Abstract

Objective:

Alzheimer’s disease (AD) is often accompanied by motor dysfunction, impaired limb strength, and gut microbiota disturbances. This study aimed to evaluate the effects of Qifuyin (QFY), a traditional Chinese medicine formula, on motor deficits, limb strength, aging, and gut microbiota composition in 3xTg-AD mice, a widely used model of AD.

Methods:

Male and female 3xTg-AD mice were administered QFY at low, medium, or high doses. Motor function was assessed using grip strength and rotarod tests. Aging was evaluated through aging scores. Gut microbiota composition was analyzed at the phylum, family, genus, and species levels. Functional profiling of microbiota was performed using KEGG, eggNOG, and carbohydrate-active enzyme (CAZyme) databases. Pearson correlation analyses were conducted to explore relationships between microbiota composition and motor performance.

Results:

QFY treatment significantly improved both absolute and normalized grip strength in male and female 3xTg-AD mice. Similarly, motor coordination, as assessed by latency to fall on the rotarod, was significantly enhanced in the groups of QFY. Aging scores were significantly reduced after the treatment of QFY. Microbiome analysis revealed that QFY treatment restored species diversity and improved the overall composition of gut microbiota, with significant increases in Muribaculaceae and decreases in Alcaligenaceae, Rhodanobacteraceae, and Spirochaetaceae. Principal component analysis (PCA) indicated that the gut microbiota composition of the QFY group resembled that of the control (Con) group. Functional analyses showed that treatment of QFY restored microbial pathways related to metabolism and genetic information processing, with significant correlations between microbial alterations and improved motor outcomes. Additionally, QFY modulated the abundance of key carbohydrate-active enzymes, including GH43 and GH35, which were positively correlated with grip strength and rotarod performance.

Conclusion:

Qifuyin improves motor function, reduces aging-related deficits, and restores gut microbiota homeostasis in 3xTg-AD mice. These findings suggest that QFY may offer therapeutic potential for addressing frailty and motor dysfunction in AD, in association with alterations in gut microbiota composition and predicted microbial functions.

1 Introduction

AD is a progressively debilitating neurodegenerative disorder and is the leading cause of dementia worldwide, posing significant challenges to public health and socioeconomic systems (Mohapatra et al., 2025; Tiwari et al., 2023). The prevalence and incidence of AD continue to rise due to global aging trends. According to the Global Burden of Disease Study 2021, there were nearly 1.92 million deaths and 52.56 million cases of AD and other dementias among individuals aged 60 and older worldwide (Yu et al., 2025). By 2050, the number of AD cases is projected to quadruple to over 82 million, imposing enormous economic and healthcare burdens globally (Ramadan, 2023; Gareri et al., 2024). The primary pathological features of AD include the accumulation of amyloid-beta (Aβ) plaques and hyperphosphorylated tau protein forming neurofibrillary tangles, leading to neuronal damage and progressive cognitive decline (Long et al., 2023; Wang et al., 2023). Thus, there is an urgent need for effective strategies to slow disease progression and improve patient quality of life.

Beyond cognitive impairment, AD is frequently accompanied by non-cognitive symptoms, including decreased limb strength, impaired motor coordination, and general physical frailty, which severely impact daily living abilities and independence (Andrade-Guerrero et al., 2024; Marogianni et al., 2025). Growing evidence suggests that motor dysfunction may precede cognitive decline and serve as an early biomarker for AD progression (Oveisgharan et al., 2024; Petkus et al., 2025). For instance, reduced gait speed has been strongly correlated with increased Aβ deposition, diminished muscle strength, and balance impairment (Marogianni et al., 2025), while a higher burden of AD biomarkers in motor cortices correlates with poorer dexterity performance (Gupta et al., 2024). The presence of motor symptoms, particularly gait disturbances and slowness, has been shown to correlate with an increased risk of rapid cognitive decline (Shaw et al., 2025). These motor dysfunctions are rooted in core AD pathological changes such as neurodegeneration and neuroinflammation (Fakorede et al., 2025). Therefore, improving limb strength and motor coordination in AD patients, thus alleviating physical frailty, has become an essential aspect of daily living ability in comprehensive AD management.

In recent years, increasing attention has been directed toward the role of the gut microbiota in mediating these non-cognitive manifestations of AD. Gut dysbiosis has been shown to induce peripheral and central inflammation (Nayak et al., 2025), increase intestinal permeability (Deleemans et al., 2021), and influence microglial activation and synaptic function through the gut–brain axis (Chen et al., 2025), thereby affecting not only cognition but also motor regulation (Marasco et al., 2022). In parallel, accumulating evidence supports a gut–muscle axis, in which microbial metabolites such as short-chain fatty acids, bile acids, and tryptophan derivatives regulate skeletal muscle protein synthesis, mitochondrial function, and energy metabolism (Zhou et al., 2023). Disruption of these pathways contributes to sarcopenia, reduced grip strength, impaired motor coordination, and ultimately physical frailty. In AD, neurodegeneration, endocrine imbalance, and microbiota-driven systemic inflammation converge, forming a mechanistic continuum linking gut dysbiosis with both motor dysfunction and frailty (Abdol Samat et al., 2025). These findings suggest that interventions capable of remodeling the gut microbiota may exert beneficial effects on both central nervous system and peripheral motor function in AD.

Currently, pharmacological interventions play an important role in the management of physical frailty, yet their application remains limited due to the complex pathogenesis and frequent coexistence of multiple comorbidities. For example, drugs targeting AD—such as cholinesterase inhibitors (donepezil, rivastigmine, and galantamine), N-methyl-D-aspartate (NMDA) receptor antagonists (memantine) and Aβ antibody (lecanemab and donanemab)—are primarily designed to improve cognitive function. However, these agents mainly provide symptomatic relief and exert limited effects on disease modification (Khalil, 2018; Singh et al., 2016). With respect to physical frailty itself, although certain pharmacological agents have been tested to alleviate muscle weakness and related symptoms, the causal relationships between polypharmacy and frailty severity remain incompletely understood, and no drugs have been specifically approved for the treatment of frailty (Campbell and Szoeke, 2009). In addition, aging-related musculoskeletal deterioration, such as osteoporosis and sarcopenia, can be treated pharmacologically, yet these treatments focus mainly on single-organ pathologies and fail to address the multisystemic nature of physical frailty (Mahindran et al., 2021). Given the multifactorial and systemic complexity of frailty, current pharmacotherapies largely aim to mitigate symptoms or manage complications rather than fundamentally reverse or halt its progression. Therefore, there is an urgent need to develop novel therapeutic strategies capable of targeting the core mechanisms of frailty to achieve more comprehensive and effective interventions.

The Chinese herbal formula Qifuyin (QFY) is composed of Panax ginseng, Rehmannia glutinosa (prepared), Angelica sinensis, stir-baked Atractylodes macrocephala, Ziziphus jujuba var. spinosa, Polygala tenuifolia (processed), and honey-fried Glycyrrhiza uralensis. Clinical studies have reported that in a trial involving 20 patients with mild cognitive impairment (MCI), administration of Qifuyin alone for 8 weeks resulted in significant therapeutic efficacy as assessed by the Mini-Mental State Examination (MMSE) score (Xiao et al., 2011). A 12-week treatment with QFY combined with memantine hydrochloride in 40 Alzheimer’s disease patients produced marked improvements in the Mini-Mental State Examination (MMSE), Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-Cog), and Activities of Daily Living (ADL) scores, indicating that QFY enhanced the therapeutic efficacy of memantine (Tianchen, 2023). A 1-month treatment with QFY combined with Butylphthalide Soft Capsules produced significant improvements in MMSE and MoCA scores among 47 Alzheimer’s disease patients. These results point to its potential to enhance the therapeutic effect of Butylphthalide (Xiaotona, 2021). A meta-analysis, which incorporated 9 randomized controlled trials (RCTs) involving 697 patients, demonstrated that QFY therapy, either as a monotherapy or as an adjunct to conventional Western medications, resulted in significantly enhanced cognitive function in patients with dementia (Wang et al., 2021). However, despite these encouraging findings, existing evidence is predominantly centered on cognitive outcomes, and the potential effects of QFY on physical frailty and motor dysfunction remain largely unexplored. Although we had found that QFY improved the ability of motor coordination, raised survival rate and prolonged the survival days under cold stress stimulation in aged APP/PS1 transgenic mice (Xiao et al., 2022), whether such effects are reproducible in other AD models, particularly those exhibiting both amyloid and tau pathology, and whether they are linked to modulation of the gut microbiota, remains unknown. In the present study, we specifically focused on physical frailty in Alzheimer’s disease. Although various AD mouse models have been widely used to explore cognitive impairment, frailty as a systemic age-related decline has received far less attention in experimental AD research. The 3xTg-AD mouse model develops both amyloid-β and tau pathology in an age-dependent manner, closely mimicking core neuropathological features of AD. However, physiological frailty per se has not been systematically evaluated in this model, and its relationship with gut microbiota remains largely unexplored. We therefore selected 3xTg-AD mice in order to investigate whether QFY could ameliorate AD-related frailty and motor dysfunction, and to determine whether these effects are associated with modulation of the gut microbiota.

2 Materials and methods2.1 The preparation of QFY

The QFY dry extract powder was purchased from Lunan Pharmaceutical Group Corporation. The specific preparation method is as follows: weigh out 3.0 kg of ginseng (Panax ginseng C. A. Mey.), 4.50 kg of prepared rehmannia root [Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. & C. A. Mey.], 4.50 kg of angelica [Angelica sinensis (Oliv.) Diels], 2.50 kg of stir-fried Atractylodes macrocephala (Atractylodes macrocephala Koidz.), 3.0 kg of sour jujube seed [Ziziphus jujuba var. spinosa (Bunge) Hu ex H.F.Chow.], 2.50 kg of processed polygala (Polygala tenuifolia Willd.), and 1.50 kg of honey-fried licorice (Glycyrrhiza uralensis Fisch). First, perform heat reflux on the ginseng with 60% ethanol twice, each for 1.5 h. Filter the mixture, set aside the residue, recover the ethanol from the filtrate, and concentrate it to a relative density of 1.03–1.9 (at 60 °C), and set it aside. Next, extract the volatile oil from angelica and stir-fried Atractylodes macrocephala using water distillation, collect the distilled aqueous solution in a separate container, and set aside the residue. The volatile oil ethanol solution is encapsulated with beta-cyclodextrin, dried, and pulverized for later use. Perform boiling of the residues from the above three herbs along with the remaining four herbs (prepared rehmannia root, etc.) in water twice, each time for 2 h. Filter the mixture and mix the filtrate with the concentrated ginseng solution. Perform concentration of the mixture to a relative density of 1.02–1.06 (at 60 °C) to obtain a clear syrup, let it stand, centrifuge, and then concentrate to a relative density of 1.22–1.28 (at 60 °C) to obtain a dense extract. Dry and pulverize the extract, then mix it with the beta-cyclodextrin encapsulated substance.

2.2 Animals and treatment

The 3xTg-AD transgenic mice [strain B6;129-Tg (APPSwe,tauP301L)1Lfa Psen1 tm1Mpm/Mmjax], carrying three mutations associated with familial Alzheimer’s disease (APP Swedish, MAPT P301L, and PSEN1 M146V), were purchased from the Jackson Laboratory. The C57BL/6J mice were purchased from Beijing Huafukang Bioscience Co., Ltd. A total of 111 mice were included in the study, comprising 53 males and 58 females. Both the C57BL/6J and 3xTg-AD transgenic mice were housed at the Experimental Animal Center of Shandong University of Traditional Chinese Medicine until they reached 10.3 months of age. All animals were maintained at a temperature of 23 ± 1 °C under a 12-h light/dark cycle with free access to food and water. Before the experiments, all mice were acclimated to the experimental environment for 6 days. All animal-related experiments have been reviewed and approved by the ethics committee of Shandong University of Traditional Chinese Medicine (Ethics No. SDUTCM202209291). All efforts were taken to minimize the number of animals used and their suffering. The 10.3-month-old C57BL/6J and 3xTg-AD transgenic mice were divided into six groups based on activity level and body weight. Each group consisted of 8–9 mice, and the treatment was administered via oral gavage: Con group (male 10, female 10) is C57BL/6J mice, Model (Mod) group (male 7, female 7) is 3xTg-AD transgenic mice, positive drug group (male 8, female 9) is 3xTg-AD + donepezil (1.0 mg/kg/day) and memantine (2.8 mg/kg/day), QFY low-dose group (male 8, female 10) is 3xTg-AD + QFY (1.06 g/kg/day), QFY medium-dose group (male 7, female 10) is 3xTg-AD + QFY (2.12 g/kg/day), QFY high-dose group (male 7, female 9) is 3xTg-AD + QFY (4.24 g/kg/day), The Con and Mod groups were administered distilled water by gavage for the duration of the study. All mice underwent behavioral tests following 305 days of treatment, and samples were collected for biochemical analysis after 328 days of treatment.

2.3 Grip strength test

The grip strength meter was placed horizontally and set to the grip strength measurement mode. Each mouse was gently placed on the grid of the apparatus, allowing it to grasp the mesh firmly with all four limbs. The mouse was then gently pulled backward by the tail in a horizontal direction until its forelimbs and hindlimbs released the grid. At this point, the instrument automatically recorded the maximal grip force. Each mouse was measured three consecutive times with a 30-s interval between trials. The mean value of the three measurements was taken as the grip strength of the mouse. To account for body weight differences, the results were normalized using the following formula: normalized grip strength = Mean grip strength/Body weight.

2.4 Rotarod test

Mice were first subjected to adaptive training on a rotarod apparatus. The training program was set to accelerate uniformly to 10 rpm within 20 s and then maintained for 280 s. Six mice were placed simultaneously in the six lanes of the rotarod apparatus. Before the start of training, each mouse was allowed to adapt on the rod for 30 s. After adaptation, the preset program was initiated until the training ended or the mouse fell off. Twenty-four hours after the adaptive training, the formal test was conducted. The program was set to accelerate uniformly to 15 rpm within 45 s and then maintained for 155 s. Six mice were placed simultaneously in the six lanes of the apparatus. Each mouse was allowed to adapt on the rod for 30 s prior to the start of the test. After adaptation, the preset program was initiated. During the test, a trial was considered terminated when the mouse fell off the rod or clung to the rod and rotated passively for three consecutive turns. The apparatus automatically recorded the latency to fall (time spent on the rod). Each mouse underwent three trials with a 30-min inter-trial interval to allow sufficient rest. The average latency across the three trials was taken as the final result, which was further normalized to body weight.

2.5 Aging score assessment

The aging assessment was conducted based on the scoring system originally established by Professors Toshio Takeda and Masanori Hosokawa at Kyoto University, combined with our group’s previous research on aging evaluation. A multidimensional aging scale was developed incorporating both behavioral and morphological characteristics of mice. The assessment included four domains: behavioral responses, skin and hair condition, ocular condition, and spinal condition. Each parameter was graded on a five-point scale (1–5), with higher scores indicating more severe aging, and a score of 5 representing the most advanced aging state.

2.6 Sample collection

Fecal samples from each mouse were collected directly after spontaneous excretion into sterile EP tubes, sealed with parafilm, immediately snap-frozen in liquid nitrogen, and subsequently stored at −80 °C until further analysis.

2.7 16 srRNA

DNA extraction: Approximately 0.25 g of sample was placed into a 2-ml centrifuge tube, followed by the addition of 500 μl Buffer SA and 100 μl Buffer SC. Eight 3-mm grinding beads and 0.2 g of 1-mm grinding beads were then added, and the mixture was homogenized using a TGrinder H24 tissue homogenizer (TIANGEN, OSE-TH-01) under the following conditions: oscillation at 6 m/s for 20 s, with a 10-s interval, for a total of two cycles. The homogenate was then incubated at 70 °C for 15 min for lysis. The lysate was centrifuged at 12,000 rpm (∼13,400 × g) for 1 min, and approximately 500 μl of the supernatant was transferred into a new 2-ml centrifuge tube. Subsequently, 200 μl Buffer SH was added, vortexed for 5 s, and incubated at 4 °C for 10 min. After centrifugation at 12,000 rpm for 2 min at room temperature, the vacuum-sealed, pre-packed 96-deep well plate from the kit was mixed by inversion several times to resuspend the magnetic beads. After removing the vacuum package, the plate was gently tapped to collect all reagents and beads at the bottom (alternatively, centrifuged at 500 rpm for 1 min). The aluminum sealing film was carefully removed prior to use to avoid spillage. Automated extraction: The TGuide S96 automated nucleic acid extraction and purification system (TIANGEN) was used to run the soil/fecal genomic DNA extraction program according to the manufacturer’s protocol. DNA quality control: The extracted nucleic acids were eluted in 35–50 μl TB buffer and stored at −20 °C until further use. DNA concentration was measured using a Qubit 3.0 fluorometer (Invitrogen) with the Qubit dsDNA HS Assay Kit, and DNA integrity was evaluated by 1% agarose gel electrophoresis.

2.8 Metagenomic sequencing

Library preparation was performed using the VAHTS® Universal Plus DNA Library Prep Kit for Illumina (ND617) according to the manufacturer’s instructions. Library quality was assessed using a Qsep-400 system for fragment analysis, and library concentration was quantified with a Qubit 3.0 fluorometer. Libraries meeting the following criteria were subjected to sequencing: concentration ≥ 1 ng/μl, fragment size distribution with a central peak of 430–530 bp and an average size of 420–580 bp, a normal peak shape, and no detectable secondary peaks. Qualified libraries were sequenced on the Illumina NovaSeq 6000 platform using a paired-end 150 bp (PE150) strategy. Metagenomic sequencing and data processing: metagenomic libraries were constructed using the VAHTS® Universal Plus DNA Library Prep Kit for Illumina (ND617) according to the manufacturer’s protocol. Library quality was evaluated using a Qsep-400 system for fragment size distribution, and library concentration was quantified with a Qubit 3.0 fluorometer. Libraries meeting the following quality control thresholds were selected for sequencing: concentration ≥ 1 ng/μl, fragment size distribution with a central peak of 430–530 bp and an average size of 420–580 bp, unimodal and normally distributed peak shape, and absence of secondary peaks. Qualified libraries were sequenced on the Illumina NovaSeq 6000 platform with a paired-end 150 bp (PE150) strategy. Raw sequencing reads were first subjected to quality control using FastQC and Trimmomatic to remove low-quality bases and adapter sequences. Host-derived sequences were filtered by aligning reads to the mouse reference genome (GRCm39) using Bowtie2, and non-host reads were retained for downstream analysis. High-quality clean reads were assembled into contigs with MEGAHIT, and open reading frames (ORFs) were predicted using Prodigal. Non-redundant gene catalogs were constructed with CD-HIT, and functional annotation was performed against the KEGG, eggNOG, and CAZy databases using DIAMOND. Taxonomic classification was carried out using Kraken2 with the NCBI RefSeq database. Relative abundances of taxa and functional categories were calculated based on mapped read counts.

3 Results3.1 QFY enhanced the limb strength of 3xTg-AD mice

Correlation analysis revealed a positive association between body weight and absolute grip strength (Figure 1D, P < 0.0001). The stronger correlation observed for normalized grip strength suggests that the weight-adjusted measure provides a more reliable indicator of neuromuscular function.

QFY enhanced the limb strength of 3xTg-AD mice. (A) Grip strength of male mice. (B) Grip strength of female mice. (C) Combined grip strength of male and female mice. (D) Correlation between grip strength and body weight. (E) Grip strength/body weight in male mice. (F) Grip strength/body weight in female mice. (G) Combined grip strength/body weight in male and female mice. Mean ± SD, n = 7–20, *P < 0.05, **P < 0.01 vs. Con, Student‘s t-test, #P < 0.05, ##P < 0.01 vs. Mod, One-way ANOVA followed by Dunnett’s multiple comparisons test, Graphpad 8.0.1.

In male mice, the Mod group exhibited a significant weaker in absolute grip strength (Figure 1A, P < 0.01) and in normalized grip strength (Figure 1E, P < 0.01) relative to Con. Compared with the Mod group, the positive drug group showed significant strength in both absolute grip strength (Figure 1A, P < 0.05) and normalized grip strength (Figure 1E, P < 0.01). The administration of QFY at a low dose (1.06 g/kg) significantly improved normalized grip strength (Figure 1E, P < 0.01). The medium dose of QFY (2.12 g/kg) further enhanced both absolute and normalized grip strength (Figures 1A, E, P < 0.01), while the high dose (4.24 g/kg) significantly improved normalized grip strength (Figure 1E, P < 0.01).

In female mice, the Mod group showed significantly decreased absolute grip strength (Figure 1B, P < 0.01) and normalized grip strength (Figure 1F, P < 0.05) compared with Con. Relative to the Mod group, the positive drug group significantly increased absolute grip strength (Figure 1B, P < 0.01) and normalized grip strength (Figure 1F, P < 0.05). Low-dose QFY significantly improved absolute grip strength (Figure 1B, P < 0.01). The medium dose significantly enhanced both absolute and normalized grip strength (Figure 1B, P < 0.01; Figure 1F, P < 0.05), and the high dose significantly enhanced the absolute grip strength (Figure 1B, P < 0.01).

When data from male and female mice were combined, the Mod group displayed significantly lower absolute grip strength (Figure 1C, P < 0.01) and normalized grip strength (Figure 1G, P < 0.01) than the Con group. Compared with the Mod group, the positive drug group significantly improved both absolute grip strength (Figure 1C, P < 0.01) and normalized grip strength (Figure 1G, P < 0.01). The treatment of QFY at low, medium, and high doses significantly increased both absolute and normalized grip strength (Figures 1C, G, P < 0.01). In a comparison of the two sexes, male mice in the medium-dose QFY group had significantly superior grip strength.

3.2 QFY improved the motor coordination ability of 3xTg-AD mice

Correlation analysis revealed no significant relationship between body weight and rotarod performance, as assessed by absolute latency to fall (Figure 2D, P > 0.05), indicating that the motor coordination deficits in 3xTg-AD mice were independent of body weight.

QFY improved the motor coordination ability of 3xTg-AD mice. (A) Latency to fall in male mice. (B) Latency to fall in female mice. (C) Combined rotating time of male and female mice. (D) Correlation between latency to fall and body weight. Mean ± SD, n = 7–20, **P < 0.01 vs. Con, Student‘s t-test, #P < 0.05, ##P < 0.01, vs. Mod, One-way ANOVA followed by Dunnett’s multiple comparisons test, Graphpad 8.0.1.

In male mice, the Mod group exhibited a significantly shorter the latency to fall on the rotating rod compared to Con (Figure 2A, P < 0.01). Compared with the Mod group, the treatment of positive drug significantly prolonged the latency to fall (Figure 2A, P < 0.01). Administration of QFY at low, medium, and high doses also significantly increased latency to fall relative to the Mod group (Figure 2A, P < 0.01).

In female mice, the 3xTg-AD Mod group showed a significant decrease in latency to fall compared with Con (Figure 2B, P < 0.01). The positive drug group demonstrated a significant extension in latency to fall relative to the Mod group (Figure 2B, P < 0.01). Both low- and medium-dose QFY significantly increased latency to fall (Figure 2B, P < 0.01), while high-dose QFY also resulted in a significant improvement (Figure 2B, P < 0.05).

When data from both sexes were combined, 3xTg-AD mice displayed significantly shorter latency to falls than Con (Figure 2C, P < 0.01). Compared to the Mod group, the positive drug group showed a significant increase in latency to fall (Figure 2C, P < 0.01). Similarly, the treatment of QFY at all tested doses significantly improved latency to fall (Figure 2C, P < 0.01).

No significant differences were observed between males and females across all groups.

3.3 QFY reduced the aging score of 3xTg-AD mice

In male mice, the 3xTg-AD group exhibited a significantly higher aging degree score compared to Con (Figure 3A, P < 0.05). Relative to the Mod group, the positive drug group, the low- and medium-dose QFY groups showed significant reductions in aging degree scores (Figure 3A, P < 0.01).

QFY reduced the aging score of 3xTg-AD mice. (A) Aging score in male mice. (B) Aging score in female mice. (C) Combined aging score of male and female mice. Mean ± SD, n = 7–20, *P < 0.05, **P < 0.01 vs. Con, Student‘s t-test, #P < 0.05, ##P < 0.01 vs. Mod, One-way ANOVA followed by Dunnett’s multiple comparisons test, Graphpad 8.0.1.

In female mice, aging degree score were also markedly elevated in the 3xTg-AD group relative to Con (Figure 3B, P < 0.01). Compared with the Mod group, the treatment of positive drug significantly lowered the aging degree score (Figure 3B, P < 0.01). The medium dose resulted in a significant decrease (Figure 3B, P < 0.05).

When data from both sexes were pooled, the 3xTg-AD group continued to demonstrate a significantly higher aging degree score than the C57 group (Figure 3C, P < 0.01). Compared to the Mod group, the positive drug group showed a significant reduction in aging degree score (Figure 3C, P < 0.05). Both low- and medium-dose QFY markedly reduced aging degree scores (Figure 3C, P < 0.01). A comparison between the sexes revealed a significantly lower aging score in females compared to males.

3.4 The treatment of QFY improved the species diversity of gut microbiota in 3xTg-AD mice

At the phylum level, the Con and QFY groups shared one common taxon, whereas the Con and Mod groups shared none (Figure 4A). At the genus level, the Con and QFY groups shared 22 taxa, compared with only 11 taxa shared between the Con and Mod groups (Figure 4E). At the species level, 26 taxa were common to the Con and QFY groups, whereas only 15 taxa were shared between the Con and Mod groups (Figure 4F). We further observed that, at taxonomic levels below the phylum, species richness in 3xTg-AD Mod mice exceeded that of Con; following QFY treatment, species number decreased and became more comparable to that of the Con group (Figures 4A–F). These results indicate that QFY exerts a restorative regulatory effect on the gut microbiota composition of 3xTg-AD mice.

The treatment of QFY improved the species diversity of gut microbiota in 3xTg-AD mice. (A) Phylum-level species count. (B) Class-level species count. (C) Order-level species count. (D) Family-level species count. (E) Genus-level species count. (F) Species-level species count.

3.5 QFY modulates gut microbiota composition and β-diversity at the family level in 3xTg-AD mice

At the family level, the top ten dominant taxa across the three groups were Muribaculaceae, Lachnospiraceae, Lactobacillaceae, Prevotellaceae, Bacteroidaceae, Erysipelotrichaceae, Helicobacteraceae, Streptococcaceae, Rikenellaceae, and Ruminococcaceae (Figure 5A). The hierarchical clustering heatmap further revealed inter-individual variations in the composition of dominant taxa (Figure 5B). At the family level, compared to the Con group, the Mod group exhibited significantly reduced levels of Muribaculaceae (Figure 5C, P < 0.01), while Lactobacillaceae (Figure 5D, P < 0.05), Helicobacteraceae (Figure 5F, P < 0.05), Erysipelotrichaceae (Figure 5G, P < 0.01), Bacteroidaceae (Figure 5H, P < 0.05) levels were significantly increased. Notably, following QFY treatment, the levels of Muribaculaceae (Figure 5C, P < 0.01) were significantly increased, whereas Erysipelotrichaceae (Figure 5C, P < 0.05) and Bacteroidaceae (Figure 5H, P < 0.01) were significantly decreased. However, no significant differences were observed in Prevotellaceae levels between the Mod and Con groups, nor between the QFY treatment group and the model group (Figure 5E). Pearson correlation analysis revealed that Muribaculaceae showed a significant positive correlation with grip strength normalized to body weight and latency to fall, whereas Bacteroidaceae exhibited a significant negative correlation with grip strength normalized to body weight (Table 1). These findings indicate that QFY effectively modulates the gut microbiota composition at the family level in 3xTg-AD mice. Principal component analysis (PCA) (Figure 5I) revealed that the β-diversity of the QFY group was similar to that of the Con group, whereas the Mod group exhibited significantly lower β-diversity compared to both Con and QFY groups. Statistical analysis of PCA scores revealed that, compared with the Con group, the Mod group exhibited a significant reduction in scores, whereas the QFY group showed a significant increase (Figure 5J).

QFY modulates gut microbiota composition and β-diversity at the family level in 3xTg-AD mice. (A) Relative abundance of gut microbiota at the family level in Con, Mod, and QFY groups. The stacked bar plot illustrates the proportion of different bacterial families, with colors representing various bacterial families. (B) Heatmap of the relative abundance of gut microbiota at the family level. The samples are clustered based on their microbial profiles, with distinct groupings for Con, Mod, and QFY samples. (C) Relative abundance of Muribaculaceae in the Con, Mod, and QFY groups. (D) Relative abundance of Lachnospiraceae in the Con, Mod, and QFY groups. (E) Relative abundance of Lactobacillaceae in the Con, Mod, and QFY groups. (F) Relative abundance of Prevotellaceae in the Con, Mod, and QFY groups. (G) Relative abundance of Helicobacteraceae in the Con, Mod, and QFY groups. (H) Relative abundance of Erysipelotrichaceae in the Con, Mod, and QFY groups. (I) Relative abundance of Bacteroidaceae in the Con, Mod, and QFY groups. (J) PCA plot illustrating the variation of gut microbiota profiles among the Con (green), Mod (blue), and QFY (orange) groups. The percentage of variance explained by the first two principal components (PC1 and PC2) is shown. (K) PCA scores for the Con, Mod, and QFY groups, with statistical significance indicated between groups. *P < 0.05, **P < 0.01 vs. Con, #P < 0.05, ##P < 0.01, vs. Mod, Student‘s t-test.

NameGrip strength/weight in grip strength testLatency to fall in rotarod testMuribaculaceaer = 0.6189
P < 0.0001**r = 0.5045
P = 0.0007**Lactobacillaceaer = −0.1232
P = 0.4372r = −0.2035
P = 0.1961Prevotellaceaer = 0.3003
P = 0.0533r = 0.07879
P = 0.6199Helicobacteraceaer = −0.2225
P = 0.1566r = −0.2218
P = 0.1581Erysipelotrichaceaer = −0.2368
P = 0.1310r = −0.1424
P = 0.3684Bacteroidaceaer = −0.3604
P = 0.0190*r = −0.1278
P = 0.4198

Correlation between microbial species abundance and frailty phenotypes in 3xTg-AD mice.

*P < 0.05, **P < 0.01, Pearson correlation analysis, Graphpad 8.0.1.

3.6 QFY modulates kEGG-associated gut microbial functions in 3xTg-AD mice

We analyzed the KO, KeggPathway3, and enzyme levels using the KEGG database (Figure 6). In this study, KeggPathway1, KeggPathway2, and KeggPathway3 refer to KEGG’s hierarchical classification of functional pathways, from broad categories (Level 1) to subcategories (Level 2) and specific metabolic or signaling pathways (Level 3), respectively. The results showed that at the KO level, the Con group and the Mod group shared 82 functions, while the Con group shared 338 functions with the QFY group (Figure 6A). At the KeggPathway3 level, the Con and Mod groups had no shared functions, but the Con group shared one function with the QFY group (Figure 6B). At the enzyme level, the Con and Mod groups shared 29 functions, whereas the Con group shared 72 functions with the QFY group (Figure 6C).

The effect of QFY on the function of intestinal microorganisms in 3xTg-AD mice based