Luteolin and Exercise Combination Therapy Ameliorates Amyloid-β 1-42 Oligomers-Induced Cognitive Impairment in AD Mice by Mediating Neuroinflammation and Autophagy

Abstract

Background:

Alzheimer’s disease (AD) disturbs many patients and family. However, little progress has been made in finding effective treatments. Given AD is a multifactorial disease, luteolin and exercise combination therapy may be more effective than monotherapy.

Objective:

To explore the therapeutic effect and underlying mechanisms of luteolin and exercise combination therapy in AD treatment.

Methods:

This study utilized a validated mouse model of AD by bilateral injection of amyloid-β (Aβ)_1-42 oligomers into the CA1 region of the hippocampus. By combining with animal behavioral test, thioflavin T detection, immunofluorescence and western blot test, the cognitive-enhancing effects of luteolin and exercise combination therapy and the underlying mechanisms were investigated.

Results:

Luteolin (100 mg/kg/d) combined with exercise could significantly improve the performance of AD model mice in novel object recognition test, and the improvement was greater than that of monotherapy. Further experiments showed that luteolin and exercise alone or in combination could reverse the increase of Aβ content, the activation of astrocytes and microglia, and the decrease of the level of autophagy in hippocampus and cortex in AD model induced by Aβ_1-42 oligomers. While the combination therapy involved more intact hippocampal and cortical areas, with greater degree of changes.

Conclusion:

Keywords

autophagy cognitive impairment luteolin and exercise combination therapy neuroinflammation

INTRODUCTION

Alzheimer’s disease (AD), the leading cause of dementia, is a complex chronic neurodegenerative brain disorder [1]. Even though N-methyl-D-aspartate receptor (NMDAR) antagonists [2] and cholinesterase inhibitors [3] are approved as treatment regimens for various degrees of AD, limited progress has been made in finding effective treatments. Current treatments focus on helping people maintain mental functioning, control behavioral symptoms, and moderate or delay symptoms of the disease. The elevated failure rate of AD treatment stems in large part from its complex pathological causes and our incomplete understanding of the relationship between the numerous pathways involved in AD development and subsequent neurodegeneration [4]. The reported causative or risk factors for AD include the presence of mutations in genes encoding presenilin 1/2, amyloid precursor protein, family history, age, low education level, and so on [5]. Given AD is a multifactorial disease, combination therapy may be more required for successful treatment than monotherapy [6].

With the discovery of elevated levels of inflammatory markers in AD patients and the identification of AD risk genes related to innate immune function, neuroinflammation is expected to become a diagnostic and therapeutic target for AD [7]. Astrocytes and microglia serve as crucial roles in the regulation of neuroinflammation [7]. As the disease progresses, activated microglia exerts deleterious effects by overexpressing proinflammatory cytokines such as interleukin (IL)-1β causing neurodegeneration in the surrounding brain regions [8]. Astrocytes dysfunction leads to increased release of cytokines and inflammatory mediators, neurodegeneration, reduced glutamate uptake and loss of neuronal synapses, ultimately leading to cognitive dysfunction in AD [9]. Hence, anti-inflammatory interventions are reported to have a certain therapeutic effect on AD. Moreover, there are numerous studies finding an interaction between neuroinflammation and autophagy in the pathogenesis of neurodegenerative diseases. LPS-induced neuroinflammation could cause autophagy impairment by dysregulating ATG genes [10]. Autophagy, as a conserved catabolic process that degrades defective proteins or organelles in lysosomes and recycles essential components in eukaryotic cells, can exhibit protective effects under normal conditions, while abnormal autophagy may cause cell death [11]. Accumulating evidence has indicated that dysfunctional autophagy contributes to AD pathogenesis. It is reported to be involved in Aβ metabolism, tau pathology, synaptic function, and mitochondrial dysfunction of AD pathogenesis [11]. Therefore, the targets to autophagy are also expected to be used in the treatment of AD.

Luteolin is a natural flavonoid from many edible plants, including broccoli, oranges, lemon, celery, parsley, green tea, carrots, apple skins, thyme, onion leaves, peppermint, rosemary, green pepper, chamomile flower, perilla, fenugreek seed, and so on [12]. It has been reported to exert various pharmacological activities such as anti-apoptosis, anti-oxidation, and anti-inflammation [13]. It can reverse the memory impairment caused by seizures and inhibit oxidative stress injury in Sprague Dawley rats [14]. It is suggested to alleviate the Aβ-induced activation of astrocytes and microglia, reduce the expression of proinflammatory factors and related kinases, and alleviate the reduction of neurotrophic factors such as brain-derived neurotrophic factor and glial cell-derived neurotrophic factor [15]. Moreover, luteolin has also been reported to inhibit Aβ_25-35-induced neuronal death in mouse cortical cultures [16]. The above evidence suggests that luteolin has great potential in the treatment of cognitive impairment caused by AD. Furthermore, exercise plays an essential role in regulating Aβ levels, inflammation, synthesis of neurotrophic factors, and cerebral blood flow [17]. It ameliorates cognitive impairment by increasing neuronal regeneration and neurotrophic factor levels in the adult hippocampus of 5×FAD mice [18]. It can stimulate the production of neurotrophins by inducing metabolic factors and muscle-derived actin to promote neurogenesis. Considerable evidence suggests that exercise may inhibit microglial activation by downregulating the levels of proinflammatory factors [19]. Studies also have shown that voluntary exercise is a physiological autophagy inducer and has a BECN1-dependent protective effect on Aβ clearance in AD mice [20]. To a large extent, exercise may confer cognitive benefits, but the results remain inconclusive and deserve further exploration. Although the mechanisms of luteolin and exercise for neuroprotection have not been fully investigated, they both have regulatory effects on neuroinflammation and autophagy. Therefore, it is reasonable to believe that luteolin and exercise combination therapy may have a better protective effect on cognitive impairment caused by AD.

In this study, it is hypothesized that the luteolin and exercise combination therapy was more effective than monotherapy in improving cognitive impairment induced by AD. Furthermore, the proteins associated with neuroinflammation and autophagy would also be examined to explore possible mechanisms for this difference in effect.

METHODS

Animals

Sixty C57BL/6J Male mice (7-8 weeks old) were obtained from the Beijing Weitong Lihua Experimental Animal Technology Company. The room was maintained at 23±1°C with 60±5% relative humidity. All mice were housed under a 12-h light/dark cycle (lights on at 08:00 AM). They were housed four per cage, with access to standard food and water ad libitum, and acclimated for a week. All animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee of Beijing Rehabilitation Hospital, Capital Medical University (Approval No. 2021bkky-014).

Aβ_1-42 oligomers-induced AD model

The Aβ_1-42 oligomers were prepared as previously described [21]. Shortly, Aβ_1-42 (A9810, Sigma, USA) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, 105228, Sigma, USA) to obtain a 1-mM solution. Subsequently, the solution was incubated at room temperature for 2 h. The HFIP was removed using a speedvac (Labconco, USA) to obtain thin clear peptide film. The film was stored at –80°C. Before use, the aliquoted film was redissolved with dimethyl sulfoxide (DMSO), followed by diluted to 100μM by phosphate buffered saline (PBS, 1×). The solution was incubated at 4°C for 16 h and then centrifuged at 13,000× g for 10 min at 4°C. Finally, the oligomeric form of Aβ_1-42 was prepared.

To obtain the Aβ_1-42 oligomers-induced AD model, the Aβ_1-42 oligomers preparation required to be stereotactic injected into the bilateral CA1 molecular layer of hippocampus of mice. After anesthesia with 2% isoflurane, a midsagittal incision was made on the scalp to sufficiently expose the skull. And then the mice were placed in a stereotactic apparatus (Benchmark, USA). To get the appropriate coordinate, the Paxinos and Watson atlas was referred to. The injection sites were±1.5 mm lateral to the midline, –2.0 mm posterior to bregma, and 1.5 mm deep from the dura. Using a nano-pump syringe with a speed of 0.4μL/min, the Aβ_1-42 oligomers were injected into the bilateral CA1 molecular layer of the hippocampus of mice (50μM in each side). After the injection, the needle was left for another 5 min and drawn out slowly. Finally, the skin was sutured and disinfected with penicillin sodium. For the mice in the Sham group, they were injected with an equivalent volume of vehicle (1% DMSO in PBS).

Experimental groups

The mice were divided into five groups (n = 12 in each group) randomly as follows: 1) sham operated group (Sham); 2) AD model group (Model), mice underwent stereotactic injections of Aβ_1-42 oligomers and were then treated with 0.5% Sodium Carboxymethyl Cellulose (CMC-Na) water solution intragastrically for 4 weeks; 3) luteolin group (Lut), mice underwent stereotactic injections of Aβ_1-42 oligomers and were then treated with 100 mg/kg/d luteolin (L11845, MEI5BIO, China) dissolved in 0.5% CMC-Na water solution intragastrically for 4 weeks; 4) exercise group (Exe), mice underwent stereotactic injections of Aβ_1-42 oligomers and then underwent wheel exercise for 1 h/d for 4 weeks, during which the mice could run on wheel with diameter of 12 cm voluntarily. Meanwhile, the mice were treated with 0.5% CMC-Na water solution intragastrically. 5) luteolin and exercise combination treatment group (Lut+Exe), mice underwent stereotactic injections of Aβ_1-42 oligomers. Then the mice were treated with 100 mg/kg/d luteolin dissolved in 0.5% CMC-Na water solution intragastrically and voluntary wheel exercise for 1 h/d for 4 weeks. Combined with the effective concentration of luteolin in the previous in vitro study [22] and the content of luteolin in the brain tissue after oral administration in the in vivo study [23], the administration concentration of luteolin in our study was set to 100 mg/kg/d. Cognitive behavioral changes were assessed using novel object recognition (NOR) test. The experimental schedule is shown in Fig. 1. The mice were anesthetized with 2% isoflurane and decapitated under unconscious state [24]. Then the brains were harvested for western blot, thioflavin T detection, and immunofluorescence assessments.

Fig. 1

Experimental schedule.

Novel object recognition (NOR) test

The NOR test was performed according to published methods [25]. Briefly, the NOR test was carried out in an open field arena, to which all mice were habituated for 10 min per day for 3 consecutive days in the absence of testing objects. On the fourth day, the training trial and testing trial were carried out successively. In the training trial, two identical objects were placed at different corners of the field arena. The mice were allowed to freely explore the arena for 5 min. After 30 min, in the testing trial, one of the familiar objects was replaced with a novel one. And the mice were reintroduced to explore the arena again for 5 min. The time that the mice spent on exploring the novel and familiar objects were measured respectively by the camcorder recording above. Object exploring was defined as the distance of the nose orientation to the object less than 2 cm, involving touching or sniffing the object. The discrimination index (DI) was calculated as the difference between the time spent on the novel object and the familiar one divided by the total exploration time. The data for DI were multiplied by 100 and expressed as a percentage.

Thioflavin T detection

Six mice in each group were anaesthetized and then perfused with PBS followed by 4% paraformaldehyde. Fixed with 4% paraformaldehyde for 24 h, the brain sections were embedded in paraffin and then cut into slices. The slices were baked at 60°C for 2 h. Dewaxed in xylene for 3 min, the slices were hydrated with ethanol of different concentrations (100%, 95%, 70% for 3 min respectively). After that, the slices were washed three times in deionized water, 3 min each. The citric acid buffer (pH = 6.0) was used in the antigen repair. To eliminate the activity of endogenous catalase, the slices were incubated with 3% H₂O₂ at room temperature for 10 min and then washed with PBS for 4 times, 3 min for each. The slices were incubated with 0.5% thioflavin T (T8320, Solarbio, China) solution at room temperature for 30 min and then washed with PBS for 3 times. The sealing tablet containing DAPI was added to the slide and the cover slide was covered. The film was kept at room temperature for at least 10 min away from light. The nail polish was applied around the cover glass so that the cover glass was fixed to the slide. Prepared films were stored at 4°C and fluorescence microscope (MF43, Guangzhou Micro-shot Technology, China) was used for observation.

Western blot

Total proteins from the hippocampus or cortex tissues were isolated using precooled RIPA buffer (Beyotime, China) and homogenized to be extracted. BCA protein assay kit (Solarbio, China) was used to determine the protein concentrations. Thereafter the protein samples were boiled and denatured. Equal amount of protein samples was separated by SDS-PAGE at suitable concentrations and transferred to the PVDF membranes (Millipore, USA). The membranes were then blocked with 5% non-fat milk for 1 h at room temperature and incubated at 4°C overnight with appropriate primary antibodies as follows: phospho-ULK1 (ser757) (YP1544, Immunoway, USA), P62 (AF5384, Affinity, USA), LC3 (14600-1-AP, Proteintech, USA), and GAPDH (ab181602, Abcam, England). After being rinsed with TBST for three times, the membranes were incubated with appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Rinsed with TBST for three times, the membranes were visualized with an ECL system (Millipore, USA), imaged in a ChemiScope Mini 3300 Chemiluminescence imaging system (ChemiScope 3300 Mini, China) and analyzed by ImageJ software.

Immunofluorescence

The section dewaxing, hydration and antigen repair were carried out according to the methods described in the Thioflavin T detection. After rinsing with PBS for 3 times, the slices were incubated with sealants containing 5% goat serum for 30 min at room temperature. Then the slices were incubated overnight at 4°C with primary antibodies GFAP (DF6040, Affinity, USA) and Iba1(DF7217, Affinity, USA) respectively. After rinsing with PBS for 3 times, each slice was added with diluted FITC-labeled goat anti-rabbit IgG and incubated at room temperature for 1 h under dark conditions. The sealing tablet containing DAPI was added to the slide and the cover slide was covered. The film was kept at room temperature for at least 10 min away from light. The nail polish was applied around the cover glass so that the cover glass was fixed to the slide. Prepared films were stored at 4°C and fluorescence microscope (MF43, Guangzhou Micro-shot Technology, China) was used for observation.

Statistical analysis

The data was analyzed with IBM SPSS 16.0. The results were expressed as mean±SEM. When the data met the equal-variance normal distribution, one-way ANOVA followed by LSD post hoc test was used for data analysis. Otherwise, Mann-Whitney U test was used. p < 0.05 was considered statistically significant.

RESULTS

The effects of luteolin, exercise, and combination treatment on cognitive deficits in Aβ_1-42 oligomers-induced AD model

NOR test was carried out to assess the effects of different treatments on the cognitive behaviors of mice. As shown in Fig. 2, the DI of the mice in AD model group was significantly decreased (p < 0.001) compared with that of the mice in sham group. Treatment with luteolin (100 mg/kg/d) could significantly elevate the DI to nearly 10% (p < 0.01), while the luteolin and exercise combination treatment could significantly increase the DI to nearly 15% (p < 0.001). However, treatment with exercise could also elevated the DI, but with no significance compared with that of the AD model group (p > 0.05). Above all, the results suggested that the combination treatment could ameliorate the cognitive deficits of Aβ_1-42 oligomers-induced AD model much better, compared with the treatment with luteolin or exercise respectively.

Fig. 2

The effects of luteolin, exercise and combination treatment on cognitive deficits in Aβ_1-42 oligomers-induced AD model. The discrimination index in testing trials of novel object recognition test. Data are presented as the mean±SEM (n = 12). ^# # #p < 0.001 compared with the sham group; ^**p < 0.01, ^***p < 0.001 compared with the AD model group.

The effects of luteolin, exercise, and combination treatment on Aβ content in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model

Aβ accumulation and plaque deposition was reported to drive the tau pathology, neuronal death, and neurodegeneration [26]. In our study, the thioflavin T detection and immunofluorescence staining were used to detect the effects of luteolin, exercise and combination treatment on Aβ in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model. As shown in Fig. 3, compared with the mice in the sham group, the Aβ content in the hippocampal CA1, CA3, and DG regions and the cortex of the mice in the AD model group was significantly increased. Both luteolin treatment and combination treatment significantly decreased Aβ in four brain regions, the latter more. Compared with the AD model group, the results showed that exercise treatment also significantly decreased Aβ in the measured brain regions, except in the cortex. Above all, the results suggested that the combination treatment could reduce the Aβ content in the Aβ_1-42 oligomers-induced AD model much better, compared with the treatment with luteolin or exercise respectively.

Fig. 3

The effects of luteolin, exercise and combination treatment on Aβ content in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model. A) The immunofluorescence of Aβ in the hippocampal CA1, CA3, and DG regions and the cortex of Aβ_1-42 oligomers-induced AD model. Scale bar = 20μm. B) The relative gray value of Aβ in the hippocampal CA1, CA3, and DG regions and the cortex between different groups. Data are presented as the mean±SEM (n = 6). ^# #p < 0.01, ^# # #p < 0.001 compared with the sham group; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 compared with the AD model group.

The effects of luteolin, exercise and combination treatment on the neuroinflammation in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model

It was reported that the accumulation of Aβ peptide in plaques is associated with chronic excessive inflammatory response [27]. Therefore, immunofluorescence staining was used to detect the activity of astrocytes (anti-GFAP antibody) and microglia (anti-Iba1 antibody) in the cerebral cortex and hippocampus of mice. As shown in Figs. 4 5, compared with the sham group, the GFAP and Iba1 expression levels in the hippocampal CA1, CA3, and DG regions were clearly increased in Aβ_1-42 oligomers-induced AD model. The Iba1 expression level was significantly increased, while there was no significantly increase of the GFAP expression level in the cortex of Aβ_1-42 oligomers-induced AD model. The activation of microglia and astrocytes by the injection of Aβ_1-42 oligomers was attenuated significantly by the combination treatment. However, the luteolin treatment could only significantly decrease the expression levels of GFAP in the hippocampal CA3 and DG regions and Iba1 in the hippocampal DG region. The exercise treatment could only significantly decrease the expression levels of GFAP in cortex and the Iba1 in the hippocampal CA3 and DG regions. Above all, the results suggested that the combination treatment could attenuate the neuroinflammation in the hippocampus and cortex of the Aβ_1-42 oligomers-induced AD model much better, compared with the treatment with luteolin or exercise respectively.

Fig. 4

The effects of luteolin, exercise and combination treatment on GFAP expression levels in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model. A) The immunofluorescence of GFAP in the hippocampal CA1, CA3, and DG regions and the cortex of Aβ_1-42 oligomers-induced AD model. Scale bar = 20μm. B) The relative gray value of GFAP in the hippocampal CA1, CA3, and DG regions and the cortex between different groups. Data are presented as the mean±SEM (n = 6). ^#p < 0.05, ^# # #p < 0.001 compared with the sham group; ^*p < 0.05, ^**p < 0.01 compared with the AD model group.

Fig. 5

The effects of luteolin, exercise and combination treatment on Iba1 expression levels in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model. A) The immunofluorescence of Iba1 in the hippocampal CA1, CA3, and DG regions and the cortex of Aβ_1-42 oligomers-induced AD model. Scale bar = 20μm. B) The relative gray value of Iba1 in the hippocampal CA1, CA3, and DG regions and the cortex between different groups. Data are presented as the mean±SEM (n = 6). ^#p < 0.05, ^# #p < 0.01, ^# # #p < 0.001 compared with the sham group; ^*p < 0.05, ^**p < 0.01 compared with the AD model group.

The effects of luteolin, exercise and combination treatment on expression levels of autophagy related proteins in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model

Autophagy, as a fundamental eukaryotic pathway, was reported to be closely related to infection, inflammation, and neurodegeneration [28]. Therefore, the autophagy related proteins were determined by western blot assay. As shown in Fig. 6, compared with the sham group, the protein expression levels of p-ULK1 (ser757) and P62 in the hippocampus and cortex of Aβ_1-42 oligomers-induced AD model were significantly upregulated, and LC3 II/LC3 I was significantly downregulated. The exercise and combination treatment could downregulate the protein expression level of p-ULK1 in the hippocampus and cortex significantly and upregulate the LC3 II/LC3 I in the cortex significantly. The combination treatment upregulated the protein expression level of LC3 II/LC3 I in the hippocampus. Notably, the luteolin, exercise and combination treatment could downregulate the expression of P62 protein to different degrees, while there was no significant difference.

Fig. 6

The effects of luteolin, exercise, and combination treatment on expression levels of autophagy related proteins in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model. A) The protein levels of p-ULK1(ser757), P62 and LC3II/I in the hippocampus and the cortex of Aβ_1-42 oligomers-induced AD model were measured by western blot. Quantitative analysis of p-ULK1(ser757), P62 and LC3II/I in the hippocampus (B) and the cortex (C) between different groups normalized by the expression levels of the sham group. Data are presented as the mean±SEM (n = 3). ^#p < 0.05, ^# #p < 0.01 compared with the sham group; ^*p < 0.05, ^**p < 0.01 compared with the AD model group.

DISCUSSION

In this study, our hypothesis has been verified that the luteolin and exercise combination therapy could improve cognitive impairment induced by AD and improved inflammation and autophagy better than monotherapy to some extent. Luteolin (100 mg/kg/d) combined with exercise could significantly improve the memory behavior of AD model mice in NOR test, and the improvement degree was greater than that of simple intervention. Further experiments showed that luteolin and exercise alone or in combination could reverse the increase of Aβ content, the activation of astrocytes and microglia, and the decrease of the level of autophagy in hippocampus and cortex in AD model induced by Aβ_1-42 oligomers. While the combination treatment involved more intact hippocampal and cortical areas, with greater degree of changes.

Aβ oligomers could evoke activation of neuroinflammation to induce neurotoxicity [28] and cognitive behavior impairment [29]. Qamar et al. found that luteolin may serve as a scaffold for the design of better inhibitors with higher affinities toward Aβ amyloid formation using molecular docking and molecular dynamics simulation [30], which may partly explain our finding that luteolin reduced Aβ levels in the hippocampus and cortex. Luteolin has been recorded to exhibit anti-inflammatory properties. In our study, however, the luteolin treatment could not significantly ameliorate the GFAP expression levels in CA1 and cortex, Iba1 expression levels in CA1, CA3, and cortex, or the autophagy-related proteins in hippocampus and cortex in AD model induced by Aβ_1-42 oligomers. Yuan et al. found that luteolin (60 mg/kg) treatment remarkably ameliorated noise-induced pro-inflammatory cytokines in the hippocampus and prefrontal cortex, whereas the luteolin (20 mg/kg) treatment could only reverse the increase of certain inflammatory cytokines [31]. Accordingly, it was suggested that luteolin (100 mg/kg) treatment could only alter inflammatory cell activation in the hippocampus sub region but not in the cortex in our study, which may be dose-dependent.

In addition, it seems like that there were lack of beneficial effects of exercise for the indicators in our study. He et al. carried out immunofluorescence or an enzyme-linked immunosorbent assay and suggested that voluntary wheel running attenuated the accumulation of amyloid plaques and neuroinflammation, which was consistent with our findings [29]. However, in our study, the exercise treatment could not significantly improve the performance of mice in NOR test, or significantly ameliorate the Aβ content in cortex, the GFAP expression levels in sub regions of hippocampus, Iba1 expression levels in CA1 and cortex, or the LC3 II/I in hippocampus. This outcome was partly supported by the previous findings. Robison et al. demonstrate that long-term voluntary wheel running does not alter vascular amyloid burden but reduces neuroinflammation in the Tg-SwDI mouse model of cerebral amyloid angiopathy [30]. Bernardo et al. also find that voluntary physical activity alone is not able to counteract the AD-related dexterous consequences [31]. As for the influence of voluntary exercise on memory, despite some studies on the topic, controversial results exist regarding the effectiveness of the onset of the access to a running wheel, the period with free access to the running wheel, the different exercise types, durations and so on, all of which may affect the outcome [32]. What is more, there were brain region specific effects of luteolin and exercise. There appeared to be synergy in hippocampus sub regions and cortex for Aβ, Iba1, and autophagy-related proteins. However, for GFAP, there appeared to be synergy in CA1 and cortex, and luteolin might drive the effects on CA3 and DG. This might be attributed to the regional specificity of astrocytes remodeling of and complex immune regulation in the context of inflammation [33, 34].

Autophagy, as a major intracytoplasmic protein degradation pathway, was reported to be correlated with the pathology of AD. Autophagy was regulated by a series of proteins, such as LC3, ULK 1, P62, and so on. The ULK complex could mediate the autophagy initiation. When the ULK 1 is phosphorylated at Ser 757, the ULK 1 kinase activity is suppressed. As a result, the autophagy initiation was inhibited [11]. The increase of LC3 II/I contributed to the autophagosome formation. P62, a prominent examples of autophagy receptors, could recognize ubiquitinated proteins or organelles targeted for degradation [35]. It was reported that Aβ may be degraded by autophagy, and upregulation of autophagy has been shown to reduce Aβ levels in a number of systems [36]. In our study, the expressions of autophagy-related proteins ULK 1 (ser757) and P62 were increased, and the expression of LC3 II/I was decreased in AD model group, which was consistent with what reported previously [35]. Luteolin could enhance the autophagy but with no significance, suggesting that luteolin alone has limited effect in promoting autophagy in AD model group. Luteolin was reported to attenuate myocardial injury by enhancing autophagy [37] but inhibit autophagy in allergic asthma [38]. The exercise treatment could increase the expression of LC3 II/I but with no significance in hippocampus. Accordingly, luteolin and exercise combination treatment could promote autophagy better.

In our study, the body weight of mice in the Exe group was significantly improved compared with that in AD model group (data not shown), which was consistent with Lim et al. [39] results. However, there are also other studies obtaining the contrary results [40]. This might be due to that the behavioral mechanisms for control of body weight during voluntary wheel exercise are related to the animal species, age, body composition before the initiation of exercise and perhaps, other complex traits as well [41, 42]. The running activity and food intake may explain these results, but they were not included in this study and will be taken into account in further research.

Both the FDA and the European Medicines Agency have published industry guidelines highlighting non-clinical and clinical data needed for combination therapy development [4], for which our study is a support. In fact, the pharmaceutical combination therapy is an increasingly common treatment for AD. For example, memantine plus donepezil showed superior results in daily activities, global assessment, and neuropsychiatric symptoms [43]. It has also been suggested that a combination of Aducanumab and ultrasound may be necessary to boost antibody levels in the brain [44]. Exercise, mobilizing most of the body’s functions, was also reported to combine with nutrition interventions to influence cognitive functioning in the elderly. An additive effect was found when docosahexaenoic acid supplementation was combined with physical exercise [45]. However, in another study, the combination of exercise training and fish oil did not confer an additional beneficial effect [46], which may be due to the duration of study, the dose of individual nutritional supplementation and the methods of assessment.

Given the poor efficacy of existing drug regimens for the treatment of AD, this study combined drug therapy and exercise rehabilitation, providing new possibilities and evidence for the treatment of AD and illustrating the advantages of this combination therapy in regulating memory behavior, neuroinflammation and autophagy. The results of this study supported further in-depth and more detailed study of this combination treatment regimen. However, this study also has its limitations. This study clarified the relationship between luteolin and exercise combination therapy and neuroinflammation and autophagy. How it works by improving neuroinflammation and regulating autophagy is worthy of further in-depth research and analysis.

Furthermore, as for the underlying mechanism of this combination therapy to improve cognitive impairment, it was reported that microglial Wnt/β-catenin signaling regulated microglial pro-inflammatory activation [47]. Accordingly, we speculate that the combination therapy might attenuate neuroinflammation through Wnt/β-catenin signaling pathway and thus improve memory impairment. Next, we will study the role of this pathway through gene editing or using inhibitors, in order to fully understand the mechanisms of action and promote clinical application.

Conclusions

Luteolin and exercise combination therapy prevented Aβ_1-42 oligomers-induced cognitive impairment, possibly by decreasing neuroinflammation and enhancing autophagy. The combination of luteolin and exercise therapy may be a useful therapeutic option for preventing and/or delaying the progression of memory dysfunction of AD. This study provides a useful example for shifting the paradigm and implementing a multivariate and personalized approach to the treatment of AD.

Footnotes

ACKNOWLEDGMENTS

The authors have no acknowledgments to report.

FUNDING

This study was supported by the science and technology development project of Beijing Rehabilitation Hospital, Capital Medical University (grant number 2020R-003, 2021-010), China Postdoctoral Science Foundation (2021M701953) and Shuimu Tsinghua Scholar Program (2021SM110).

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

DATA AVAILABILITY

The data supporting the finding of this study are available on request from the corresponding author. The data are not publicly available due to privacy.

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Luteolin and Exercise Combination Therapy Ameliorates Amyloid-β 1-42 Oligomers-Induced Cognitive Impairment in AD Mice by Mediating Neuroinflammation and Autophagy

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

METHODS

Animals

Aβ1-42 oligomers-induced AD model

Experimental groups

Novel object recognition (NOR) test

Thioflavin T detection

Western blot

Immunofluorescence

Statistical analysis

RESULTS

The effects of luteolin, exercise, and combination treatment on cognitive deficits in Aβ1-42 oligomers-induced AD model

The effects of luteolin, exercise, and combination treatment on Aβ content in hippocampus and cortex in Aβ1-42 oligomers-induced AD model

The effects of luteolin, exercise and combination treatment on the neuroinflammation in hippocampus and cortex in Aβ1-42 oligomers-induced AD model

The effects of luteolin, exercise and combination treatment on expression levels of autophagy related proteins in hippocampus and cortex in Aβ1-42 oligomers-induced AD model

DISCUSSION

Conclusions

Footnotes

ACKNOWLEDGMENTS

FUNDING

CONFLICT OF INTEREST

DATA AVAILABILITY

References

Aβ_1-42 oligomers-induced AD model

The effects of luteolin, exercise, and combination treatment on cognitive deficits in Aβ_1-42 oligomers-induced AD model

The effects of luteolin, exercise, and combination treatment on Aβ content in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model

The effects of luteolin, exercise and combination treatment on the neuroinflammation in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model

The effects of luteolin, exercise and combination treatment on expression levels of autophagy related proteins in hippocampus and cortex in Aβ_1-42 oligomers-induced AD model