https://orcid.org/0000-0002-7362-9972
Abstract
Xanthohumol (XAN) is a unique component of Humulus lupulus L. and is known for its diverse biological activities. In this study, we investigated whether Xanthohumol could ameliorate memory impairment of APP/PS1 mice, and explored its potential mechanism of action.
APP/PS1 mice were used for in vivo test and were treated with N-acetylcysteine and Xanthohumol for 2 months. Learning and memory levels were evaluated by the Morris water maze. Inflammatory and oxidative markers in serum and hippocampus and the deposition of Aβ in the hippocampus were determined. Moreover, the expression of autophagy and apoptosis proteins was also evaluated by western blot.
Xanthohumol significantly reduced the latency and increased the residence time of mice in the target quadrant. Additionally, Xanthohumol increased superoxide dismutase level and reduced Interleukin-6 and Interleukin-1β levels both in serum and hippocampus. Xanthohumol also significantly reduced Aβ deposition in the hippocampus and activated autophagy and anti-apoptotic signals.
Xanthohumol effectively ameliorates memory impairment of APP/PS1 mice by activating mTOR/LC3 and Bax/Bcl-2 signalling pathways, which provides new insight into the neuroprotective effects of Xanthohumol.
Introduction
With the aggravation of social ageing problems, senile neurodegenerative diseases have become increasingly prominent.[1] Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by a decline in memory and cognitive ability, and β-amyloid deposition in the brain is the most important pathological feature due to its diversity of neuronal toxicity.[2–4] The abnormal Aβ deposition affects the physiological activities of neurons in the brain and induces a series of complex reactions such as neuroinflammation, oxidative stress, loss of transmitters and metabolic disorders, leading to neuronal damages.[5–7] The pathogenesis of AD is complex, and the current drugs used in its treatment only delays the onset of the disease, which has great limitations.[8] Therefore, it is of great clinical interest that new drugs and therapeutic targets are developed.
Xanthohumol (XAN) is a unique active ingredient of hops and is recognized as an effective anticancer medicine,[9] it also exerts other beneficial effects including anti-oxidation, antibiosis, anti-inflammation and cardiovascular protection.[10, 11] Additionally, the neuroprotective effect of XAN also has been demonstrated in many in vitro and in vivo studies. XAN is reported to attenuate obesity-induced neurocognitive metabolic damage,[12] protect against ageing-related brain damage,[13] reduce glutamate excitotoxicity[14] and improve cognitive function in young animals.[15] XAN also reduces Aβ-induced reactive oxygen species (ROS) generation in SH-SY5Y cells,[16] and inhibits the high phosphorylation of tau in N2a stably transfected with amyloid precursor protein (N2a/APP) cell.[17, 18] It is also reported to alleviate H2O2-induced oxidative stress injury and improve ischemia-induced brain damage.[19–21] Nevertheless, it is not clear whether XAN can raise learning ability and memory reproduction in APP/ Presenilin-1 (APP/PS1) mice and the underlying mechanism of action by which XAN exerts its effects needs to be elucidated.
Autophagy plays a crucial role in degrading intracellular denatured proteins and damaged organelles.[22] Studies have confirmed that abnormal autophagy is one of the key pathological features in the AD pathological process,[23] and activating autophagy is the main way to clear Aβ deposition in the AD brain.[24, 25]. Therefore, it is considered to be a potential way to treat AD by effectively regulating autophagy, thus enhancing the clearance ability of Aβ. A mammalian target of rapamycin (mTOR) signalling pathway is the focus of autophagy research, and inhibition of mTOR protein is the beginning of the autophagy pathway.[26] Rapamycin, an autophagy activator, has been found to markedly decrease the deposition of Aβ protein and restore the learning and memory function in AD patients, which is related to its antioxidant stress and anti-inflammatory activities.[27] Maiese et al. have also found that inhibition of mTOR phosphorylation can repair and consolidate memory and enhance synaptic plasticity.[28] Beclin1 is am autophagy-related gene necessary to initiate autophagy and Beclin1 reduction leads to autophagy inhibition, Aβ peptide accumulation and neurodegeneration.[29] Cheng et al. have shown that isovitexin improves spatial memory impairment in AD mice via decreasing inflammatory factor levels and promoting the expression of light chain 3-Ⅱ (LC3-Ⅱ) and beclin-1 in the hippocampus. Ubisol-Q10 supplementation is also reported to up-regulated the expression of beclin-1 in the brain of AD mice.[30] Furthermore, abnormal neuronal apoptosis cycle and over-expression of apoptotic genes lead to apoptosis and loss of a large number of neurons.[31] One study has shown that hops ameliorate brain injury in mice by reducing neuronal apoptosis.[32]
Therefore, the present study is the first to report whether XAN can inhibit Aβ deposition and raise learning and memory levels of APP/PS1 mice by regulating mTOR/LC3II and B-cell lymphoma-2-Associated X/B-cell lymphoma-2 (Bax/Bcl-2) signalling pathways, with a view to opening avenues for XAN application and AD therapy.
Materials and Methods
Reagents
XAN was purchased from Liding Biotechnology (Shanghai, China). N-acetylcysteine (NAC) and bicinchoninic acid (BCA) protein assay kits were supplied by Beyotime Biotechnology (Shanghai, China). Carboxymethyl cellulose (CMC-Na) was purchased from Dalian Meilun Biotechnology (Dalian, China). Enzyme-linked immunosorbent assay (ELISA) for the measurement of Interleukin-6 (IL-6), Interleukin-1β (IL-1β) and biochemistry kits for superoxide dismutase (SOD) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Paraformaldehyde (4%) was purchased from Beijing Labgic Technology Co., Ltd. (Beijing, China). Rabbit anti-Beclin1 (AB_2692326, 1 : 2000) and anti-LC3B (AB_2827794, 1 : 2000) were provided by Abcam (Cambridge, MA, USA). Rabbit anti-Bax (AB_2833304, 1 : 1000) and anti-Bcl-2 (AB_2835021, 1 : 1000) were obtained from Affinity Biosciences (Cincinnati, OH, USA). Rabbit anti-p-mTOR (AB_10691552, 1 : 1000), anti-GAPDH (AB_561053, 1 : 1000) and anti-rabbit IgG (AB_2099233, 1 : 1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).
Animals and treatment
40 nine-month-old male APP/PS1 mice and 10 male wild-type C57BL/6J mice weighing 30±1 g were used in this study. Mice were purchased from Nanjing Institute of Biomedicine (Jiangsu, China) (Certificate No.: 201802415, license No.: SYXK (Su) 2015-0001) and fed in the Laboratory Animal Center of the Second Military Medical University. Animals were maintained in a temperature-controlled environment (21℃ ± 2℃), on a 12 : 12 h light-dark cycle and had free access to food and tap water. All animal experiments were performed according to the animal care and user guidelines issued by the Second Military Medical University, China, and were approved by the Committee on Ethics of Medical Research Second Military Medical University (approved No. 201802415).
Mice were classified equally into five groups of 10 mice namely : CONTROL (CON), APP/PS1, APP/PS1 + NAC (100 mg/kg/d), APP/PS1+XAN-L (30 mg/kg/d) and APP/PS1 + XAN-H (90 mg/kg/d). NAC was used as the positive control drug, and NAC and XAN were dissolved in 0.5% CMC-Na. Mice in the treatment groups were given NAC and XAN, while mice in the CON and APP/PS1 groups were given an equal volume of 0.5% CMC-Na by gavage. The selection of the doses for drugs in the experiment was based on our previous study.[33] The experimental dosage was adjusted according to the weight of 0.1mL/g, and all drugs are given 6 days a week for 2 months. The feeding cycle of mice was determined pre-experimentally.
Morris water maze test
Morris water maze (MWM) test was used to evaluate the learning and spatial memory of mice and was implemented as previously described.[34] The water maze consists of a biological cylindrical pool with a radius of 120 cm, filled with a white liquid prepared by titanium dioxide, and the temperature is controlled at 25 ± 1°C. The experiment consists of two parts: directional navigation tests and spatial probe tests. In the oriented navigation test, Escape latency was recorded. The mice were trained daily to search for a platform hidden underwater using different colours and shapes of references on the wall of the pool in more than three experiments for more than five continuous days. On day 6, the platform was taken for probe test, and all mice were assessed for their learning and spatial memory via the number of platform traverses and the swimming time of mice spent in the quadrants where the platform is located.
Enzyme-linked immunosorbent assays
The IL-6, IL-1β, and SOD levels in hippocampus and serum were measured by ELISA Kits according to the manufacturer’s protocol.
Immunohistochemistry
The brain sections were sequentially immersed in xylene and ethanol of different concentrations (100%, 85%, 75%, 0%) for dewaxing and hydration. The brain sections were immersed in sodium citrate buffer (pH = 6.0) and heated in a microwave oven. The sections were then incubated in 3% hydrogen peroxide for 25 min and then sealed with 3% bovine serum albumin (BSA) for 30 min at room temperature. After sealing, the sections were incubated with prepared primary antibody overnight at 4°C and then incubated with secondary antibody for 50 min at room temperature. Finally, fresh DAB chromogenic solution was added for colouration and hematoxylineosin was used to stain the sections, the positive section is stained brown-yellow. Image-pro plus software was used to calculate positive coverage.
Tissue preparation and Congo red staining
First, anaesthetized mice (pentobarbital, 100 mg/kg, i.p.) were perfused with saline (0.9%) at 4℃, and then the mice were fixed with 4% paraformaldehyde by heart perfusion. Following this, the skull of the mouse was gently stripped off on a clean operating table to expose the brain tissue which was fixed overnight in 4% paraformaldehyde (4℃). Brain tissue was then dehydrated and precipitated by immersing in a 30% sucrose solution before sectioning.
The hippocampus slices were sequentially by placing them in xylene i (20 min), xylene ii (20 min), absolute ethanol i (5 min), absolute ethanol ii (5 min),75% alcohol (5 min) and tap water. The slices were immersed in Congo red dye for 1 h, then washed for 2 min with tap water and differentiated by potassium hydroxide solution. When the positive plaque was obvious and the background was colourless, they were washed with tap water. The hippocampus slices were then stained with hematoxylin and dehydrated before microscopic examination and image analysis.
Western blot
The expression of p-mTOR, Beclin1, LC3, Bcl-2 and Bax in the hippocampus were measured.
150 μl cell lysate containing 1% protease phosphatase inhibitor and 1% Phenylmethylsulfonyl fluoride (PMSF) was added to the hippocampus and homogenized at low temperature. The homogenate was placed on ice for 30 min and centrifuged at 4°C to obtain the supernatant. The protein concentrations in samples were analysed using BCA test kits. Each protein sample was then mixed with 5× loading buffer, and left in a boiling water bath for 10 min. The proteins were separated by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to the 0.45 μm polyvinylidene fluoride membranes by the wet transfer method.[35] After sealing in 5% BSA blocking solution for 1 h at room temperature, membranes were incubated with the corresponding prediluted primary antibodies overnight at 4°C. The membranes were then washed three times with tris-buffered saline containing 1% Tween-20 (TBST) for 10 min, and then incubated with secondary antibody horseradish peroxidase-labelled goat anti-rabbit IgG (1 : 2000) for 1 h at room temperature. Following this, the membranes were again washed three times with TBST for 10 min and then coloured with a luminescent solution for 30 s. Finally, the membranes were exposed to Tanon-5200 chemiluminescence instrument and the band intensity was quantified by optical density using Image J software.[36] glyceraldehyde-3phosphate dehydrogenase (GAPDH) protein was used as the internal reference for data analysis.
Statistical analyses
Data presented in this study are the mean ± standard error of the mean (SEM). All results were analyzed by using GraphPad Prism 5 and performed with one-way analysis of variance followed by Tukey’s post-hoc comparisons tests. P < 0.05 was deemed statistically significant.
Results
Xanthohumol significantly improves the learning and spatial memory of APP / PS1 mice
Compared with APP/PS1 mice, normal mice exhibited shorter escape latency during training sessions (P < 0.05). On the fifth day of XAN treatment, the learning and memory ability of APP/ PS1 mice was markedly improved (P < 0.01), and the effect of a high dose of XAN was superior to that of the low dose, showing a lower latency (Figure 1a). Also, NAC significantly alleviated the learning and memory impairment of APP/PS1 mice, and there was a significant difference in latency between NAC group and APP/PS1 group (P < 0.01). The longer the stay duration and the higher platform crossing times, the stronger is the memory ability of mice. Our results showed that mice in the CON group had significant higher platform swimming time and crossing times compared to mice in the APP/PS1 group (P < 0.05), whereas the swimming time and platform crossing times of NAC and XAN-treated (30 and 90 mg/kg) mice in the target quadrant group increased significantly compared to the APP/PS1 mice (P < 0.05) (Figure 1b and c). Similarly, the test results of mice in the XAN-H group were better than those in the XAN-L group. In addition, there was no significant difference in swimming speed in each group, indicating that the swimming ability of mice was approximately the same (Figure 1d).
Effects of XAN on spatial learning and memory of APP/PS1 mice (n = 7). (a) Escape latency time of mice in each group; (b) Crossing platform times of mice in each group; (c) Target quadrant residence time of mice in each group; (d) Average swimming speed of mice in each group. The data are expressed as means ± SEM. *P < 0.05, ***P < 0.001 versus CON; #P < 0.05 versus APP/PS1.
Xanthohumol effectively attenuates the neuroinflammation in APP/PS1 mice
Inflammatory levels in the hippocampus and serum are also important indicators for AD assessors. As shown in Figure 2(a and b), the levels of IL-1β and IL-6 in the hippocampus of APP/PS1 mice were markedly higher than those in the CON group (P < 0.001), whereas NAC and XAN (30 and 90 mg/kg) treatments significantly reduced the IL-1β and IL-6 levels in the hippocampus of APP / PS1 mice (P < 0.001). Furthermore, the IL-1β and IL-6 levels of serum in APP/PS1 mice were markedly increased compared to those in normal mice. Also, similar to the positive drug, XAN (30 and 90 mg/kg) significantly reduced serum IL-1β and IL-6 levels in APP/PS1 mice (Figure 2c and d). Moreover, XAN decreased inflammatory cytokines in the hippocampus and serum of APP/PS1 mice in a dose-dependent manner.
Anti-inflammatory effect of XAN on APP/PS1 mice (n = 7). The IL-1β and IL-6 levels both in the hippocampus (a, b) and serum (c, d). The data are expressed as means ± SEM. ***P < 0.001 versus CON; ###P < 0.001 versus APP/PS1.
Xanthohumol effectively reduces the level of oxidative stress in APP/PS1 mice
Furthermore, SOD levels in the hippocampus and serum were detected. As shown in Figure 3(a and b), the SOD levels in hippocampus and serum of APP/PS1 mice were significantly decreased compared with those in the CON group (P < 0.001), whereas NAC and XAN (30 and 90mg/kg) treatments significantly increased the SOD levels in hippocampus and serum of APP/PS1 mice (P < 0.001).
Antioxidant effect of XAN on APP/PS1 mice. (a, b) SOD levels both in hippocampus and serum (n = 7); (c) the expression of SOD in hippocampus(n = 3). The data are expressed as means ± SEM. *P < 0.05 versus CON; #P < 0.05 versus APP/PS1.
Also, to further determine the effect of XAN on SOD level in the hippocampus of APP/ PS1 mice, the expression of SOD was determined by Western blot (Figure 3c). In accordance with the above results, the expression of SOD in the hippocampus of APP/PS1 mice was significantly down-regulated compared with that of normal mice, while NAC and XAN (30, 90 mg/kg) treatments significantly up-regulated the expression of SOD (P < 0.01).
Xanthohumol reduced Aβ deposition in the hippocampus of APP/PS1 mice
Senile plaques formed by Aβ are one of the main signs of AD, and the accumulation of amyloid plaques in brain tissue is the main cause of spatial learning and memory disorders.[37] After Congo red staining, the brownish red or orange precipitates were positive for Aβ, suggesting the presence of Aβ aggregation in the brain. As Figure 4 showed, compared with normal mice, a large number of subcircular orange plaques of varying sizes were observed in the hippocampus of APP/PS1 mice (P < 0.01), and the plaques were surrounded by blue activated glial cells bands. After treatment with NAC and XAN (30 and 90 mg/kg), the number of orange-red precipitates in the hippocampus of APP/PS1 mice decreased significantly (P < 0.01), indicating that NAC and XAN can improve the deposition of Aβ in the hippocampus of APP/PS1 mice.
Effect of XAN on amyloid plaques in APP/PS1 mice (n = 3). (a) Congo red-positive plaque in the hippocampus of APP/PS1 mice; (b) Quantitative analysis of numbers of Aβ. The data are expressed as means ± SEM. **P < 0.01 versus CON; ##P < 0.01 versus APP/PS1.
Xanthohumol activates autophagy and apoptosis pathways
To further study the relationship between XAN improving the learning and memory ability of APP/PS1 mice and autophagy signal pathway, the expression of autophagy key proteins including p-mTOR, beclin-1 and LC3 were detected via Western blot. Our data showed that compared with normal mice, the expression of p-mTOR in the hippocampus of APP/PS1 mice was significantly up-regulated, and the expression of beclin-1 and LC3 was significantly down-regulated (P < 0.05) (Figure 5). XAN (30, 90 mg/kg) significantly activated the autophagy pathway mainly manifested by the decrease of p-mTOR expression (P < 0.01) and the increase of beclin-1(P < 0.01) and LC3 II (P < 0.01) protein expression (Figure 5). Moreover, the results of the high concentration treatment group (XAN-H) were better than that of the low concentration group (XAN-L), which was consistent with the results of previous experiments.
Western blot analysis of autophagy protein (n = 3). (a) Western blot of p-mTOR, LC3, Beclin-1 and GAPDH was used as an internal control; (b-d) Quantitative analysis of p-mTOR, LC3 and Beclin-1. Western blot data were representative of three independent experiments with a similar result. The data are expressed as means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 versus CON; #P < 0.05, ##P < 0.01 and ###P < 0.001 versus APP/PS1.
We also studied the relationship between the neuroprotective effect of XAN and its anti-apoptotic effect. The findings suggested that the expression of pro-apoptotic protein Bax in the hippocampus of APP/PS1 mice was significantly increased, and the expression of anti-apoptotic protein Bcl-2 was significantly reduced compared with those of normal mice. (P < 0.05) (Figure 6). XAN (30 and 90 mg/kg) significantly decreased Bax expression and increased Bcl-2 expression in a dose-dependant manner compared with APP/PS1 mice (P < 0.05), indicating the strong anti-apoptotic ability of XAN.
Western blot analysis of apoptotic protein (n = 3). (a) Western blot of Bcl-2 and Bax, GAPDH was used as internal control; (b-c) Quantitative analysis of Bcl-2 and Bax. Western blot data is representative of three independent experiments with similar result. The data are expressed as means ± SEM. *P < 0.05 and **P < 0.01 versus CON; #P < 0.05 and ##P < 0.01 versus APP/PS1.
Discussion
There are many natural compounds derived from traditional Chinese medicine which have been reported to display neuroprotective effects that inhibit the deposition of Aβ in the brain, including dihydroartemisinin, albiflorin, and osthole.[38–40] However, there have been no studies to clarify the effect of XAN on Aβ deposition in the brain of APP/PS1 mice before this work. Preceding studies have shown that XAN could prevent cerebral ischemia caused by middle cerebral artery occlusion in rats,[41] which may rely on the α, β-unsaturated ketone structure in XAN.[42] In this study, we reveal for the first time that XAN displays potent improvement against Aβ-mediated memory impairment in mice via activating autophagy and anti-apoptotic signalling pathways.
The deposition of Aβ in the brain is critically affected by autophagy. Under physiological conditions, autophagy is the key to the homeostasis of neuronal energy metabolism by recycling damaged organelles and defective proteins. Accumulation of immature autophagic vacuoles (AVs) in the neurites of brain dystrophy in patients with AD indicates that the autophagy process is disrupted,[43,44], which suggests the impairment of autophagy in AD. As a result, regulation of autophagy may be a new approach to AD therapy. As an important serine‐threonine protein kinase, mTOR is a classical negative regulator of autophagy[45] which mainly depends on the regulation of mTOR pathway, which is activated early in AD patients.[46,47]. In this study, we found that transgenic mice exhibited hyperphosphorylated mTOR compared with the control group, while XAN treatments could inhibit the abnormal phosphorylation, and thus promoted autophagy. LC3 II is specifically localized in autophagosome membranes,[48] and Beclin-1 is necessary for the formation of autophagosome, both of which are specific autophagy markers.[49] Study has shown that Beclin-1 overexpression reduces Aβ deposition in transgenic mice[50] and our results show that the expression of both Beclin-1 and LC3 proteins were significantly decreased in the XAN treatment group, indicating that XAN activated the autophagy pathway. Our results showed that XAN inhibits Aβ levels in APP/PS1 transgenic mice via activating mTOR/LC3II signalling pathway, thereby ameliorating memory dysfunction.
Apoptosis is a kind of active and orderly cell death mode determined by genes, which exists in the process of organism development. Fossati et al. confirmed the differential expression of apoptosis-related genes caused by Aβ in endothelial cells (EC).[51] Bcl-2 protein has high homology with the survival gene Ced-9 of Caenorhabditis elegans. Bcl-2 enhances cell survival and Bax promotes cell death,[52] both of which are key apoptosis regulators in the apoptosis signalling transduction pathway. Our study showed that XAN attenuated deposition of Aβ in vivo in the hippocampus of APP/ PS1 mice via decreasing the expression of Bax and increasing the expression of Bcl-2.
Treatment of APP/PS1 mice with XAN significantly regulates a set of mTOR-associated autophagy and apoptosis proteins as well as the corresponding inflammatory and oxidative factors, including the antioxidant enzymes SOD and pleiotropic cytokine IL-6 and IL-1β. A mTOR signalling pathway is closely connected to apoptosis, and mTOR regulation of apoptosis is different in different diseases. The regulation of mTOR on apoptosis may be related to its downstream related apoptotic proteins, such as Bcl-2.[53] Our results suggest that XAN may inhibit Aβ-induced neuronal apoptosis by activating the mTOR/ LC3 pathway, thereby improving the learning and memory of transgenic mice. In addition, it is worth noting that oxidative stress represented by lipid peroxidation can be observed in the brain of AD patients,[54] and the increase of endogenous reactive oxygen species will lead to neuronal apoptosis. The production of SOD antioxidant enzymes can protect the body from apoptosis caused by Aβ, which is consistent with our results in this study (Figure 3). These data provide evidence for the role of XAN in improving the learning and memory of APP/PS1 mice, which is of great significance for further research on the potential neuroprotective effect of XAN. However, there are limitations to our study, for example, although both doses of XAN significantly increased the learning and memory ability of APP/PS1 mice while improving the levels of inflammation and oxidation, the effect of high-dose XAN was only slightly higher than that of low-dose XAN, and there was no significant difference between the two doses. Therefore, it allows us to consider whether higher concentrations of XAN will possess higher neuroprotective effects on APP/PS1 mice, which we will explore further in future studies.
Conclusion
In conclusion, XAN manifests effective improvement of memory impairment in APP/PS1 mice through activating mTOR/LC3 and its downstream pathway Bcl-2/Bax, inhibiting apoptosis, and thus reducing Aβ accumulation in brain tissues. In addition, it is suggested that the neuroinflammation reaction of APP/PS1 mice was reduced, and the activity of the antioxidant enzyme was increased significantly after treatment with XAN. Although XAN is a potential compound in vivo, in-vitro experiments are still needed to further certify it as a potent neuroprotective drug. Besides, the relationship between autophagy and apoptosis signalling pathway should be further investigated using pharmacological inhibitors or gene silencing technique.
Author’s contributions
Xiaolei Sun, Jiabao Zhang, and Yunxiang Guo carried out the pharmacological tests; Tianshuang Xia, Guoping Wang and Xiaojin Li participated in the experimental work; Xiaolei Sun and Jiabao Zhang performed the statistical analysis; Xiaolei Sun wrote the manuscript; Khalid Rahman corrected English grammar mistakes; Ting Han, Nani Wang and Hailiang Xin performed the design of the study and contributed with reagents and analytical tools.
Funding
This work was supported by the National Natural Science Foundation of China (U1603283).
Conflict of interest
The authors declare that they have no competing interests.
References
Author notes
These authors contributed equally to this work.
