Di Huang Yi Zhi Fang improves cognitive function in APP/PS1 mice by inducing neuronal mitochondrial autophagy through the PINK1-parkin pathway

Abstract

Background

Alzheimer's disease (AD) is an irreversible age-related neurodegenerative condition characterized by the deposition of amyloid-β (Aβ) peptides and neurofibrillary tangles. Di Huang Yi Zhi (DHYZ) formula, a traditional Chinese herbal compound comprising several prescriptions, demonstrates properties that improve cognitive abilities in clinical. Nonetheless, its molecular mechanisms on treating AD through improving neuron cells mitochondria function have not been deeply investigated.

Objective

This study administered DHYZ to APP/PS1 mice to explore its potential therapeutic mechanisms in AD treatment.

Methods

APP/PS1 transgenic mice were given DHYZ (L, M, H), donepezil, or distilled water for a consecutive 12-week period. The Morris water maze test was used to assess memory capacity, transmission electron microscopy was used to observe mitochondrial and synaptic structures, immunohistochemistry and western blot detected proteins involved in the mitochondrial autophagy pathway, ELISA measured serum Aβ content, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assessed neuronal cell apoptosis.

Results

DHYZ demonstrates a notable therapeutic impact on mice with AD, effectively improving cognitive and memory impairments. DHYZ decreases Aβ accumulation in the hippocampus by reducing BACE1 activity and enhancing Aβ clearance through the blood-brain barrier. Additionally, DHYZ significantly suppresses neuronal apoptosis, enhances synaptic structure, and increases synapse numbers, processes strongly linked to the activation of mitochondrial PINK1-Parkin autophagy.

Conclusions

DHYZ enhances cognitive function in APP/PS1 mice by stimulating neuronal mitochondrial autophagy through the PINK1-Parkin pathway.

Keywords

Alzheimer's disease amyloid-β autophagy Di Huang Yi Zhi mitochondrial

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder affecting the central nervous system, characterized by high morbidity, disability, and mortality rates, accompanied by symptoms like anxiety, depression, and impaired daily functioning.¹ Currently, it is estimated that over 40 million people worldwide are affected by AD. With a continuously aging population, the incidence rate of AD is expected to increase significantly.^2–5 Currently available medications for treating AD include lecanemab,⁶ donepezil,⁷ rivastigmine,⁸ and so on. However, current pharmaceutical interventions may lead to side effects that compromise human health.⁹ Therefore, the pursuit of a safe and effective treatment approach that directly targets neuroprotection is crucial.

Mitochondria are vital, dynamic organelles crucial for energy production, essential in cell death, apoptosis, reactive oxygen species, and calcium regulation.¹⁰ The impairment of mitochondrial function is closely linked to neuronal dysfunction,¹¹ while mitophagy plays a crucial role in maintaining mitochondrial quality.¹² Studies have shown that the administration of mitophagy inducers to APP/PS1 mice enhances the phagocytic activity of microglial cells, which could be vital in clearing Aβ plaques outside neurons. Additionally, mitophagy could inhibit tau phosphorylation, thereby improving neurofibrillary tangles-induced neuronal dysfunction.¹³ Besides, tau inhibits mitophagy in neuronal systems of mouse neuroblastoma cells by sequestering Parkin in the cytoplasm, accumulating dysfunctional mitochondria and aggravating the disease's progression.¹⁴ The results mentioned above indicate that impaired mitophagy plays a significant role in the pathogenesis of AD. Therefore, targeting mitochondrial autophagy and removing Aβ plaques is crucial to enhance the effectiveness of AD therapy.

Traditional Chinese Medicine (TCM) plays a vital role in both preventing and treating AD, renowned for its gentle approach and minimal side effects on the human body. Harnessing the strengths of TCM, we have developed a Chinese herbal formulation known as Di-Huang-Yi-Zhi (DHYZ). The DHYZ formula comprises Shu-Di (prepared root of Rehmannia glutinosa), Yi-Zhi-Ren (fruits of Alpinia oxyphylla Miq.), Shi-Chang-Pu (root of Acorus tatarinowii Schott), Fu-Shen (Poria with hostwood), and Dan-Shen (root of Salvia miltiorrhiza Bunge) (Chinese patent ZL2008102047153.3). It has been clinically validated for its effectiveness in managing AD. Besides, the therapeutic mechanism for AD has been investigated and confirmed at the neuronal cell level. For instance, DHYZ can decrease the excessive phosphorylation of the tau protein in the neurons of rats with dementia induced by Aβ_1–40, while also regulating the abnormal expression of apoptosis-related proteins.¹⁵ Besides, DHYZ has the ability to safeguard PC12 cells from Aβ-induced reduction in mitochondrial membrane potential and prevent cell apoptosis. From the aforementioned research, it is evident that DHYZ functions to improve mitochondrial quality and reduce neuronal apoptosis. Nonetheless, the molecular mechanism underlying its enhancement of mitochondria remains inadequately investigated, which will be our study's central focus.

This study explored the impact of DHYZ on the behavior and cognitive function of individuals with AD. Additionally, it conducted an in-depth investigation into how DHYZ enhances neuronal cell mitochondrial function in AD. The results demonstrated that DHYZ significantly enhanced the behavioral and cognitive abilities of mice with AD, lowered Aβ levels and deposition, inhibited neuronal apoptosis, improved synaptic function, and enhanced mitochondrial quality by activating the mitochondrial autophagy pathway (PINK1-Parkin pathway) to eliminate damaged mitochondria, thus safeguarding cells from oxidative stress damage (Figure 1). These results demonstrate that DHYZ emerges as a promising supplement to TCM treatments for the management and delay of AD progression by enhancing mitochondrial function.

Figure 1.

DHYZ could effectively improve the behavior and cognitive function of AD mice.

Methods

DHYZ preparation

In the preparation of Di Huang Yi Zhi Fang, Acorus calamus and Alpinia oxyphylla were soaked in water at 8 times their weight, followed by heating and refluxing for 8 h. The volatile oil and filtrate were then collected while preserving the residue. Radix rehmanniae praeparata, Cornus officinalis, Salviae miltiorrhizae, and Poria cocos were decocted twice with the Acorus calamus and Alpinia oxyphylla residue for 1 h each time. The two filtrates along with the extraction filtrate of Acorus calamus and Alpinia oxyphylla were consolidated, concentrated, and supplemented with 50% ethanol. Subsequently, the ethanol was retrieved, and the concentration was adjusted to a relative density of 1.240–1.270 at 60°C, followed by vacuum drying and pulverization to produce a dry extract. As per the volatile oil quantity, β-cyclodextrin was weighed at six times the amount, dispersed in double the volume of water, gradually added to the volatile oil, dispersed, and dried to produce the volatile oil inclusion complex. The powdered Tortoise-shell glue was combined with the mentioned dry extract and volatile oil inclusion complex to create a brown powder of the Chinese herbal extract. Each gram of the extract powder contains 3.52 g of raw herbs and is stored at −20°C Table 1.

Table 1.

Di Huang Yi Zhi.

English name	Chinese name	Amount required (g)
Rehmannia glutinosa Libosch	Shu Di Huang	15
Dogwood	Shan Zhu Yu	9
Salvia miltiorrhiza	Dan Shen	15
Acorus gramineus	Shi Chang Pu	9
Tortoise-shell glue	Gui Jia Jiao	3
Poria cum Ligno Hospite	Fu Shen	9
Alpinia oxyphylla	Yi Zhi	6

Animal experiments and methods

The animal feeding and care were conducted by YouShu Life Science & Technology (Shanghai) Co., Ltd (Permission Number YS-m202305001). Sixty healthy SPF male mice, consisting of fifty APP/PS1 mice (C57BL/6J background, the gene identification report can be found in the Supplemental Material) and ten C57BL/6J mice aged 3 months, were procured from Shanghai Southern Model Biotechnology Company and housed in a facility with controlled temperature (20−23°C) and humidity (40−70%). After a 2-week acclimation period, the mice were randomly assigned to six groups: WT: C57BL/6J mice receiving distilled water (n = 10); AD: APP/PS1 transgenic mice receiving distilled water (n = 10); Donepezil: APP/PS1 transgenic mice receiving 0.65 mg/kg body weight (bw) donepezil hydrochloride (n = 10); DHYZ-L: APP/PS1 transgenic mice receiving 1.6 g/(kg bw) DHYZ (n = 10); DHYZ-M: APP/PS1 transgenic mice receiving 3.2 g/(kg bw) DHYZ (n = 10); DHYZ-H: APP/PS1 transgenic mice receiving 6.4 g/(kg bw) DHYZ (n = 10). Drug treatment was administered daily for a consecutive 12-week period. With advancing age in APP/PS1 mice, senile plaques resembling those seen in AD patients develop in their brains. Notably, clear AD symptoms were detected in our AD group after 12 weeks, marking the successful establishment of the model.

On the last day, all mice underwent a 12-h fast with free access to water, then the mice underwent eye blood sampling, the samples were centrifuged at 3500 g for 15 min at 4°C to isolate the serum, which was then stored at −80°C for future use. Subsequently, the mice were anesthetized with chloralhydrate, and they underwent immediate cardiac perfusion with a 0.9% saline solution, followed by perfusion with 4% paraformaldehyde in 0.1 M PBS (pH 7.4). Subsequently, the brains were excised, and the hippocampal tissue was dissected from them.¹⁶ The hippocampal tissue of some mice (n = 3) was fixed in paraformaldehyde for 24 h for immunohistochemistry (IHC). For the other mice (n = 3), the hippocampal tissue was sectioned into approximately 1 mm³ pieces and preserved in 2.5% glutaraldehyde for transmission electron microscopy (TEM). The hippocampal tissue of the remaining mice (n = 4) was stored in liquid nitrogen for western blotting analysis.

Morris water maze

The Morris water maze method was utilized to assess the orientation navigation and spatial exploration experiments twelve weeks post the drug intervention. Each test spanned seven consecutive days, preceded by an adaptation test the day prior, with the orientation navigation experiment conducted over the ensuing six days. The circular pool, divided into four quadrants, had water maintained at a temperature of 26 ± 1°C, with the platform positioned 1 cm below the water surface. Upon completion of the navigation experiment, the platform was removed for the spatial exploration phase. Animals were individually placed in the quadrant where the original platform was positioned, and their swimming paths were monitored for 90 s. The total swimming distance during the latency period, as well as the frequency of crossing the platform within 90 s, were recorded for each mouse over six days of training.

Transmission electron microscopy

The hippocampal tissues from the aforementioned mice were fixed in a solution containing 2.5% glutaraldehyde and 1% osmium tetroxide at 4°C for 2 h. Subsequently, the tissues underwent dehydration using ethanol gradients of 50%, 70%, and 90%. After dehydration, the tissues were embedded in epoxy resin and sectioned into ultra-thin slices of 90 nm. These slices were then stained with 2% uranyl acetate for 5 min followed by 2% lead citrate for 15 min. The synapses and mitochondria in the mouse hippocampus were visualized through transmission electron microscopy (H-7650, Hitachi, Japan), Statistical analysis of synaptic numbers and observation of synaptic structures refer to the work of Zhang et al.¹⁷

Immunohistochemistry

The 5 μ m thick specimen slices underwent immunostaining using seven antibodies: OPTN, PINK1, LC3, Cyt-c, Parkin, BACE1, and amyloid-β (Aβ). Following overnight fixation in anhydrous ethanol at 4°C, the slices were briefly washed in running water for 1 min, followed by a 5-min rinse with phosphate-buffered saline (pH 7.3). Antibodies (OPTN, PINK1, LC3, Cyt-c, Parkin, BACE1, and Aβ) purchased from Abcam were diluted at a 1:200 ratio in antibody dilution solution, then applied to the specimens, and incubated overnight at 4°C. Following that, the specimens were subjected to three 5-min washes with phosphate-buffered saline. Subsequently, they were incubated with a goat anti-rabbit IgG secondary antibody from Abcam at room temperature for 30 min. Following the secondary antibody staining, the specimens were washed thrice with phosphate-buffered saline for 5 min each. Subsequently, they underwent DAB (Solibao, Beijing, China) color development for 30 s and counterstaining with hematoxylin for 2 min. This was followed by a 10-min rinse in water before sequential dehydration in 75%, 85%, 95%, and 100% ethanol for 5 min each. Post-ethanol dehydration, the specimens were clarified twice with xylene for 5 min each and then mounted using neutral resin. Positive antibody expression results in dark brown staining of the hippocampal sections, whereas in the absence of the antibody, no specific staining occurs in the hippocampal sections.

Detection Aβ content

The serum Aβ levels were quantified using ELISA kits following the manufacturer's guidelines (Invitrogen, Carlsbad, CA, USA), with absorbance measured at 450 nm.

Western blot

The hippocampal tissues of mice were lysed with RIPA buffer, centrifuged, and the resulting supernatant was collected for subsequent analysis. The protein concentration was determined using the BCA (Nanjing Jiancheng Biology Engineering Institute, Nanjing, China) method. Subsequently, the samples were mixed with loading buffer and the proteins were denatured at 95°C for 10 min in a water bath. The proteins were separated on a 6%-10% SDS-PAGE gel, then electroblotted onto polyvinylidene fluoride (PVDF) membranes. The membranes were subsequently blocked with 3% BSA for 1 h, washed three times for 15 min each with TBST buffer, and finally incubated with primary antibodies (OPTN, PINK1, LC3, p62, Cyt-c, Parkin, BACE1, Aβ, and GAPDH) overnight at 4°C. Antibodies against OPTN, PINK1, LC3, p62, Cyt-c, Parkin, BACE1, Aβ, and GAPDH were obtained from Cell Signaling Technology (Massachusetts) and used at a dilution of 1:1000. The membranes, washed with a TBST buffer, were subsequently incubated with secondary antibodies. These antibodies, sourced from ZenBioScience Technology in Chengdu, China, were horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies at a dilution of 1:5000 for at least 1 h. Subsequently, the membranes were rinsed with TBST buffer and then infiltrated with ECL. Development was carried out using the Tocan 240 gel imaging system from Bio-Rad (California). Subsequent analysis of protein quantification was conducted with ImageJ software.

Data statistics

Statistical analysis was conducted using GraphPad Prism 8.0 software. The results are presented as mean ± SE. Group differences were evaluated using the Student t-test, where p < 0.05 was considered statistically significant.

Results

DHYZ improved spatial learning and memory deﬁcits in APP/PS1 mouse

The impact of DHYZ intervention on spatial behavior and cognitive deficits in AD mice was evaluated following a 12-week treatment period using the Morris water maze (MWM) test (Figure 2). During the 7-day training period for mice, the platform was removed in the final trial, and the mice were placed in the water from the opposite quadrant of the original platform location. Various parameters including escape latency time (the duration for the mice to locate the submerged platform after entering the water), platform crossings, total distance covered, and time spent on the platform were recorded. Results from the MWM test indicated that mice in the AD group exhibited more pronounced spatial impairments compared to the WT and experimental groups. They showed circular movements without traversing the target quadrant, alongside diminished discrimination indexes such as time spent in the target quadrant and interactions with the target platform (Figure 2A). Treatment with varying doses of DHYZ demonstrated an improvement in cognitive spatial abilities by reducing spatial errors and decreasing avoidance latency to locate the platform site. Following 7 days of training, both DHYZ (L, M, H) and Donepezil treatments notably reduced escape latency. However, in the AD group after the same period of training, minimal improvement was observed, with no significant difference in escape latency compared to the initial day. Additionally, DHYZ intervention led to enhanced spatial navigation skills, evidenced by decreased escape latency time, increased target platform crossings and time spent within the target quadrant on the final day of testing (Figure 2B). These findings support the effectiveness of DHYZ treatment in ameliorating spatial behavior and memory impairments in AD mice.

Figure 2.

DHYZ improved spatial behavior and memory deficits in AD mouse (n = 10). (A) The representative mouse trajectories at No. 12 week: red circle indicated the total swimming area, blue circle in the upper right corner indicated the target platform, red dots indicated the start position and blue dots indicated the end position. (B) Total distance travelled by mice in the water maze test at No. 12 week (mm), crossing the target platform, time in the target quadrant (s) and escape latency(s). *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the AD group.

DHYZ impaired Aβ production and accumulation in AD mouse

IHC assessment in Figure 3A revealed distinct changes in BACE1 and Aβ protein expression in hippocampal slices following DHYZ treatment. Compared to the WT group, the AD group exhibited elevated protein expression, indicating increased BACE1 and Aβ levels during AD progression. Treatment, particularly with DHYZ-H, led to noticeable reductions in BACE1 and Aβ expression (p < 0.01) compared to the AD group. In Figure 3B, Aβ content in plasma post-DHYZ treatment was examined. The WT group had significantly higher plasma Aβ levels than the AD group (p < 0.001). Notably, DHYZ-M (p < 0.05) and DHYZ-H (p < 0.01) groups exhibited increased blood Aβ content compared to the AD group. The impact of DHYZ on key proteins implicated in AD pathogenesis was further investigated through western blotting, revealing significantly higher BACE1 and Aβ protein expression in the AD group compared to the WT group (p < 0.001). Notably, DHYZ effectively reduced the expression of these proteins compared to Donepezil (p < 0.001), indicating its superior therapeutic efficacy.

Figure 3.

DHYZ decreased the production and accumulation of Aβ in the hippocampus of APP/PS1 mouse. (A) Immunohistochemical observed BACE1, Aβ expression in mouse hippocampus (400×), n = 3. (B) Aβ content in mouse plasma, n = 4. (C) Western blot detected BACE1, Aβ expression in mouse hippocampus, n = 4. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the AD group.

DHYZ alleviated nerve damage and improved synaptic structure in AD mouse

In Figure 4A, tunnel data revealed a significant increase in neuronal apoptosis in the AD group compared to the WT group (p < 0.001). While DHYZ-L, DHYZ-M and DHYZ-H notably reduced apoptosis (p < 0.01). Cognitive function in AD mice was linked to synaptic number and integrity in the hippocampus. TEM findings indicated that the WT group exhibited increased synaptic numbers, larger synaptic areas, and a well-defined structure of presynaptic and postsynaptic membranes with abundant synaptic vesicles compared to the AD group (p < 0.05). Conversely, the DHYZ-L (p < 0.05), DHYZ-M (p < 0.001) and DHYZ-H (p < 0.001) group demonstrated enhanced synaptic numbers, improved synaptic structures, and increased synaptic vesicles (Figure 4B).

Figure 4.

DHYZ alleviated nerve damage and improved synaptic structure in the hippocampus of AD mouse. (A) Apoptosis of mouse hippocampal neurons was observed using TUNEL staining (400×), n = 3. (B) Transmission electron microscopy observed mouse hippocampal synaptic structures (5300×), n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the AD group.

DHYZ modified mitochondrial function in hippocampus of AD mouse

In Figure 5A, the expression of PINK1, Parkin, OPTN, and LC3 proteins in hippocampal sections displayed distinct differences in IHC between the AD and WT groups (visible as brown particles, indicating positivity). After 12 weeks of DHYZ treatment, a significant increase in the number of positive brown particles was observed in the hippocampal tissue sections of the high-dose groups compared to those in the AD group (Figure 5A). Protein quantification was performed using the ImageJ software for analysis. In Figure 5B, the expression level of these proteins (PINK1, Parkin, p62, OPTN, and LC3) in AD mice exposed to AD showed a significant reduction (p < 0.001), consistent with the observations in Figure 5A which depicted higher negative cell counts in the AD group. Subsequently, after DHYZ treatment, the expression levels of PINK1, Parkin, p62, OPTN, and LC3 significantly increased compared to the AD group (p < 0.05), indicating DHYZ's potential in modulating mitochondrial function-related proteins (Figure 5B). In AD and the DHYZ group, the protein expression level of Cyt-c and p62 exhibited an inverse trend compared to PINK1, Parkin, OPTN, and LC3 proteins (Figure 5A, B). This disparity arises from the promotion of apoptotic body formation and the initiation of cellular apoptosis by the release of Cyt-c protein from mitochondria. Alterations in mitochondrial morphology were visualized using TEM in Figure 5C. The WT group exhibited numerous mitochondria with intact nuclear membranes, visible nucleoli, normal chromatin structure, abundant ribosomes, and glycogen granules. Conversely, AD onset resulted in reduced organelles, compromised mitochondrial density, and structural integrity decline, presenting disrupted cellular morphology with cytoplasmic swelling and the formation of autophagic vesicle structures. Post-DHYZ treatment, mitochondrial membranes showed fusion with reduced severity compared to the AD group, increased autophagic lysosomes, and enhanced autophagy function, particularly evident in the high DHYZ group. Notably, these findings demonstrated DHYZ's capacity to ameliorate mitochondrial structural damage, reduce swelling, and enhance autophagy function in the brain tissues of APP/PS1 mice, ultimately improving behavioral cognition in AD.

Figure 5.

DHYZ improved the mitochondrial function in the AD mouse hippocampus. (A) Immunohistochemical observed the expression of PINK1, Parkin, OPTN, LC3 and Cyt-C related proteins in the hippocampus (400×), n = 3. (B) Western Blot detected the expression of PINK1, Parkin, OPTN and LC3, Cyt-C related proteins, n = 4; (C) Transmission electron microscopy observed the changes of mitochondrial function in the hippocampus after DHYZ treatment (and the red arrows indicate autophagy) (6700×), n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the AD group.

Discussion

In the twenty-first century, mental health disorders, especially AD, have emerged as a global health concern.¹⁸ The main causes of AD are the accumulation of Aβ plaques, neurofibrillary tangles, and neuronal apoptosis, which involves cognitive dysfunction.¹⁹ Multiple studies have shown that lecanemab, a drug targeting Aβ protein, represents a pioneering targeted therapy for AD, addressing the root cause of the disease to fundamentally tackle the etiology and decelerate cognitive decline.²⁰ However, this medication may also lead to dizziness, increased confusion, brain swelling, bleeding, and brain atrophy. Consequently, safe and effective traditional Chinese medicine therapy has emerged as a novel treatment modality.²¹ Previous study indicated the DHYZ can enhance cognitive function and alleviate clinical symptoms in AD patients, decrease aberrant phosphorylation of tau protein in Aβ-induced dementia rats, and modulate the irregular expression of apoptosis-related proteins.²² In our current research, APP/PS1 transgenic mice was used to represent AD model, DHYZ was administrated to investigate whether it can attenuate AD. Our results indicate that DHYZ treatment significantly improved memory function, reduced Aβ deposition, enhanced mitochondrial structure, and promoted mitophagy in APP/PS1 transgenic mice.

Numerous studies have indicated that the excessive accumulation of Aβ serves as the primary pathological hallmark of AD, resulting from the abnormal breakdown of AβPP.²³ Additionally, studies indicate that controlling the activity of β-secretase enzymes can effectively inhibit Aβ deposition, with BACE1 identified as the critical rate-limiting enzyme for Aβ production.^24,25 Our findings confirmed the marked elevation in BACE1 expression, a key protein in the amyloid cascade, detected in the hippocampi of AD mouse brains. Treatment with DHYZ led to a noteworthy decrease in BACE1 protein expression levels as anticipated (Figure 3), with DHYZ-M and DHYZ-H significantly reducing BACE1 expression compared to AD conditions (p < 0.001). The removal of excessive Aβ accumulation is primarily aided by two pathways: the central pathway, involving Aβ phagocytosis or enzymatic breakdown by microglia and astrocytes, and the peripheral pathway, allowing the transfer of Aβ from the brain to the periphery through the blood-brain barrier, cerebrospinal fluid, interstitial fluid efflux, or lymphatic circulation.²⁶ Our results show a significant reduction in hippocampal Aβ levels after DHYZ treatment (p < 0.05), suggesting effective Aβ accumulation reduction by DHYZ. Furthermore, Aβ concentrations in the bloodstream notably increased in the DHYZ-M and DHYZ-H groups compared to the AD group, indicating that DHYZ predominantly promotes Aβ transfer from the hippocampus into the bloodstream through the blood-brain barrier.

In AD, the hyperphosphorylation of tau protein plays a role in the formation of intracellular neurofibrillary tangles, which disrupt the normal neuronal transport system.²⁷ Moreover, the accumulation of Aβ and tau proteins in neuron cells can also impede normal neuronal function, culminating in neuronal demise.²⁸ Hence, assessing neuronal apoptosis in the hippocampal tissue post-treatment served as a tangible measure of the effectiveness of the AD treatment. The outcomes of this study concerning hippocampal neurons were as anticipated, with DHYZ notably diminishing nerve damage and neuronal apoptosis (Figure 4). The decrease in neuronal apoptosis corresponded with enhancements in memory, learning, thinking, and behavior in the mice, as evidenced by improved cognitive performance in the Morris water maze, particularly in the frequency of crossing the target platform and the time spent in the target quadrant (Figure 2). Synapses are the locations where neurons form functional connections, playing a crucial role in information transmission. Damage to synapses and a reduction in their numbers can impair neural information transmission function, thereby worsening cognitive dysfunction.²⁹ Our findings revealed a reduction in synapse count and disrupted synaptic structure in AD mice. DHYZ intervention effectively mitigated age-related synaptic damage by increasing synapse numbers and correcting ultrastructural changes in neurons and synapses (Figure 4). In conclusion, our study highlights the significant advancements in reducing nerve damage and neuronal apoptosis, as well as improving synaptic structure, quantity, and synaptic vesicle counts post-DHYZ treatment.

Mitochondrial autophagy, a specific form of cellular autophagy, plays a crucial role in maintaining cellular health by degrading and eliminating damaged or aged mitochondria. In AD, the induction of mitochondrial autophagy is implicated in removing intracellular waste in hippocampal tissues, facilitating cellular organelle self-repair, and concurrently degrading dysfunctional mitochondria.³⁰ The activation of mitochondrial autophagy engages multiple protein pathways. Research has identified key proteins, including PINK1, Parkin, BNIP3, LC3, NIX, OPTN, p62, FUNDC1, TBK1, SIAH1, among others, crucially implicated in this autophagic process.³¹ Specifically, PINK1 phosphorylates and recruits Parkin, triggering a phosphorylation cascade that functions as a signal for the autophagy process. Moreover, junction proteins like P62, OPTN, and NDP52 identify phosphorylated polyubiquitin chains on mitochondrial proteins, initiating autophagosome formation around impaired mitochondria.³² Subsequently, OPTN was recruited downstream of autophagosome initiation via the LC3 interaction region (LIR) motif to enhance mitochondrial autophagy.³³ Protein quantification results demonstrated that protein expressions of OPTN, p62, PINK1, Parkin (p < 0.001), and LC3 (p < 0.05) were reduced in the AD group. Conversely, following DHYZ intervention, expressions of PINK1, Parkin, OPTN, p62, and LC3 were upregulated in the DHYZ group compared to the AD group (p < 0.05 or p < 0.01), indicative of DHYZ's ability to modulate the PINK1-Parkin pathway activation through OPTN, thereby promoting mitochondrial autophagy. Furthermore, TEM was used to observe macroscopic changes in mitochondrial ultrastructure. In comparison to the control group, the AD group showed reduced mitochondrial organelles, lower mitochondrial crisis density, and incomplete organelle structure (Figure 5C), indicating cognitive impairments in AD mice (Figure 2). Conversely, following DHYZ intervention, there was an increase in autophagic lysosomes and notable cellular autophagy observed in the mitochondria.

In conclusion, our findings demonstrate that DHYZ exerts a significant therapeutic effect on AD mice, effectively ameliorating cognitive and memory dysfunction. DHYZ reduces Aβ accumulation in the hippocampus by downregulating BACE1 activity and promoting Aβ clearance via blood-brain barrier. Moreover, DHYZ markedly inhibits neuronal apoptosis, improves synaptic structure, and boosts synapse numbers, processes closely associated with the activation of mitochondrial PINK1-Parkin autophagy. Our study provides valuable insights into the holistic mechanisms of Chinese herbal remedies for AD treatment.

Supplemental Material

sj-docx-1-alz-10.1177_13872877241299832 - Supplemental material for Di Huang Yi Zhi Fang improves cognitive function in APP/PS1 mice by inducing neuronal mitochondrial autophagy through the PINK1-parkin pathway

Supplemental material, sj-docx-1-alz-10.1177_13872877241299832 for Di Huang Yi Zhi Fang improves cognitive function in APP/PS1 mice by inducing neuronal mitochondrial autophagy through the PINK1-parkin pathway by Limin Zhang, Hongmei An, Rongrong Zhen, Tong Zhang, Minrui Ding, Mengxue Zhang, Yiguo Sun and Chao Gu in Journal of Alzheimer's Disease

Footnotes

Acknowledgments

This experiment was made possible by the joint efforts of all the authors and was supported by the fundings mentioned above.

ORCID iDs

Rongrong Zhen

Yiguo Sun

Chao Gu

Author contributions

Limin Zhang (Conceptualization; Data curation; Formal analysis; Methodology; Software); Hongmei An (Supervision); Rongrong Zhen (Investigation; Writing – original draft; Writing – review & editing); Tong Zhang (Investigation; Writing – original draft; Writing – review & editing); Minrui Ding (Data curation; Investigation; Methodology); Mengxue Zhang (Data curation; Investigation; Methodology); Yiguo Sun (Supervision); Chao Gu (Resources; Supervision).

Funding

This work was supported by the National Natural Science Foundation of China project (82104744), Shanghai Science and Technology Commission Domestic Scientific and Technological Cooperation Project (22015820900).

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Supplemental material

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