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Research Article

Effects of Qi-fu-yin on Improving Cognitive Impairment and Reducing Cellular Senescence in the Brains of 5xFAD Mice

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DOI:

10.3791/72118

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September 8th, 2026

 ,  ,  ,  ,  ,  , 
1Innovative Institute of Chinese Medicine and Pharmacy, Shandong University of Traditional Chinese Medicine

* These authors contributed equally

In This Article

Summary

This study evaluates Qi-fu-yin (QFY) treatment in 5xFAD mice. The findings show that QFY administration is associated with enhanced learning and memory performance, reduced cerebral Aβ deposition and cellular senescence, and attenuated synaptic structural damage in the cerebral cortex.

Abstract

Although the mechanism of Qi-fu-yin (QFY), a traditional Chinese medicine prescription, remains incompletely understood, it has shown therapeutic potential in Alzheimer's disease (AD). This study aimed to investigate the association between QFY treatment and cognitive impairment in 5xFAD mice. We used grip strength, gait, colony nesting, shuttle box, Morris water maze test, and ELISA to evaluate the levels of Amyloid-β protein 1-42 (Aβ1-42), Aβ1-40, Growth-associated protein 43 (GAP-43), Synaptophysin (SYN), and Postsynaptic density protein 95 (PSD-95) in the brains of 5xFAD mice. Luminex cytokine analysis was used to quantify Senescence-associated secretory phenotype (SASP) levels in the cortex. The amount of Senescence-associated β-galactosidase (SA-β-Gal), Aβ deposition, and dendritic spines in the animals' brains was measured by SA-β-Gal staining, immunohistochemistry, and Golgi-Cox staining, respectively. QFY treatment dramatically reduced cortical Aβ plaque burden and decreased p21 expression in 5xFAD mice, according to immunofluorescence tests. In 5xFAD mice, QFY therapy markedly enhanced learning and memory performance, alleviated aging, and increased the amounts of GAP-43, PSD-95, and SYN proteins in the mice's cortex. Following QFY treatment, there was a significant decrease in Aβ plaque and SA-β-Gal activity in the brain, as well as Aβ1-42 and Aβ1-42/Aβ1-40 levels in the hippocampus. The levels of IL-1α, IL-1β, IL-6, IL-17A, and IFN-γ were considerably reduced in the cortex of the 5xFAD mice. PCA and Pearson correlation analyses demonstrated a strong negative association between SASP cytokines and synaptic protein levels. These findings suggest an association between QFY treatment and improvements in age‑related cognitive decline and cellular senescence phenotypes in 5xFAD mice, alongside mitigated synaptic structural damage. These findings support further investigation of QFY as a potential therapeutic candidate for Alzheimer's disease.

Introduction

Alzheimer's disease (AD)1 is the most prevalent form of dementia, characterized by amyloid-β protein (Aβ), hyperphosphorylated tau proteins (P-Tau), synaptic loss, and chronic inflammation. Currently available medications for AD have a number of side effects and are unable to stop the disease's progression2,3,4,5.

Senescence, which manifests as organism-wide disruptions across cellular and interstitial microenvironments rather than localized organ-specific alterations, is a key risk factor in AD pathogenesis. This systemic dysregulation distinguishes AD progression from single tissue-pathology models by fundamentally changing homeostatic mechanisms across biological systems6,7. Although cellular senescence initially serves as a homeostatic regulatory mechanism, the accumulation of senescent cells eventually causes progressive tissue degradation and persistent inflammatory cascades, fundamentally disrupting physiological homeostasis8. The ensuing inflammation may harm synapses and impair cognitive function9,10.

Cellular senescence is a complex cellular stress response characterized by telomere shortening, permanent cell-cycle arrest, and altered secretory activity11. Additional hallmarks include enlarged, flattened cell morphology, positive senescence-associated β-galactosidase (SA-β-Gal) staining, lipofuscin accumulation, and secretion of SASP factors12. Memory formation is affected by the notable decline in the levels of proteins associated with synaptic plasticity of axons and dendrites, which occurs with cellular senescence13,14. Because synaptic plasticity is essential for learning and memory, disruptions in synaptic proteins contribute to cognitive impairment. These proteins include GAP-43, SYN, and PSD-95. Undoubtedly, synaptic function is affected by reductions in dendritic numbers and functional abnormalities in the brains of the elderly15.

QFY is a traditional Chinese medicine formula derived from Jing Yue Quan Shu. It is composed of Panax ginseng C.A.Mey (Araliaceae), Polygala tenuifolia Willd (Polygalaceae), Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C. A. Mey (Orobanchaceae), Angelica sinensis (Oliv.) Diels (Apiaceae), Glycyrrhiza uralensis Fisch. ex DC. (Fabaceae), Atractylodes macrocephala Koidz (Asteraceae), Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H.F. Chou (Rhamnaceae). Research has demonstrated that QFY significantly ameliorates cognitive dysfunction in rats with diabetes-induced brain injury16. Our previous study found that QFY could improve cognitive learning and memory functions impairment in 5xFAD and APP/PS1 mice. In addition, QFY demonstrated high safety in non-clinical studies, and no obvious acute, subacute, or long-term toxic reactions were observed17,18,19. Analysis of the clinical therapeutic effect of QFY showed that AD patients experienced a significant increase in their capacity for activities of daily living and MMSE scores after QFY treatment20. Following three consecutive days of QFY administration in male Sprague-Dawley rats, ten prototype QFY-derived components, including butylidenephthalide, butylphthalide, 20(S)-ginsenoside Rh1, 20(R)-ginsenoside Rh1, and zingibroside R1, were detected in cerebrospinal fluid21,22. However, the therapeutic mechanism of QFY in AD is still unclear. In this study, we investigated the association between QFY treatment, cellular senescence, and AD-related pathological changes in 5xFAD mice.

Although previous studies have demonstrated the neuroprotective effects of QFY in AD models, the specific involvement of cellular senescence in mediating these effects remains unexplored. Moreover, whether QFY confers cognitive benefits through senescence‑associated synaptic preservation, rather than solely through amyloid‑β reduction, has not been addressed. The present study was therefore designed to investigate whether QFY treatment attenuates cellular senescence and whether this attenuation is associated with improved synaptic plasticity in 5xFAD mice.

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Protocol

All animal experiments were approved by the Ethics Committee of Experimental Animal Welfare of Shandong University of Traditional Chinese Medicine (SDUTCM20221101001).

Drugs

QFY granule dry cream powder was prepared and tested by Lunan Pharmaceutical Houpu Research and Development Center. Donepezil and Memantine were purchased from Shanghai Yuanye Technology. Male 5xFAD mice with an initial body weight range of 19–27 g were used in this study. The QFY dosage was derived from the clinical human dose of 43 g/d, which corresponds to 16.297 g/d after adjusting for the QFY granule dry cream powder. The medium dose for the present experiment was calculated based on the clinical human dose (16.297 g/70 kg x 9.1 = 2.11861 g/kg/d). The high dose was set to twice the medium dose (4.24 g/kg/d), and the low dose to half the medium dose (1.06 g/kg/d). All drug solutions were administered at a volume of 0.1 mL per 10 g of body weight.

For the positive control, combined administration of donepezil and memantine hydrochloride was employed. The dosages were determined with reference to their current clinical application doses (10 mg/d and 20 mg/d, respectively) and previous data from our research group, resulting in a regimen of donepezil at 1.0 mg/kg/d plus memantine at 2.8 mg/kg/d, yielding a total combined dose of 3.8 mg/kg/d. The final concentration of the donepezil–memantine mixture was prepared as 0.38 mg/mL, calculated as 3.8 mg/kg divided by the administration volume of 0.1 mL per 10 g body weight.

QFY solutions at low, medium, and high doses were prepared as follows: 5.3 g, 10.6 g, and 21.2 g of QFY dry powder were each placed into separate 100 mL beakers, mixed with 30 mL of purified water with thorough stirring, transferred to graduated cylinders, and brought to a final volume of 50 mL with purified water, yielding final mass concentrations of 0.106 g/mL, 0.212 g/mL, and 0.424 g/mL, respectively. For the positive control, 5 mg of donepezil and 14 mg of memantine were dissolved in purified water and adjusted to a final volume of 50 mL. Animals in each group were given the corresponding drug solution once daily via oral gavage.

Animals

5xFAD transgenic mice [B6. Cg-Tg (APPSwFlLon, PSEN1* M146L* L286V)6799Vas/Mmjax] were bred in the laboratory. The animals were all maintained in an SPF environment at Shandong University of Traditional Chinese Medicine, with a temperature of 22–24 oC, a humidity of 50–60%, and a 12 h light-dark cycle. Food and water were provided to the animals at all times.

At 2.5 months of age, male 5xFAD mice were randomly allocated to five groups: untreated 5xFAD, donepezil plus memantine, low-dose QFY, medium-dose QFY, and high-dose QFY. Age-matched male wild-type (WT) littermates served as the WT control group. Treatments were administered once daily for 220 days. (Figure 1).

Behavioral tests

Grip strength test

The forelimb skeletal muscle strength of the mice was measured using a grip strength meter23. During the experiment, the testing device was positioned horizontally, ensuring the mouse remained parallel to the table surface. After the device was turned on, the mouse was gently placed on the grip plate. When the mouse had securely grasped the plate, its tail was pulled backward with equal force until the mouse released its grip. The maximum grip strength was documented. This measurement was repeated three times, and the average of these values was used to determine the mouse’s grip strength.

Gait test

Mice's gait regularity was measured using CatWalk XT. The experiment was divided into a training period and a test period24. One day earlier, the experimental mice were transferred to the experimental room to allow them to adapt to the new environment. During the training period, the mice were placed at the starting point and ran along the channel to the end point. Each mouse was trained three times a day. After 3 days of training (until each mouse could quickly pass through the channel), the mice were returned to the cage. The test period was the same as the training period, and the computer software would automatically analyze each movement trajectory. Green dots were displayed if the trajectory was qualified, orange dots were unqualified, and at least three green dots were qualified for each mouse, which were analyzed by CatWalkXT 10.0 software.

Shuttle box test

The shuttle box test was used to assess conditional active avoidance ability in mice25. The apparatus consisted of a shuttle box (18 cm x 18 cm) housed within a sound‑attenuating chamber.  The experiment comprised a 4‑day learning period followed by a 1‑day test period.  On each learning day, mice were allowed a 120‑s adaptation period in the shuttle box before training began. Each trial started with a 6‑s conditioned stimulus (70‑dB, 1000‑Hz tone combined with a 500‑lux light), followed by a 3‑s, 0.3‑mA foot shock delivered through the grid floor, and concluded with a 12 s inter‑trial interval. If the mouse crossed to the opposite compartment during the conditioned stimulus, the shock was omitted, and the response was recorded as active avoidance. If the mouse did not cross during the conditioned stimulus but crossed during the foot shock, it was recorded as a passive avoidance response. If the mouse failed to cross throughout the trial, it was recorded as an escape failure. Training was repeated for 4 consecutive days with 30 trials per day. During the test period (day 5), the foot shock was omitted while all other parameters remained the same as in the learning period. The number of active avoidance responses was recorded as the primary measure of learning and memory.

Morris water maze

The Morris water maze test was used to assess the spatial learning and memory abilities of experimental animals26. The experiment was carried out in a tank that had a diameter of 120 cm and a depth of 50 cm. The tank was filled with turbid white water, and the temperature was maintained at 21 °C. A platform with a diameter of 10 cm was installed 1 cm below the water surface in one of the quadrants. Visual clues were placed around the pool as spatial landmarks for the mice. The learning process was conducted over four consecutive days, with each day's session beginning in one of three quadrants. Start positions varied pseudo-randomly among the four cardinal points. In each trial, the procedure ended when the animal successfully reached the platform. A maximum trial duration of 60 s was established; if the animal failed to locate the platform within this timeframe, it was carefully guided to the platform. After reaching the platform, the animal was given a brief 10 s respite before being returned to its home cage. After the learning period ends, the test period begins 24 h later. Mice are placed from the opposite quadrant of the platform on Day 5. The escape latency of the test period, the movement time of the target quadrant, and the number of crossings of the original underwater platform area were recorded.

Colony nesting test

The daily activities of experimental animals were assessed using the colony nesting test27. The experiment lasted 24 h, with three animals per group. After the drug administration on the same day, new cages were set up and lined with 6 g soft paper for nesting, and photographs were taken at 6 h, 12 h, and 24 h. At the end of the experiment, the paper was scattered throughout the cage, and no bite was given. 1 point. The paper pieces were clustered to the side of the cage, but loosely, with no formed nests, no obvious tearing or folding for 2 points; The paper pieces gathered and folded into a formed but relatively flat nest without obvious tearing was 3 points; A deep nest with extensively shredded paper was assigned a score of 4.  At the end of the experiment, the remaining untorn tissue (defined as paper fragments weighing >0.1 g that were not shredded or incorporated into the nest) was carefully collected, air‑dried, and weighed using an electronic balance (precision: 0.01 g). 

Sampling and sample collection

We collected samples only from the WT, 5xFAD, and QFY (2.12 g/kg/d) groups. After CO2 asphyxiation, the heads were cut off, the right hemisphere was quickly opened to separate the hippocampus and cortex, rinsed with phosphate-buffered saline (PBS), and placed in 1.5 mL frozen tubes in liquid nitrogen. It was stored at -80 oC for biochemical tests. The left hemisphere was fixed by immersion in 4% paraformaldehyde for 24 h, dehydrated by immersion in absolute ethanol, embedded in paraffin wax, sectioned at 5 µm, and deparaffinized for immunohistochemical detection.

Enzyme-linked immunosorbent assay (ELISA)

ELISAs were performed to quantify target protein levels in brain tissues. Aβ1–40 and Aβ1–42 contents in hippocampal homogenates were measured using commercial kits, whereas cortical levels of GAP-43, PSD-95, and SYN were determined using kits sourced from Jiangsu Enzyme Immunoassay Industry. All procedures were carried out strictly in accordance with the manufacturers' protocols. Standard series were prepared by serial dilution following the kit instructions. The microplate layout included blank wells, standard wells, 5xFAD sample wells, QFY‑treated (2.12 g/kg/d) sample wells, and wild‑type wells. Prior to loading, all samples were diluted twofold with the provided diluent. Reagents were added with the pipette tip held perpendicular to the bottom of the well, then gently agitated to ensure thorough mixing.

After plate sealing, the initial incubation was conducted at 37 °C for 30 min. During this period, the wash buffer was prepared by diluting the concentrated stock with ultrapure water at a 1:29 ratio (1 mL concentrate to 29 mL water). Following incubation, the sealing film was removed, the liquid discarded, and the plate blotted dry. Each well was then filled with 200 µL of wash buffer, allowed to stand for 30 s, and emptied; the cycle was repeated 5 times. Enzyme‑labeled reagent (50 µL) was subsequently added to all wells except the blanks, and the plate was resealed and incubated again at 37 °C for 30 min, followed by an identical five‑cycle washing step.

For color development, 50 µL of chromogen solution A was added to each well, followed by 50 µL of chromogen solution B, with gentle mixing after each addition. The plate was incubated at 37 °C in the dark for 10 min. The enzymatic reaction was terminated by adding 50 µL of stop solution per well, which promptly converted the blue color to yellow. Absorbance (optical density) was measured at 450 nm using a microplate reader, with blank wells serving as the reference; all readings were obtained within 15 min after stopping the reaction.

Luminex

Cortical levels of SASP factors were measured using the Bio‑Plex Pro Mouse Cytokine 23‑plex Assay kit, with all procedures performed according to the manufacturer's instructions. Reagents were removed from 4 °C storage and allowed to equilibrate to room temperature for 30 min prior to use. Standard stocks were serially diluted to generate a seven‑point standard curve, plus one blank well, according to the kit protocol. Magnetic beads were premixed and diluted to the recommended working concentration; detection antibodies were prepared as working solutions; and the wash buffer concentrate was diluted to 1x with deionized water. PE‑streptavidin conjugate was also diluted to 1x using the supplied dilution buffer.

For the assay, 50 µL of bead suspension, standards, quality controls, and diluted tissue samples (5.6 µg/µL) were added sequentially to designated wells according to the plate layout. The plate was then sealed and incubated overnight at 4 °C with orbital shaking at 800 rpm. Following incubation, the beads were washed three times with 200 µL of wash buffer per well. Subsequently, 50 µL of the prepared detection antibody was added to each well, and the plate was incubated at room temperature for 1 h with shaking at 800 rpm. After another three wash cycles, 50 µL of PE‑streptavidin working solution was introduced, and the plate was incubated for an additional 30 min at room temperature under the same shaking conditions. A final wash step (three times with 200 µL wash buffer) was performed, after which 150 µL of wash buffer was added to each well, and the plate was shaken at room temperature for 2 min at 800 rpm before being read on the Bio‑Plex instrument.

SA-β-Gal staining and immunohistochemistry

SA-β-Gal staining was performed according to the Senescence β-Galactosidase Staining Kit. After SA-β-Gal staining, sections were subjected to antigen retrieval by heating in citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubating the sections in a peroxidase-blocking solution for 10 min at room temperature. The sections were then incubated with a rabbit monoclonal anti‑Aβ₁-₄₂ antibody (1:400) at 4 °C overnight. After washing, the sections were incubated with an anti‑rabbit/mouse IgG secondary antibody for 20 min at room temperature. Following further washing, immunoreactivity was visualized with DAB for 2 min. Finally, the sections were dehydrated, cleared, and mounted with neutral resin for microscopic examination.

Immunofluorescence

For immunofluorescence staining, paraffin sections were deparaffinized in xylene (3 x 15 min) and rehydrated through a graded ethanol series (100%, 95%, and 75%; 2 x 5 min each). Antigen retrieval was performed by immersing the sections in 1x sodium citrate buffer at 95 °C for 30 min, then gradually cooling to room temperature. After three rinses with phosphate-buffered saline with Tween 20 (PBST) (5 min each), sections were permeabilized and blocked with 10% normal goat serum containing 0.3% Triton X‑100 for 90 min at room temperature. Sections were then incubated with primary antibodies against p21/CIP1/CDKN1A (1:200) and Aβ₁‑₄₂ (1:400) at 37 °C for 2 h. Following four washes with PBST (5 min each), sections were incubated with species‑matched Alexa Fluor‑conjugated secondary antibodies (1:500) at 37 °C for 2 h in the dark. After additional washing (4 x 5 min in PBST), sections were mounted with DAPI‑containing antifade mounting medium and imaged using a fluorescence microscope.

Golgi-Cox staining

Golgi‑Cox staining was performed using a commercial kit according to the manufacturer’s instructions. Solutions A and B were mixed 24 h prior to the experiment. After mice were euthanized, brain tissues were rapidly removed, rinsed with PBS, and immersed in the mixture of Solutions A and B. Tissues were stored at room temperature in the dark for 6 h, after which the impregnation solution was replaced with fresh solution and incubation continued for an additional 2 weeks at room temperature in the dark (at least 2 mL of impregnation solution per brain tissue). Tissues were then transferred to Solution C and incubated for 24 h, then replaced with fresh Solution C and incubated for an additional 7 days at room temperature in the dark. Brain tissues were sectioned at 100 µm thickness using a vibratome. Sections were mounted onto gelatin‑coated slides and allowed to dry at room temperature for 3 days. Slides were rinsed twice with distilled water (5 min each), then immersed in a mixture of Solution D, Solution E, and distilled water (1:1:2 ratio) for 15 min. After two additional rinses with distilled water (5 min each), sections were dehydrated through graded ethanol solutions (50%, 75%, and 95%; 5 min each), followed by four rinses in absolute ethanol (4 min each) and three rinses in xylene (5 min each). Finally, sections were coverslipped with mounting medium. Images were captured with a microscope, and dendritic spine density was quantified using ImageJ.

Statistical analysis

All results were reported as mean ± SD. The data were analyzed and graphed using GraphPad Prism 8.1.0. The Student’s t-test was used to compare two groups. For comparisons involving multiple groups, a one-way ANOVA was used, with pairwise comparisons performed using Dunnett's test. Statistical significance was set at p < 0.05.

Multivariate and correlation analyses

Principal component analysis (PCA) was performed using GraphPad Prism 8.0.1 to visualize group-wise clustering of SASP factors (IL-1α, IL-1β, IL-6, IL-17A, IFN-γ, CCL3, CCL4, and CCL5) among the WT, 5xFAD, and QFY (2.12 g/kg/day) groups (n = 8 per group). The score plot was generated based on the first two principal components. No data transformation was applied prior to PCA. Pearson correlation analysis was conducted to evaluate linear relationships between SASP factors and synaptic proteins (PSD-95, GAP-43, and SYN), as well as Aβ₁₋₄₀, Aβ₁₋₄₂, and the Aβ₁₋₄₂/Aβ₁₋₄₀ ratio. The analysis was performed using GraphPad Prism, with a two-tailed significance threshold of p < 0.05. Correlation matrices and a heatmap were generated to visualize the strength and direction of associations.

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Results

Administration of QFY enhances learning and memory in 5xFAD mice

After QFY administration, a shuttle box test was conducted to assess the conditional active avoidance response of mice (Figure 2A–C). The results showed the number of active avoidances on days 1-4 (Figure 2A, p < 0.05) and the area under the curve (AUC) of active avoidance (Figure 2B, p ...

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Discussion

Neural synaptic plasticity plays a key role in cognitive function. In the brains of AD patients, Aβ deposits spread across different regions, directly causing synaptic structural damage, loss of excitatory synapses, and cognitive decline28. GAP-43 (a presynaptic membrane protein)29, PSD-95 (a scaffold protein located in the postsynaptic membrane)30, and SYN (a phosphoprotein associated with synaptic vesicles)31 play an im...

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Disclosures

The authors have no conflicts of interest to declare.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (82205078, 82374062), Shandong Provincial Natural Science Foundation (ZR2026MS1293, ZR2021QH157), Shandong Province Traditional Chinese Medicine Science and Technology Project (M20251730), Science and Technology Projects of Xizang Autonomous Region, China (XZ202601ZY0167), and Shandong Province Technology Innovation Guidance Program (Central Government-Guided Local Science and Technology Development Fund) (YDZX2023137), with the project titled 'Development and Industrialization Research of Tongli Enteric‑Coated Capsules as a Class 1 New Traditional Chinese Medicine.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Drugs
DonepezilShanghai Yuanye Technologylot.J15HB188725
MemantineShanghai Yuanye Technologylot. J21HB189391
Qi-fu-yinLunan Pharmaceutical Houpu Research and Development Centerlot. 2209001
Behavioral tests
CatWalk XTNoldusCatWalkXT 10.0 software
Grip strength meterBileadbiosciBLD-ZL-20
Morris water mazeShanghai Xinruan Information Technology Co., Ltd120cm
Shuttle boxShanghai Xinruan Information Technology Co., LtdXR-XC404
Antibodies
Anti-Aβ1-42 antibodyAbcamCat# ab201061
Anti-p21/CIP1/CDKN1A antibodyNovus BiologicalsCat# NBP3-15661
Assay Kits
Aβ1-40 ELISA kitThermo Fisher ScientificCat# KHB3482
Aβ1-42 ELISA kitThermo Fisher ScientificCat# KHB3442
Bio-Plex Pro Mouse Cytokine 23-plex Assay kitR&D SystemsCat# LXLBM23-1
GAP-43 ELISA kitJiangsu Enzyme Immunoassay IndustryCat# MM-47506M1
Golgi-Cox staining kitBeiJing Biolead Biology Sci & Tech CoCat# PK401
PSD-95 ELISA kitJiangsu Enzyme Immunoassay IndustryCat# MM-45953M1
Senescence β-Galactosidase Staining kitBeyotimeCat# C0602
Synaptophysin (SYN) ELISA kitJiangsu Enzyme Immunoassay IndustryCat# MM-46504M1
Chemicals & Reagents
Anti-rabbit and mouse IgG (secondary antibody)StarterCat# S0C1001
DAB chromogenStarterCat# S0C1001
Software
CatWalk XTNoldus Information TechnologyVersion 10.0
ImageJNational Institutes of Health (NIH)Version 1.53

References

  1. Joe E, Ringman JM. Cognitive symptoms of Alzheimer's disease: clinical management and prevention. BMJ. 2019;367:l6217.
  2. Scheff SW, et al. Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology. 2007;68(18):1501-8.
  3. Muralidar S, et al. Role of tau protein in Alzheimer's disease: the prime pathological player. Int J Biol Macromol. 2020;163:1599-617.
  4. Hardy JA, Higgins GA. Alzheimer's disease: the amyloid cascade hypothesis. Science. 1992;256(5054):184-5.
  5. Calsolaro V, Edison P. Neuroinflammation in Alzheimer's disease: current evidence and future directions. Alzheimers Dement. 2016;12(6):719-32.
  6. Campisi J. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell. 2005;120(4):513-22.
  7. Yao R. An in-silico assessment suggests the potential effects of goji (fruit of Lycium barbarum L.) against aging-related diseases. Food Med Homol. 2025;2(2):9420036.
  8. Campisi J, d'Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007;8(9):729-40.
  9. Salminen A, Kaarniranta K, Kauppinen A. ER stress activates immunosuppressive network: implications for aging and Alzheimer's disease. J Mol Med (Berl). 2020;98(5):633-50.
  10. Mirabella F, et al. Prenatal interleukin 6 elevation increases glutamatergic synapse density and disrupts hippocampal connectivity in offspring. Immunity. 2021;54(11):2611-31.e8.
  11. Baker DJ, Petersen RC. Cellular senescence in brain aging and neurodegenerative diseases: evidence and perspectives. J Clin Invest. 2018;128(4):1208-16.
  12. Lucas V, Cavadas C, Aveleira CA, Hook V. Cellular senescence: from mechanisms to current biomarkers and senotherapies. Pharmacol Rev. 2023;75(4):675-713.
  13. Bloss EB, et al. Evidence for reduced experience-dependent dendritic spine plasticity in the aging prefrontal cortex. J Neurosci. 2011;31(21):7831-9.
  14. Ye XS, et al. The food and medicine homologous Chinese medicine from Leguminosae species: a comprehensive review on bioactive constituents with neuroprotective effects on nervous system. Food Med Homol. 2025;2(2):9420033.
  15. Dickstein DL, Weaver CM, Luebke JI, Hof PR. Dendritic spine changes associated with normal aging. Neuroscience. 2013;251:21-32.
  16. Wang SY, et al. Qifu-Yin attenuates AGEs-induced Alzheimer-like pathophysiological changes through the RAGE/NF-kappaB pathway. Chin J Nat Med. 2014;12(12):920-8.
  17. Yang X, et al. Qi-fu-yin attenuated cognitive disorders in 5xFAD mice of Alzheimer's disease animal model by regulating immunity. Front Neurol. 2023;14:1183764.
  18. Xiao QY, et al. Effects of Qi-Fu-Yin on aging of APP/PS1 transgenic mice by regulating the intestinal microbiome. Front Cell Infect Microbiol. 2023;12:1048513.
  19. Chen K, et al. Safety and toxicity of Qi-Fu Yin, a classical traditional Chinese medicine prescription: a nonclinical research. J Appl Toxicol. 2025;45(9):1811-38.
  20. Wang L, Qiao P, Yue L, Sun R. Is Qi Fu Yin effective in clinical treatment of dementia? A meta-analysis of 697 patients. Medicine (Baltimore). 2021;100(5):e24526.
  21. Li H, et al. Identification of chemical components of Qi-Fu-Yin and its prototype components and metabolites in rat plasma and cerebrospinal fluid via UPLC-Q-TOF-MS. Evid Based Complement Alternat Med. 2021;2021:1995766.
  22. Lei X, et al. Integrating network pharmacology and component analysis to study the potential mechanisms of Qi-Fu-Yin Decoction in treating Alzheimer's disease. Drug Des Devel Ther. 2023;17:2841-58. doi:10.2147/DDDT.S402624.
  23. Takeshita H, et al. Modified forelimb grip strength test detects aging-associated physiological decline in skeletal muscle function in male mice. Sci Rep. 2017;7:42323.
  24. Caggiano V, et al. Midbrain circuits that set locomotor speed and gait selection. Nature. 2018;553(7689):455-60.
  25. Ehret B, et al. Population-level coding of avoidance learning in medial prefrontal cortex. Nat Neurosci. 2024;27(9):1805-15.
  26. Curdt N, et al. Search strategy analysis of Tg4-42 Alzheimer mice in the Morris water maze reveals early spatial navigation deficits. Sci Rep. 2022;12:5451.
  27. Deacon RMJ. Assessing nest building in mice. Nat Protoc. 2006;1(3):1117-9.
  28. Tu S, Okamoto SI, Lipton SA, Xu H. Oligomeric Aβ-induced synaptic dysfunction in Alzheimer's disease. Mol Neurodegener. 2014;9:48.
  29. Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci. 1997;20(2):84-91.
  30. Dore K, et al. PSD-95 protects synapses from β-amyloid. Cell Rep. 2021;35(9):109194.
  31. Kokotos AC, et al. Synaptophysin sustains presynaptic performance by preserving vesicular synaptobrevin-II levels. J Neurochem. 2019;151(1):28-37.
  32. Martin SB, et al. Synaptophysin and Synaptojanin-1 in Down syndrome are differentially affected by Alzheimer's disease. J Alzheimers Dis. 2014;42(3):767-75.
  33. Namioka N, et al. Oxidative stress and inflammation are associated with physical frailty in patients with Alzheimer's disease. Geriatr Gerontol Int. 2016;17(6):913-8.
  34. Kojima G, Liljas A, Iliffe S, Walters K. Prevalence of frailty in mild to moderate Alzheimer's disease: a systematic review and meta-analysis. Curr Alzheimer Res. 2017;14(12):1256-63.
  35. Dimri GP, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92(20):9363-7.
  36. Baker DJ, et al. Naturally occurring p16Ink4a-positive cells shorten healthy lifespan. Nature. 2016;530(7589):184-9.
  37. Zhang P, et al. Senolytic therapy alleviates Abeta-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer's disease model. Nat Neurosci. 2019;22(5):719-28.
  38. Ota H, Ogawa S, Ouchi Y, Akishita M. Protective effects of NMDA receptor antagonist, memantine, against senescence of PC12 cells: a possible role of nNOS and combined effects with donepezil. Exp Gerontol. 2015;72:109-16.
  39. Herdy JR, et al. Increased post-mitotic senescence in aged human neurons is a pathological feature of Alzheimer's disease. Cell Stem Cell. 2022;29(12):1637-52.e6.
  40. Hu Y, et al. Replicative senescence dictates the emergence of disease-associated microglia and contributes to Abeta pathology. Cell Rep. 2021;35(10):109228.
  41. Tchkonia T, et al. Cellular senescence and the senescent secretory phenotype: therapeutic opportunities. J Clin Invest. 2013;123(3):966-72.
  42. Guerrero A, De Strooper B, Arancibia-Cárcamo IL. Cellular senescence at the crossroads of inflammation and Alzheimer's disease. Trends Neurosci. 2021;44(9):714-27.
  43. Acosta JC, et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Biol. 2013;15(8):978-90.
  44. Wang B, Han J, Elisseeff JH, Demaria M. The senescence-associated secretory phenotype and its physiological and pathological implications. Nat Rev Mol Cell Biol. 2024;25(12):958-78.
  45. Gaikwad S, Senapati S, Haque MA, Kayed R. Senescence, brain inflammation, and oligomeric tau drive cognitive decline in Alzheimer's disease: evidence from clinical and preclinical studies. Alzheimers Dement. 2024;20(1):709-27.
  46. Si Z, Sun L, Wang X. Evidence and perspectives of cell senescence in neurodegenerative diseases. Biomed Pharmacother. 2021;137:111327.
  47. Chen X, et al. Senescence-like changes induced by expression of p21Waf1/Cip1 in NIH3T3 cell line. Cell Res. 2002;12(3-4):229-33.
  48. Engeland K. Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ. 2022;29(5):946-60.
  49. Choi I, et al. Autophagy enables microglia to engage amyloid plaques and prevents microglial senescence. Nat Cell Biol. 2023;25(7):963-74.

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