The combination of β-asarone and icariin inhibits amyloid-β and reverses cognitive deficits by promoting mitophagy in models of Alzheimer’s disease

doi:10.21203/rs.3.rs-411952/v1

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The combination of β-asarone and icariin inhibits amyloid-β and reverses cognitive deficits by promoting mitophagy in models of Alzheimer’s disease

https://doi.org/10.21203/rs.3.rs-411952/v1

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published 29 Nov, 2021

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Background: β-Asarone is the main constituent of Acorus tatarinowii Schott and exhibits important effects in diseases such as neurodegenerative and neurovascular diseases. Icariin (ICA) is a major active ingredient of Epimedium that has attracted increasing attention because of its unique pharmacological effects in degenerative disease. In this paper, we primarily explored the effects of the combination of β-asarone and ICA in clearing noxious proteins and reversing cognitive deficits. The accumulation of damaged mitochondria and mitophagy are characteristics of ageing and age-related neurodegeneration, including AD.

Methods: APP/PS1 mice and in Aβ_1-42-induced PC12 cell models were used. The groups were divided into five: normal group, model group (above animals and cells), icariin combined with β-asarone group, A3 and CSA group. Transmission electron microscopy was used to observe brain tissues (hippocampus and temporal lobe)/cell mitochondria, autophagosomes and other structures. IF and WB were used to detect the expression of Beclin-1, LC3, p62, Pink1, Parkin and p-Parkin. Dal-autophagy combined with LSCM was used to detect the occurrence of Autophagy. Mitophagy Detection Kit combined with LSCM was used to detect the occurrence of mitochondrial autophagy, and the effects of icariin combined with β-asarone on mitochondrial autophagy in AD animal and cell model were investigated.

Results: We showed evidence that autophagy/mitophagy is damaged in the hippocampus of APP/PS1 mice and in Aβ_1-42-induced PC12 cell models. Then, we found that the combination therapy can reduce the cytotoxicity caused by Aβ_1-42, increase cell viability, inhibit the expression of Aβ, APP, PS1 and BACE1, and promote the expression of SYN. The efficacy of the combination is better than that of the single drug. What’s more, it also promoted the expression of Beclin-1, LC3 Ⅰ/ in, Pink1, Parkin and p-parkin while inhibited p62.

Conclusion: These conclusions suggest that weakened removal of defective mitochondria is a pivotal event in AD pathogenesis and that combination treatment with mitophagy inducers represents a potential strategy for therapeutic intervention.

Neurobiology of Disease

β-Asarone

Icariin

APP/PS1

Mitophagy

Autophagy

BACE1

Acorus tatarinowii Schott is commonly used herb for nervous system diseases, the main effective fraction is the volatile oil. β-Asarone is a fundamental component of the volatile oil that can go through the "blood-brain" barrier into the brain. Our group has been working on β-asarone for nearly 30 years, and we found that it has a very good pharmacodynamic effect in AD. This monomer improved the behavioural symptoms of Alzheimer’s rats, and improved learning and memory abilities by inhibiting the PI3K/Akt/mTOR pathway [1,2,3]. Icariin (ICA) is a flavonoid part isolated from Epimedium that has been extensively researched in the field of degenerative diseases [4,5]. In our previous research, we treated Aβ₄₂-induced PC12 cells with icariin and β-asarone at gradient concentrations. By exploring the effects of drug concentration and combination treatment, we found that the combination treatment had better therapeutic outcomes than the individual treatments [6]. AD is a complex disease, and intervention from a single mechanism of action may not be the optimal principle of drug therapy. Just like a blood pressure medication that combines Calcium channel blockers with diuretics.

Dementia describes an individual pattern of impaired memory and thinking that impairs at least two cognitive domains [7]. Alzheimer's disease (AD) is an irreversible, age-related neurodegenerative disease characterized by gradual memory loss and cognitive decline [8]. Extracellular senile plaques are one of the main neuropathological features of AD which is also called Aβ (amyloid beta) [9,10]. Although there is genetic sign and evidence that Aβ participates in the induction of synaptic dysfunction, destroys neural connections, and is associated with neuronal death in a specific manner, the number and distribution of Aβ deposition are only weakly correlated with the clinical manifestations of the disease [11].

Autophagy is the most important cellular system that degrades and circulates intracellular proteins and organelles under different physiological and pathological conditions [12]. Mitophagy is a selective autophagy in which mitochondria are modified by multiple ubiquitin chains, phagocytized by autophagosomes and degraded after lysosomal fusion [13]. This process engages targeting damaged or redundant mitochondrial to lysosomes, where mitochondrial components are degraded and recycled. PTEN induced kinase 1 (PINK1) and parkin are essential proteins for mitotic phagocytosis in mammals [14].

The mitochondrial membrane potential (ΔΨm), it is produced by oxidation-reduction alterations related with Krebs cycle activity and acts as an intermediate form of energy storage for ATP synthase to produce ATP. These transitions produce not only potential (because of custody separation) but also proton gradient, which together create transmembrane potential [15,16]. Ca²⁺ is an significant molecule that affects a extensive array of physiological and pathological processes. Unlike other subordinate couriers, Ca²⁺ is not manufactured or metabolized but is kept and swiftly discharged by channels/pumps that preserve the concentrations in different cell sections [17]. The calcium hypothesis also reformulates one of the important theories about AD and offers evidence for a role of Ca²⁺ in AD pathogenesis [18]. Importantly, mitochondrial quality control plays critical effect of maintaining healthy mitochondrial pool and inhibiting neuronal death on neuronal function and survival [19.20].

Our previous analysis provided evidence that defects in autophagy have a significant function in AD development. The autophagy-lysosome pathway is another chief Aβ clearance road as well as the different degrading enzymes [21]. Therefore, maintaining autophagy at the level of neuron and organism may be a promising therapeutic method. Nevertheless, the role of mitophagy in AD progression remains indistinct.

This is the first time we have used the combination of β-asarone and ICA to investigate AD. Based on the evidence cited above, we detected changes in mitochondria and mitophagy in the APP/PS1 mouse and PC12 cell models and showed defective mitophagy in AD. Then, we analyzed the pharmacological effect of the combination therapy using behavioral tests and measuring the levels of AD-related proteins. Furthermore, we used antimycin A (A3) and cyclosporine A (CSA) with the combination treatment as an activator and an inhibitor of mitophagy, respectively, to investigate the mechanism. As far as we know, our data show that this combination can protect against damage from toxic pathology and mitochondrial disruption and that this process occurs via promotion of mitophagy, which could act as a potential key contributor to therapeutic action.

Chemicals and reagents

The Aβ_1-42 used in these experiments was acquired from Life Technologies (USA); β-asarone was purchased from NIFDC (Beijing, China,112018-201601), and the purity value is 96.8%; icarrin donepezil was purchased from NIFDC (Beijing, China,110737-201516), and the purity value is 94.2%; donepezil was obtained from the First Affiliated Hospital of Jinan University (Guangzhou, China); A3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); CSA was purchased from Selleck (Houston, Texas, USA). 3MA, high-glucose DMEM, FBS, trypsin, and PBS were obtained from Gibco (Gaithersburg, MD, USA); Cell Counting Kit-8 (CCK-8), Cytotoxicity LDH Assay Kit—WST (LDH), and Cellular

Senescence Detection Kit—SPiDER-bGal were obtained from Dojindo Molecular Technologies, Inc. (Tokyo, Japan); the bicinchoninic acid (BCA) protein assay kit, Immunol Fluorescence Staining Kit, and Immunohistochemistry Staining Kit were obtained from Beyotime (Shanghai, China); anti-Beclin 1 antibody [EPR19662] (1:100, catalog no. ab207612; Abcam); anti-BACE1 antibody [EPR19523] (1:50, catalog no. ab183612; Abcam); anti-Synapsin I antibody-Synaptic Marker (1:800, catalog no. ab64581; Abcam); anti-SQSTM1/p62 antibody (1:800, catalog no. ab91526; Abcam); anti-Presenilin 1/PS-1 antibody [EP2000Y] (1:100, catalog no. ab76083; Abcam); anti-APPL antibody (1:500, catalog no. ab180140; Abcam); anti-PINK1 antibody [EPR20730] (catalog no. ab216144; Abcam); anti-Parkin antibody [PRK8] (1:100, catalog no. ab77924; Abcam).

The experimental mice were provided by the Institute of Model Animal Science, Nanjing University, certificate number: SCXK (SU 2010 0001, B6C3 TG APPSWE PSEN1DE9 85DBO /J strain, SPF grade, 10, half male and half female, body weight (20±2) g 3 months old; C57BL/6 mice, SPF grade, 10, half male and half female, body weight (20±2 g) 3 months old.

PC12 (highly differentiated) rat adrenal pheochromocytoma cells (highly differentiated purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Table No. TCR 9 (highly differentiated).

Cell culture and handing

Aβ_1-42 is the component found in amyloid plaques, and it has 42 amino acids. We used gradient concentrations of Aβ_1-42 (0, 2.215, 4.43, 8.86, 17.72 μM) induced PC12 cell for 6, 12, 24 and 48 h. Cellular viability and toxicity were detected by CCK8 and LDH assays, and the final condition we set was 7μM Aβ_1-42 interference for 12h.

Cell proliferation and cytotoxicity

Cell viability was detected using CCK-8 assay. For the CCK-8 assay, PC12 cells were seeded in 96-well plates and were cultured for 24 h. The cells were then treated with CCK-8 reagent and further cultured for 2h according to the manufacturer’s instructions. The absorbances were measured at 450 nm. Each group of experiments included 6 replicates and repeated three times. Cell injury was assessed through measuring the LDH activity in the supernatant of PC12 cells using an LDH kit according to the manufacturer's protocol.

Electron microscopy

Electron microscopy was used to observe the autophagosome. For mouse hippocampus, the tissue was clean with PBS once and fixed for 24 h at 4°C in 2.5% glutaraldehyde and another 2 h in 1% osmium tetroxide. Then were made into ultrathin electron microscope sections. The cells were counted and adjusted to a density of 1 × 106 cells/ml and then fixed for 24 h at 4°C in 2.5% glutaraldehyde and another 2 h in 1% osmium tetroxide.

MSD assay analysis of Aβ₃₈, Aβ₄₀ and Aβ₄₂

The analytical performance of the measurement of Aβ38, Aβ40 and Aβ42 levels in serum and in the hippocampal and prefrontal cortex tissues by MSD assay was assessed in terms of parallelism, sensitivity, accuracy, precision, lot-to-lot variability, selectivity and freeze/thaw stability according to published guidelines (Meso QuickPlex SQ 120).

Flow cytometric evaluation of MMP and Ca²⁺

PC12 cells were cultured in DMEM as described above, seeded (2.4x10⁵ cells/mL, 200 μL) on a μ-slide 8-well plate (ibidi) and cultured at 37 °C in a 5% CO2 incubator overnight. The medium was removed, treatments were added to each well, and then the cells were cultured at 37 °C for 90 minutes in the 5% CO₂ incubator. After the supernatant (100 μL) was removed, JC-1 working solution (4 μmol/L, 100 μL) was added, and then the cells were cultured at 37 °C for 30 minutes in the 5% CO2 incubator. Then, the cells were washed twice with 200 μL of HBSS. Imaging buffer solution (200 μL) was added, and the cells were observed under a laser confocal microscope (green: 488 nm (Ex), 500–550 nm (Em); red: 561 nm (Ex), 560–610 nm (Em)) and flow cytometer (green: 488 nm (Ex), 515–545 nm (Em); red: 488 nm (Ex), 564–604 nm (Em)).

Immunofluorescence (IF) analysis

Briefly, approximately 9–10 slices from each tissue were incubated in blocking buffer (3% H₂O₂) for 10 min at room temperature. PC12 cells were cultured on the microscope slide at a density of 5×10⁵ cells/cm² and were treated as described above. Then, endogenous peroxidase activity was quenched for 10 min in PBS containing 3% H₂O₂, chilled in water, and then immersed for 5 min in PBS. Thereafter, samples were incubated overnight with the primary antibody at 4 °C and then incubated with the appropriate fluorescent probe-conjugated secondary antibodies for 1 h at room temperature while protected from light. Then set the slides with SABC-FITC (diluted 1:200) and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) at a 1:5,000 dilution. Then the slices were mounted with an anti-fluorescent quencher. Pictures were taken with an Olympus microscope (BX53). Specific primary antibodies used 1:100, Plaque numbers per ROI were counted and quantified with image J.

Western Bolt analysis

The hippocampus and prefrontal cortex of mice were obtained from APP/PS1 brain tissue. Following electrophoresis, gels were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore Ltd, Ireland) at 200 mA for 60 min. The membrane was incubated for 10 min in Tris-Buffered Saline Tween-20 (TBST) buffer containing 5% skimmed milk on the shaker. Antibodies used at a 1:1,000 dilution and Secondary antibodies used at a 1:3,000 dilution.

DALGreen autophagy detection and mitophagy detection

PC12 cells were incubated at 37oC for 30 minutes with 250 μl of 1 μmol/l DALGreen working solution. After the cells were washed with the culture medium twice, the culture medium or amino acid-free mediumthe (Wako Pure Chemical Industries, Ltd., Code: 048-33575) was added to the well. After 6 hours incubation, the cells were washed with Hanks’ HEPES buffer twice and then DALGreen was observed by confocal fluorescence microscopy. Finally observed by confocal fluorescence microscopy. Ex: 561 nm, Em: 650 nm. Lysosomes, Ex: 488 nm, Em: 502-554 nm.

Mouse learning memory tests

All the experimental mice were provided by Institute of Model Zoology, Nanjing University, certificate number: SCXK (SU 2010 0001). Ethical code: SYXK (Guangdong 2018 0092). Mice were trained in the water maze with four trials per day for 5 days. They were placed in a pool of water containing a platform just below the surface of the water. They escaped from the maze (i.e., they were removed from the pool) when they found the platform. Distal visual cues were arrayed around the room, and in general, the mice were able to learn the location of the hidden platform based on these cues. Water maze performance was analyzed by the amount of time spent in the target quadrant (sec). When a mouse exceeded 300 s, it was recorded as 300 s.

All mice were allowed one day of rest after the water maze test and the passive avoidance test before the step-down test was conducted using a step-down recorder (Jinan Yiyan Technology Development Co., Ltd.) and a previously described method [22].

Statistical analysis

All data were expressed as means ± standard deviation. Differences between two groups were analyzed using the t-test, and differences among three or more groups were analyzed using single-factor analysis of variance (one-way ANOVA) with SPSS (version 16.0 for Windows). Differences were considered statistically significant at P< 0.05.

The composition of icariin combined with β-asarone was determined by ¹³C-NMR

¹³C-NMR spectrum is shown in Figure 1, which clearly shows the residual dissolution peak containing the dissolution of CDCl₃. As can be seen from the detection of icariin in (Fig. 1A). 37 different ¹³C chemical shifts were presented in this spectrum. As can be seen from the detection of (Fig. 1B). β- asarone, 19 different 13C chemical shifts were presented in this spectrum. From the results of icariin + β-asarone in (Fig. 1C), it can be seen that 45 different ¹³C chemical shifts are presented in the spectrum.The number of ¹³C above is consistent with the sum of the two monomers and CDCl₃, and the spectral peaks are clearly separated. Fig. A and Fig. B are splicing according to the chemical shift, and it can be seen that no new ¹³C structure is formed after the mixing of the two.This also provides a certain pharmacological basis for the follow-up experiments.

Defective mitochondrial function and mitophagy in the APP/PS1 mouse and in the PC12 cell model

To uncover the cellular and molecular causes of mitochondrial impairment and the accumulation of damaged mitochondria in AD, we examined the changes in mitochondria in the hippocampus of APP/PS1 mice and in the PC12 cell model. The hippocampal neurons in the APP/PS1 samples displayed altered mitochondrial morphology characterized by reduced size and excessive mitochondrial damage compared to those in the healthy control samples (Fig. 2A). The cell model also showed the same changes, including swollen and deformed mitochondria (Fig. 2B). ΔΨm is not only used for ATP synthesis but is also a factor that determines the viability of mitochondria participating in the elimination of disabled mitochondria [23]. In PC12 cells, we detected a decrease in mitochondrial viability in the model group from both flow cytometric detection and laser confocal microscopy. JC-1, a cationic carbocyanine dye (green), exhibits potential-dependent accumulation in mitochondria where it forms J-aggregates (red), whereas if it remains as monomer, it shows green fluorescence. The concentration was reduced, the monomer emitted more green fluorescence in the AD group, and the ΔΨm remained at a lower level (Fig. 2C). Accurate measurements of intracellular Ca²⁺ concentration allow for a more comprehensive understanding of Ca²⁺-regulated cell functions and signalling pathways. Fluo-4 is a calcium-specific fluorescent dye that exhibits a greater than 100-fold increase in fluorescence upon binding Ca²⁺ ions. The AD cells showed increased intracellular calcium levels compared to those of the control cells (Fig. 2D). Combined with the results of our previously published articles, we have demonstrated that autophagy is inhibited in these AD models, but we also need to systematically examine the protein levels of major factors involved in mitochondrial metabolism and mitophagy. The expression levels of the mitochondrial quality control protein PINK1 and the autophagy proteins LC3 I/II and Beclin-1 were decreased in both the hippocampal and PC12 cell samples. P62, a substrate degraded by autophagy, showed the opposite tendency (Fig. 2E, F). These findings showed the accumulation of damaged mitochondria in the hippocampus of APP/PS1 mice and the PC12 cell model accompanied by defects in autophagy and mitophagy.

The combination treatment ameliorates cognitive decline and AD pathology in APP/PS1 mice

Based on the relationship between the effective dose and the dose used in the animal experiments, we chose to treat the mice in the combination group with 15 mg/kg/d β-asarone and 20 mg/kg/d icariin. To investigate the effect of the combination treatment on AD, we tested the behaviour of APP/PS1 mice (Fig. 3A) and analysed the levels of AD-related proteins (Fig. 3B–D). The transgenic AD mice exhibited a longer swimming time (P<0.001) and a greater number of errors (P<0.01) in the water maze test and longer latency in the step-down and step-through tests (P<0.05). Treatment with either β-asarone, icariin, combined β-asarone and icariin or donepezil all improved learning and memory retention in the AD mice, and the combination treatment showed a better effect than the individual treatments (P<0.05) (Fig. 3A). The MSD assay was used to test the three proteins Aβ₃₈/ Aβ₄₀/ Aβ₄₂in the serum, hippocampus and PFC; from the data in Fig 2B, it can be seen that Aβ₃₈ was only detected in the serum, while Aβ₃₈ was not detected in hippocampal and PFC tissues. The overall expression of Aβ₄₀ was higher than that of Aβ₄₂, which is consistent with the existing knowledge that in human cerebrospinal fluid and blood, Aβ₄₀ expression is 10 times and 1.5 times higher than that of Aβ₄₂, respectively; however, Aβ₄₂ is more toxic and more likely to aggregate, thereby forming a toxic precipitate and triggering neurotoxicity, so Aβ₄₂ is the main focus among the three similar proteins in AD research. The results of the simultaneous comparison of the three proteins Aβ₃₈, Aβ₄₀, and Aβ₄₂ suggest that the expression analysis of Aβ₄₂ is more meaningful for AD than those of Aβ₃₈ and Aβ₄₀. The analysis of Aβ₄₂ is shown separately. In the normal control mice, a small amount of Aβ₄₂ was detected in only the serum, whereas no expression of Aβ₄₂ was detected in the hippocampus or PFC. The contents of Aβ₄₂ in the serum, hippocampus, and PFC in the AD group were significantly higher than those in the normal group (P<0.01, P<0.001), and the contents of Aβ₄₂ in the three tissues decreased after the application of icariin or the combination treatment (P<0.05). Western blotting was used to simultaneously detect APP, PS1, Aβ, Syn and BACE1.

The combination treatment did not change the expression level of PS1 but decreased the levels of APP, Aβ, Syn and BACE1. This result demonstrated that the combination therapy effectively attenuated the expression of AD-related pathological metabolites and toxic proteins. Furthermore, the expression of synaptophysin was most pronounced in the combination group (Fig. 3C). These results indicate that the combination treatment ameliorated memory deficits in the APP/PS1 mouse model, promoted the degradation of toxic proteins and increased the expression of synaptic proteins.

The combination treatment inhibits cell damage in the Aβ_1-42PC12 cell model of AD

We cultured PC12 cells in vitro and found that 7 μM Aβ_1-42 for 12 h was the optimal Aβ_1-42 concentration for this model. In the initial experiments, we investigated the effects of gradient concentrations of β-asarone (12, 24, 36, 72, and 144 μM) or icariin (3, 6, 12, and 24 μM). We found that the protective effect of β-asarone and icariin on cell proliferation was dose-dependent; when the β-asarone/icariin concentrations were increased to 72/12 μM, the proliferation capacity of the PC12 cells started to decrease. At the β-asarone/icariin concentrations of 144/24 μM, viability was further reduced (Fig. 4A). We chose 36 μM β-asarone and 6 μM icariin as the working concentrations. The concentration of donepezil was also calculated according to the effective doses in humans and in cells, and the final concentration was determined by means of a preliminary experiment.

After we established a stable AD cell model, we investigated the effects of 6 μM icariin, 36 μM β-asarone, the combination of 36 μM β-asarone and 6 μM icariin, and 10 μM donepezil. Compared with the individual icariin and β-asarone treatments, the combination treatment improved cell proliferation and decreased cell damage (Fig. 4B). We also analysed APP, PS1, Syn and BACE1 by western blotting (Fig. 4C, D). The combination treatment suppressed the levels of APP and PS1 and increased the expression of Syn. Furthermore, we observed the expression of APP, PS1 and Syn with IF (Fig. 4E, F). Collectively, these data suggested that the combination treatment showed a better effect in inhibiting the accumulation of toxic proteins and metabolites and promoting the secretion of synaptophysin, thus exerting a protective effect in AD pathology.

The combination treatment activated autophagy and mitophagy

There is substantial evidence that dysregulation of autophagy occurs in both AD animal models and AD patients [24]. To determine the occurrence of autophagy, we performed electron microscopy on hippocampal slices of APP/PS1 mice. Electron microscopy found that the combination treatment promoted the formation of autophagic vacuoles (autophagosomes, AU) and multilamellar bodies (MLB) and increased the density of synapses (Fig. 5A). Mitophagy defects lead to the accumulation of damaged mitochondria, energy deprivation and eventually neuronal loss. Hence, compromised mitophagy could contribute substantially to AD pathology and cognitive decline. PINK1 triggers signals for mitophagy initiation and parkin-mediated signal amplification. When we detected the expression of Beclin-1, LC3 I/II, p62, PINK1 and Parkin in the hippocampus of APP/PS1 mice, we clearly found that the combination treatment induced autophagy and mitophagy (Fig. 5B, C). Then, western blotting and IF were used to measure the above proteins in PC12 cell models (Fig. 5D–G). Moreover, we used DAL-Green to measure autophagy (Fig. 5H–I). DAL-Green is a small fluorescent molecule with unique properties that emits fluorescence under hydrophobic and acidic conditions and can detect autolysosomes. The combination and A3 groups displayed a decreased amount of p62, while the other proteins tended to increase (Fig. 5F, G). Taken together with the DAL-Green findings, all the results indicated that the combination treatment significantly promoted the occurrence of autophagy (Fig. 5H, Autophagy). Furthermore, we detected mitophagy, mitochondria and lysosomes with Mtphagy Dye (Fig. 5H, Mtphagy). The combination drug led to an increase in the fluorescence intensities of the Mtphagy, Mitochondria Deep Bright and Lysosome dyes. This indicates that the combination treatment promoted mitochondrial autophagy while improving mitochondrial function. However, A3 promoted mitophagy but had little effect on mitochondria (Fig. 5H, Mtphagy). Thus, the combination treatment acts as a specific autophagy/mitophagy activator and has a certain protective effect against the destruction of mitochondrial function in the AD state.

Mitophagy induced by the combination treatment inhibits amyloid-β and reverses cognitive deficits

To obtain a systematic, unbiased overview of how this combination treatment affects memory-related neuronal functions in AD, we performed further exploration into to the relationship between the combination treatment, autophagy/mitophagy and AD. We performed behavioural tests after treatment with the mitophagy-activating agent A3 and the mitophagy-inhibiting agent CSA. The AD mice showed a significant decrease in behavioural indicators compared with those of the control mice (Fig. 6A). In the water maze test, the time spent in the target quadrant in the combination treatment and A3 groups was reduced compared with that in the AD group. Moreover, the amount of time the CSA group spent in the target quadrant was extended. The results of the platform test were consistent with those of the water maze. In the step-through test, only the combination treatment group exhibited reduced time, while there were no obvious changes in the other groups. We also used the MSD assay to detect the proteins Aβ₃₈, Aβ₄₀, and Aβ₄₂in the serum, hippocampus and PFC (Fig. 6B). As shown in Figure 5B, Aβ₃₈ was only detected in the serum, while hippocampal and PFC tissues expressed lower levels of Aβ₃₈. In the normal control mice, only a small amount of Aβ₄₂ was detected in the serum, while Aβ₄₂ expression was not detected in the hippocampus or PFC. The content of Aβ₄₂ in the serum, hippocampus and PFC in the AD group was significantly higher than that in the normal group (P<0.05, P<0.001). After icariin was applied in combination with β-asarone or activator A3, the contents of Aβ₄₀ and Aβ₄₂ were significantly reduced. The ability of the activator treatment to inhibit Aβ₄₀ in the prefrontal cortex was better than that of the combination treatment (P<0.05). Next, we examined the changes in AD-related indicators in the hippocampus of the above groups. BACE1 is the putatively rate-limiting initiating enzyme in Aβ generation, and it is considered a prime drug target for lowering cerebral Aβ levels in the treatment and/or prevention of AD [24]. LC3 is a central protein in the autophagy pathway that functions in autophagy substrate selection and autophagosome biogenesis. PINK1 and Parkin are established mediators of mitophagy, the selective removal of damaged mitochondria by autophagy [25]. We found that the combination treatment led to decreased expression of APP and BACE1, while LC3 I/II and PINK1 expression increased. Moreover, A3 treatment promoted the levels of LC3 I/II and PINK1, and the levels of APP and BACE1 were both suppressed. Western blot experiments showed the same results (Fig. 6C, D). Then, we also used the cell model to analyse the levels of the above proteins in the same groups. The results were basically consistent with those from the APP/PS1 mice. The combination treatment and the A3 treatment promoted the expression of LC3 I/II and PINK1, thus inducing autophagy/mitophagy and inhibiting BACE1 and APP (Fig. 6E, F). We can conclude from the above results that the combination treatment reduced the deposition of toxic proteins by promoting autophagy/mitophagy.

In the current study, we describe a combination treatment of icariin and β-asarone with efficacy in a cross-species model of AD. This is the first combination of these two monomers in AD research. We divided the research into three parts: 1) the effect of the combination treatment on AD models; 2) the effect of the combination treatment on autophagy/mitophagy; and 3) the effect of the combination treatment on AD by affecting autophagy/mitophagy on PINK1/Parkin. Through the above analysis, we aimed to systematically examine the effect and mechanism of combined icariin and β-asarone treatment on AD.

Enhanced metaphorical activity has been reported to promote Aβ and tau clearance in in vitro and in vivo models. Meanwhile, there is growing evidence that treatment of AD should be preceded by intervention before the formation of pathological products. The efficacy of the combination therapy was better than that of the individual therapies applied separately.

Based on our previous years of research, we obtained the optimal concentration of the two monomers and combined the two active ingredients to generate a stable, single-dose form. The combination of β-asarone and icariin showed a better effect in inhibiting the accumulation of toxic proteins and metabolites in vivo and in vitro, and we found that the expression of BACE1 was significantly inhibited. In the past decade, the therapeutic potential of BACE1 inhibitors has been studied. Nevertheless, although BACE1 inhibitors can effectively decrease the number of Aβ in the brain, clinical trials still fail to prove the improvement of cognitive function in patients with mild to moderate AD, which calls into question the real value of these potential anti AD drugs and the plan of clinical trials [26]. Therefore, in addition to the basic pathological changes, we also examined the effect of the combination on autophagy/mitophagy. We identified the changes in both in vivo and in vitro models. We compared the combination treatment with mitophagy activator and mitophagy inhibitor treatments, and the experimental results suggested that the combination treatment effectively promoted the occurrence of autophagy and protected against mitochondrial damage. Furthermore, promoting autophagy also exerted a protective effect by clearing metabolic products and promoting the secretion of synaptophysin, thus exerting a protective effect in AD pathology.

AD is a varied disease with multiplex pathobiology. There are about 40 million patients over the age of 60 suffering from AD all around the world, and the number is increasing in minutes, supposed to doubling every 20 years [27,28]. Several topical original discoveries have highlighted significant pathological roles of additional serious cellular and molecular processes. However, the most important neuropathological criteria for the diagnosis of AD are the deposition of extracellular Aβ as neuritic plaques and the accumulation of intracellular hyperphosphorylated tau as neurofibrillary tangles [29]. Currently, clinical drug therapy is generally alienated into two classifications: acetylcholinesterase inhibitors (AChEIs) and N-methyl-D-aspartic acid (NMDA) receptor antagonists. As neurotransmitter modulators, these medicines can get rid of signs in a short time but cannot postponement the progression of the problem [30]. Recently, a new drug, aducanumab, has been sent to the FDA after analysis yielded positive results in early Alzheimer’s Disease [31]. Donepezil is a representative cholinesterase inhibitor that improves the ability of impaired nerve endings to transmit messages between nerve cells [32]. These drugs are based on a single compound with a single target. There are also numerous new evocative hypotheses about the etiology of AD, including mitochondrial cascade hypothesis, neurovascular hypothesis, inflammation hypothesis, calcium homeostasis hypothesis, metal ion hypothesis and lymphatic system hypothesis. However, the final cause of AD remains unclear. Among the clinical trials to test various hypotheses of AD by 2019, the amyloid hypothesis was the most heavily tested (22.3% of trials), followed by the neurotransmitter hypothesis (19.0% of trials). Autophagy dysfunction followed by metabolic or oxidative stress as a result of aging leads to the accumulation of indigestible or incompletely degraded materials containing the components for Aβ generation, including APP and its processing enzymes [33,34]. Some studies have also shown that promoting mitochondrial autophagy can ameliorate the cognitive impairments characteristic of both Aβ and tau pathologies [35].

The current data broaden our understanding of the effect of the combination of β-asarone and icariin, which promotes the clearance of toxic proteins such as APP, PS1 and BACE1, promotes the formation of autophagosomes and increases the expression of Beclin-1, PINK1 and p/Parkin. These structural and functional enhancements help to explain the improved learning and reversed cognitive deficits observed after administration. Our team has long been committed to the study of neurodegenerative diseases. We believe that the pathogenesis of AD is very complicated, and it is possible that important links have not yet been revealed. Maybe because the pathophysiological mechanism of AD is not clear, and the combination therapy improved the pathological results caused by a combination of various factors through different molecular mechanisms. Therefore, we should pay attention to multiple links and targets and try to explore the use of combined drugs, thus accelerating the study of recommending AD.

Limitations of the current study include these results also raise many important questions, such as (1) If AD is a progressive disease, when is the best time for drug application? (2) Since mitophagy is also a dynamic and ongoing process, should the effectiveness of a treatment be evaluated based on the time at which it is given to the patient? While it is difficult to address these questions with only AD models, we are conducting more related clinical trials to further explain this problem.

The evidence reported here in powerfully indicates that mitochondrial function and mitophagy may be defective in AD. The combination of β-asarone and icariin modulated mitophagy and improved the pathological damage of AD, potentially by improving the function of mitochondria and promoting the occurrence of mitophagy. Thus, targeting the regulation of autophagy at both the neuronal and organismal levels may be a promising therapeutic approach. New autophagy/mitophagy activator-based therapies might represent an innovative and attractive strategy in regenerative medicine.

Ethical Approval and Consent to participate

All the experiments complied with ARRIVE guidelines and were carried out in strict accordance with the recommendations in the Guide for the Care Use of Laboratory Cells of Jinan University. Efforts were made to minimize the number of animals used.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by National Natural Science Foundation of China (81903971), National Natural Science Foundation of Guangdong (2021A1515011470), National Key R&D Program of China (2018YFC2002500), General Program of China Postdoctoral Science Foundation (2018M643377).

Authors' contributions

Study design: Ronghua Zhang and Nanbu Wang. Study conduct: Nanbu Wang, Haoyu Wang, Qi Pan, Jian Kang. Data collection: Haoyu Wang , Qi Pan and Ziwen Liang. Data analysis: Nanbu Wang, Haoyu Wang. Data interpretation: Nanbu Wang. Drafting manuscript: Nanbu Wang, Haoyu Wang and Qi Pan. Approving the final version of the manuscript: All authors. The authors declare that there is no conflict of interests regarding the publication of this article.

Acknowledgements

We thank to the Experimental animal center of Guangzhou University of Chinese Medicine. Thanks to Jinan University School of Life Science and Technology, Analytical Testing Center. Special thanks to Prof. Yongqi Fang for his help in the research and development of the project. Thanks to Prof. Yiwen Ruan for her guidance in electron microscopy analysis, Prof. Hailan Tang for her guidance in laser confocal experiments, associate Prof. Ling Li for her support of animal experiment, Prof. Jichun Zhang for his guidance in cell study.

Data availability statement

The data that support the findings of this study are available from the author upon reasonable request.

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The combination of β-asarone and icariin inhibits amyloid-β and reverses cognitive deficits by promoting mitophagy in models of Alzheimer’s disease

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