cancer-related signaling pathways in Alzheimer’s disease etiology. Biochim Biophys Acta Rev Cancer, 2017; 1868, 341−58. doi: 10.1016/j.bbcan.2017.07.001 [35] Schellenberg GD, Montine TJ. The genetics and neuropathology of Alzheimer’s disease. Acta Neuropathol, 2012; 124, 305−23. doi: 10.1007/s00401-012-0996-2 [36] Reitz C, Mayeux R. Alzheimer disease: epidemiology, diagnostic criteria, risk factors and biomarkers. Biochem Pharmacol, 2014; 88, 640−51. doi: 10.1016/j.bcp.2013.12.024 [37] Ahmad A, Manjrekar P, Yadav C, et al. Evaluation of ischemia-modified albumin, malondialdehyde, and advanced oxidative protein products as markers of vascular injury in diabetic nephropathy. Biomark Insights, 2016; 11, 63−8. [38] Bao LP, Li JS, Zha DQ, et al. Chlorogenic acid prevents diabetic nephropathy by inhibiting oxidative stress and inflammation through modulation of the Nrf2/HO-1 and NF-ĸB pathways. Int Immunopharmacol, 2018; 54, 245−53. doi: 10.1016/j.intimp.2017.11.021 [39] Hou YN, Peng SJ, Li XM, et al. Honokiol alleviates oxidative stress-induced neurotoxicity via activation of Nrf2. ACS Chem Neurosci, 2018; 9, 3108−16. doi: 10.1021/acschemneuro.8b00290 [40] Kim J, Kundu M, Viollet B, et al. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol, 2011; 13, 132−41. doi: 10.1038/ncb2152 [41] Wang WP, Zhao H, Chen BH. DJ-1 protects retinal pericytes against high glucose-induced oxidative stress through the Nrf2 signaling pathway. Sci Rep, 2020; 10, 2477. doi: 10.1038/s41598-020-59408-2 [42] Han T, Liu MH, Yang SB. DJ-1 alleviates angiotensin II-induced endothelial progenitor cell damage by activating the PPARγ/HO-1 pathway. J Cell Biochem, 2018; 119, 392−400. doi: 10.1002/jcb.26191 [43] Loboda A, Damulewicz M, Pyza E, et al. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell Mol Life Sci, 2016; 73, 3221−47. doi: 10.1007/s00018-016-2223-0 [44] Klionsky DJ, Petroni G, Amaravadi RK, et al. Autophagy in major human diseases. EMBO J, 2021; 40, e108863. [45] Wirawan E, Walle LV, Kersse K, et al. Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis, 2010; 1, e18. doi: 10.1038/cddis.2009.16 [46] Ouyang CH, You JY, Xie ZL. The interplay between autophagy and apoptosis in the diabetic heart. J Mol Cell Cardiol, 2014; 71, 71−80. doi: 10.1016/j.yjmcc.2013.10.014 [47] Li MY, Zhu XL, Zhao BX, et al. Adrenomedullin alleviates the pyroptosis of Leydig cells by promoting autophagy via the ROS-AMPK-mTOR axis. Cell Death Dis, 2019; 10, 489. doi: 10.1038/s41419-019-1728-5 [48] Zhang M, Teng CH, Wu FF, et al. Edaravone attenuates traumatic brain injury through anti-inflammatory and anti-oxidative modulation. Exp Ther Med, 2019; 18, 467−74. [49] Pajares M, Jiménez-Moreno N, García-Yagüe ÁJ, et al. Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes. Autophagy, 2016; 12, 1902−16. doi: 10.1080/15548627.2016.1208889 [50] Kaushal GP, Chandrashekar K, Juncos LA. Molecular interactions between reactive oxygen species and autophagy in kidney disease. Int J Mol Sci, 2019; 20, 3791. doi: 10.3390/ijms20153791 [51] Jain A, Lamark T, Sjøttem E, et al. p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription. J Biol Chem, 2010; 285, 22576−91. doi: 10.1074/jbc.M110.118976 [52] Fan SN, Zhang B, Luan P, et al. PI3K/AKT/mTOR/p70S6K pathway is involved in Aβ25-35-induced autophagy. BioMed Res Int, 2015; 2015, 161020. [53] González-Rodríguez Á, Mayoral R, Agra N, et al. Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD. Cell Death Dis, 2014; 5, e1179. doi: 10.1038/cddis.2014.162 [54] Petrović A, Bogojević D, Korać A, et al. Oxidative stress-dependent contribution of HMGB1 to the interplay between apoptosis and autophagy in diabetic rat liver. J Physiol Biochem, 2017; 73, 511−21. doi: 10.1007/s13105-017-0574-0 [55] Xiong YJ, Deng ZB, Liu JN, et al. Enhancement of epithelial cell autophagy induced by sinensetin alleviates epithelial barrier dysfunction in colitis. Pharmacol Res, 2019; 148, 104461. doi: 10.1016/j.phrs.2019.104461 [56] Pang J, Li FZ, Feng X, et al. Influences of different dietary energy level on sheep testicular development associated with AMPK/ULK1/autophagy pathway. Theriogenology, 2018; 108, 362−70. doi: 10.1016/j.theriogenology.2017.12.017 [57] Arab HH, Al-Shorbagy MY, Saad MA. Activation of autophagy and suppression of apoptosis by dapagliflozin attenuates experimental inflammatory bowel disease in rats: Targeting AMPK/mTOR, HMGB1/RAGE and Nrf2/HO-1 pathways. Chem Biol Interact, 2021; 335, 109368. doi: 10.1016/j.cbi.2021.109368 [58] Deng J, Zeng LS, Lai XY, et al. Metformin protects against intestinal barrier dysfunction via AMPKα1-dependent inhibition of JNK signalling activation. J Cell Mol Med, 2018; 22, 546−57. doi: 10.1111/jcmm.13342 [59] Hu QY, Knight PH, Ren YH, et al. The emerging role of stimulator of interferons genes signaling in sepsis: Inflammation, autophagy, and cell death. Acta Physiol, 2019; 225, e13194. [60] Shen BY, Feng HH, Cheng JQ, et al. Geniposide alleviates non-alcohol fatty liver disease via regulating Nrf2/AMPK/mTOR signalling pathways. J Cell Mol Med, 2020; 24, 5097−108. doi: 10.1111/jcmm.15139