Housefly (Musca domestica) larvae powder, preventing oxidative stress injury via regulation of UCP4 and CyclinD1 and modulation of JNK and P38 signaling in APP/PS1 mice

Abstract

Housefly (Musca domestica) Larvae powder (HL) is rich in antioxidants. As oxidative stress is considered as one of the main pathogenesis in Alzheimer's Disease (AD), this study was designed to explore the protective effects of HL as an antioxidant on APP/PS1 mice. 2-Month-old APP/PS1 mice were divided into a model control (MC) group, a Donepezil group and a HL group, and C57BL/6 mice were used as the normal control (NC) group. After 180 days of treatment, the memory ability was measured by Morris Water Maze (MWM). The presence of Aβ and the expression of Uncoupling Protein 4 (UCP4) and CyclinD1 were detected by immunohistochemistry. The expressions of Superoxide Dismutase 1 (SOD1), Catalase (CAT) and Mitogen-activated Protein Kinase (MAPK) signal pathways were measured by western blotting. Compared with untreated APP/PS1 mice, the memory abilities of the HL-treated mice were significantly improved. Furthermore, the HL treatment not only down-regulated the deposition of Aβ and the expression of CylinD1, but also increased both the mRNA and protein levels of SOD, CAT, and UCP4, and enhanced the phosphorylation of JNK and P38 MAPK activation. In conclusion, these results suggest that HL may have a protective effect against memory impairment and prevent oxidative stress-induced injury via the regulation of UCP4 and CyclinD1 and the modulation of JNK and P38 MAPK signaling in AD.

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1. Introduction

Alzheimer's disease (AD) is a degenerative disease of the central nervous system, which is characterized by progressive dementia and cognitive impairments. The main pathological features are senile plaques composed of extracellular amyloid-β (Aβ) peptide, intracellular neurofibrillary tangles (NFTs) composed of hyper-phosphorylated tau, and loss of neurons.^1,2 Since the exact pathogenesis of AD is unclear currently, there are no effective medications or supplements for it.

Oxidative stress is one of the most important factors contributing to the underlying pathology of AD.^3,4 Some studies have shown that oxidative stress impairs neurons in the early stages of AD.⁵ Uncoupling proteins (UCPs) are a family of mitochondrial inner membrane proteins which are transporters, including UCP1 to UCP5. Particularly, UCP4 is detected mainly in the central nervous system.^6,7 UCP4 can maintain the function of mitochondria in neural tissue, enhance neuronal survival⁸ and relieve oxidative stress.^9,10

Adult neurons are believed to be in a quiescent state after they are differentiated. Cell cycle reactivation (CCR) means mature neurons re-enter various phases of the cell cycle, resulting from the abnormal activation of cell cycle regulators, such as Cyclin.¹¹ The expression of Cyclin D and Cyclin B in the affected neurons of patients suffering from AD was shown to be abnormally higher than those of healthy people at the same age.¹² In addition, some studies have suggested that oxidative stress induces neuronal injury via CCR,¹³ and antioxidants decreased the expression of Cyclin D1 to prevent cell cycle disorder.¹⁴ Therefore, CCR may be associated with AD and oxidative stress, however the molecular mechanism has not yet been demonstrated.

Housefly (Musca domestica) is a common insect in nature and its larvae (HL) are rich in nutrition. HL has been shown to exhibit antiviral and^15,16 antifungal activities,¹⁶ anti-atherosclerosis pro-inflammatory responses¹⁷ and antitumor¹⁸ effects. Furthermore, some studies have shown that HL is rich in antioxidants¹⁹ and is able to scavenge oxidative radicals.^20,21 Our previous study has shown that HL contains several anti-oxidative proteins, including cuticle proteins, catalase proteins, AhpC/TSA family proteins, antibacterial peptide, heat shock proteins etc., which contribute to the anti-oxidative protection of HL.¹⁹ However, there are limited studies available on the antioxidant protection of HL in AD. Therefore, this study aimed to investigate the neuro-protective effect of HL on APP/PS1 mice, and explored the possible molecular mechanism involved in it. We also compared the effects of HL on Donepezil, which was included as a positive control.

2. Materials and methods

2.1 Drugs and reagents

Donepezil was obtained from Eisai Co., Ltd (China). SOD1 and CAT antibodies were from OriGene Technologies Co., Ltd (USA). UCP4 and CyclinD1 antibodies were from Novus Biologicals Co., Ltd (USA). Horseradish peroxide-labeled goat-anti-rabbit and goat-anti-mouse IgG were obtained from Cell Signaling Technology Co., Ltd (USA). DAB staining solution was obtained from Sangon Biotech Co., Ltd (China). Other reagents were obtained from Beyotime Biotechnology Co., Ltd (China) or Tianjin Baishi Chemical Industry Co., Ltd (China).

2.2 Preparation of HL powder

HL was purchased from Yaodetang (China) and cleaned with distilled water and disinfected with 70% ethanol. Physiological saline was added to a mass-to-volume ratio of 1 : 1. HL was turned into a mixture by using a grinder. To obtain HL powder, the mixture was defatted by the Soxhlet extraction method,²² and was filtered through a 100-mesh gauze, and the filtered liquid was vacuum-dried. A moderate amount of HL powder was dissolved in physiological saline before using for daily gastric gavage. According to previous research, HL has no oral toxicity in mice^23–25 and 200 mg kg⁻¹ of HL was effective in delaying aging in vivo;²⁶ we chose 200 mg kg⁻¹ as the dosage of HL in our study.

2.3 Animals and drug treatment

Thirty 2-month-old APPSwe/PS1dE9 mice were provided by Nanjing Biomedical Research Institute of Nanjing University. These mice were randomly divided into three groups: a model control (MC) group, a Housefly Larvae (HL) group and a Donepezil group (the positive control). Ten 2-month-old C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center, and were considered as the normal control (NC) group. Drug treatment started from 2-month-old to 8-month-old for a total period of 180 days. Drugs (HL: 200 mg kg⁻¹; Donepezil: 5.7 mg kg⁻¹) were administered intra-gastrically. The other groups were given the same volume of saline. The mice were housed at a specific-pathogen-free facility at Guangdong Pharmaceutical University, and were maintained on a 12 h light/dark cycle, with free access to food and water. All the study protocols involving animals in this study were approved by the Guangdong Pharmaceutical University Animal Care and Use Committee (NO. gdpulac20150157).

2.4 Morris water maze (MWM)

The MWM was used to evaluate the learning memory abilities in mice.^27,28 Briefly, a circular pool was filled with a 24 ± 1 °C opaque water–milk mixture. A video camera with a tracking system (Chengdu Techman Software, China) was automatically used to record data. Mice were trained to find the platform for five days in a water maze every day. A maximum time of 90 s was provided to each mouse. If they failed to find the platform in 90 s, the mice would be guided to the platform and kept on the platform for 10 s. The system recorded the time needed for each mouse to reach the platform (latency). The platform was removed on the sixth day. The times covered in the previous platform position by each mouse was recorded to assess the memory ability.

2.5 Immunohistochemical (IHC) staining

Paraffin-embedded brain tissues were prepared and the sections were incubated with primary antibodies (dilution 1 : 400 for Aβ, 1 : 400 for CyclinD1 and 1 : 100 for UCP4) at 4 °C overnight, followed by incubation with horseradish peroxide-labeled goat-anti-mouse (for Aβ and CyclinD1) and goat-anti-rabbit (for UCP4) IgG working solution. Then sections were stained in DAB and counter-stained with hematoxylin. Finally, each cover-slipped section was examined under a microscope at 100× magnification.

2.6 Western blotting

The proteins were extracted from hippocampus tissue. The protein concentration was determined using a Bradford assay (Sangon, Shanghai, China). The protein extract (10 μg) was resolved by SDS-PAGE according to standard protocols and transferred to a nitrocellulose membrane. The membranes were blocked in fat-free milk. Primary antibodies including SOD1 (1 : 1000, Origene, USA), CAT (1 : 200, Origene, USA), Tubulin (1 : 2000, Seebio, China) and GAPDH (1 : 500, Xianzhi, China) were incubated at 4 °C overnight. Secondary antibodies were goat anti-mouse or goat anti-rabbit IgG (1 : 2000, Cell Signaling Technology, USA).

2.7 Real time reverse transcription-polymerase chain reaction (RT-PCR)

RT-PCR was used to analyze the levels of CAT, SOD1, UCP4 and CyclinD1 mRNA. The brains were placed on ice. A general RNA extraction kit (Dongsheng Biotech) was used for the preparation of total RNA in the hippocampus tissue. A HiScript II Q RT SuperMix Kit (Vazyme Biotech, China) was used to prepare cDNA from 1 μg RNA. The corresponding primer sequences of each detected protein are shown in Table 1.

Table 1 Primer pairs used in RT-PCR

Gene	Primers	Sequences	Amplicon (bp)
UCP4	Forward	5′-TCG AGA CAA ACA AGG AAG GGG-3′	132
	Reverse	5′-GAC CAA GGG GTC ATT CTC AGC-3′
CAT	Forward	5′-CCC CTA TTG CCG TTC GAT TCT-3′	211
	Reverse	5′-TTC AGG TGA GTC TGT GGG TTT-3′
SOD1	Forward	5′-GGG TTC CAC GTC CAT CAG TA-3′	132
	Reverse	5′-AGT CAC ATT GCC CAG GTC TC-3′
CyclinD1	Forward	5′-TCC TGC TAC CGC ACA ACG-3′	175
	Reverse	5′-GAC CAG CCT CTT CCT CCA C-3′
β-Actin	Forward	5′-AGA GGG AAA TCG TGC GTG AC-3′	138
	Reverse	5′-CAA TAG TGA TGA CCT GGC CGT-3′

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2.8 Statistical analysis

All data were analyzed using Prism 5 software and expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA followed by Tukey's tests for multiple comparisons. Differences with P < 0.05 were considered statistically significant.

3. Results

3.1 Effects of HL on escape latency of APP/PS1 mice in the MWM test

APP/PS1 mice were trained in the MWM to test their spatial learning and memory abilities. As shown in Fig. 1a, the escape latency (the time to find the hidden platform) was not significantly different between groups on the first day. After 3 days of training, the escape latency was significantly shortened in the NC, HL and Donepezil groups compared to that of the MC group (P < 0.05) (Fig. 1b). The results showed that the administration of HL or Donepezil caused a significant reduction in escape latency compared with the MC group (P < 0.05). In addition, the escape latency in the HL group was significantly less than that of the Donepezil group (P < 0.05).

Fig. 1 The mean of latencies to find a hidden platform in the MWM. (a) Escape latency on the first day; (b) escape latency on the third day after training. Data are presented as mean ± S.E.M. (A: P < 0.05 vs. NC; B: P < 0.05 vs. MC; C: P < 0.05 vs. Donepezil).

3.2 Effect of HL on the deposition of Aβ in the hippocampus

The photomicrograph of IHC results showed that there was no deposition of Aβ in the NC group. By contrast, the MC group had a massive deposition of Aβ in the hippocampus (Fig. 2). In the groups treated with Donepezil and HL, the deposition of Aβ was notably decreased compared with that of the MC group (P < 0.05).

Fig. 2 IHC staining of Aβ deposition in hippocampus tissue of mice in the experimental groups. (a) Photomicrograph of Aβ deposition in all groups (scale bar = 50 μm). (b) The analysis data of Aβ deposition in the hippocampus of all groups by Image J. Data are presented as mean ± S.E.M. (A: P < 0.05 vs. NC; B: P < 0.05 vs. MC; magnification 100×).

3.3 Effect of HL on UCP4 and CyclinD1 expression

As illustrated in Fig. 3(a, c and e), the percentage of immuno-positive neurons of UCP4 in the hippocampus was remarkably lower in the MC group (P < 0.05) than that in the NC group. After treating with HL, the expression of UCP4 was significantly enhanced compared with that of the MC group (P < 0.05) while the expression of UCP4 was not statistically different between the Donepezil group and the MC group. Moreover, as shown in Fig. 3(b, d and f), the percentage of immuno-positive neurons of CyclinD1 in the hippocampus was dramatically higher in the MC group than that in the NC group (P < 0.05). After treating with HL and Donepezil, the expression of CyclinD1 was significantly decreased compared with that of the MC group (P < 0.05). The above-mentioned results were all verified from the levels of both protein and mRNA.

Fig. 3 IHC staining of UCP4 and CyclinD1 in hippocampus tissue of mice in the experimental groups. (a and b) Photomicrograph of UCP4 and CyclinD1 in the hippocampus (scale bar = 50 μm). (c and d) The analysis data of UCP4 and CyclinD1 protein levels in the hippocampus by Image J. (e and f) The UCP4 and CyclinD1 mRNA levels in the hippocampus (A: P < 0.05 vs. NC; B: P < 0.05 vs. MC; magnification 100×).

3.4 Effect of HL on CAT and SOD1 levels

As shown in Fig. 4(c–f), the levels of CAT and SOD1 in the MC group were remarkably lower than those in the NC group (P < 0.05). In addition, both levels of CAT and SOD1 in the HL group were significantly increased compared with those in the MC group (P < 0.05), while the level of SOD1, not CAT, in the Donepezil group was significantly higher than that in the MC group (P < 0.05). Besides, the level of CAT in the HL group was significantly higher than that in the Donepezil group (P < 0.05).

Fig. 4 Expression of SOD1 and CAT in the hippocampus of mice in the experimental groups. (a and b) Western blotting results of the expression of CAT and SOD1. (c and d) The relative density-analysis of western blotting using a Tanon Gel Imaging system. (e and f) The analysis of CAT and SOD1 mRNA levels. (A: P < 0.05 vs. NC; B: P < 0.05 vs. MC; C: P < 0.05 vs. Donepezil).

3.5 Effect of HL on the expression of p-JNK and p-P38

As shown in Fig. 5(c and d), the expression levels of p-JNK and p-P38 in the MC groups were significantly lower than those in the NC group (P < 0.05). Compared with the MC group, the levels of p-JNK and p-P38 in the HL group were significantly increased (P < 0.05). However, Donepezil-treatment only improved JNK (P < 0.05), but not P38 activation, which suggests that HL enhanced JNK and P38 MAPK activation in APP/PS1 mice, while Donepezil only enhanced JNK activation. Otherwise, the degree of activation in JNK and P38 MAPK signaling in the HL group was significantly higher than that in the Donepezil group (P < 0.05).

Fig. 5 Expression of p-JNK and p-P38 in the hippocampus of mice in the experimental groups. (a and b) Western blotting results of the expression of p-JNK and p-P38. (c and d) The relative density-analysis of western blotting using a Tanon Gel Imaging system. (A: P < 0.05 vs. NC; B: P < 0.05 vs. MC; C: P < 0.05 vs. Donepezil).

4. Discussion

MWM is widely used to evaluate the memory and learning abilities in mouse models of AD.²⁷ A short escape latency could be associated with the good memory of mice.²⁹ In the present study, we observed a significantly longer escape latency in the MC group than that in the NC group after training. Donepezil and HL significantly shortened the escape latency. The results revealed that HL was protective against learning and memory impairment, which was as effective as Donepezil.

The aggregation of beta-amyloid peptide (Aβ) is characteristic of AD, which leads to senile plaques.³⁰ The deposition of Aβ in the hippocampal region can induce oxidative stress, contributing to neuronal death.³¹ Many studies have demonstrated that Aβ promotes reactive oxygen species (ROS) production, and oxidative damage also induces the aggregation of Aβ in neuronal cells.^32–34 In the present study, we found that HL and Donepezil significantly decreased the accumulation of Aβ in the hippocampus. Furthermore, HL, an antioxidant,¹⁹ inhibited Aβ-induced oxidative damage, and oxidative stress induced Aβ deposition through decreasing the oxidative stress level in hippocampal neurons.

Adult neurons are terminally differentiated and remain in the G0 phase of the cell cycle. Cell cycle-related proteins participate in cell cycle regulation, such as Cyclin, CDK, etc. However, it has been reported that the expression of cell cycle-related proteins was identified in the AD brain.³⁵ It was reported that Aβ induced neuronal damage and CDK5 over-activation.³⁶ Aβ promoted ROS production and CDK5 activation through disrupting the antioxidant system, leading to cell death.³⁷ Cyclin D is a member of the cyclin protein family regulating cell cycle progression, including CyclinD1, CyclinD2, and CyclinD3. Among the proteins, Cyclin D1 is a crucial factor, which provokes neurons to leave the G0 phase and enter into the G1 phase. Some studies indicated that CyclinD1 can be detected in the affected neurons from the AD brain.³⁵ In the present study, we found that the level of CyclinD1 was significantly higher in the MC group than that in the NC group. HL and Donepezil treatment significantly down-regulated the CyclinD1 level, alleviating neuronal damage. However, there are no significant differences between HL and Donepezil groups. HL is a natural antioxidant with non-toxicity^23,25 and can be taken as a precaution against AD, which is better than Donepezil in animal models.

Uncoupling proteins (UCPs) are the family of mitochondrial anion transporters, including UCP1 to UCP5. UCP2 and 4 mainly express in the brain tissue of mammals. Both of them play an important role in the function of the central nervous system. UCPs can be neuro-protective agents in neurodegenerative diseases through reducing the production of ROS.^38–40 Indeed, it has been observed that the mRNA level of UCP4 was remarkably decreased in the brain of AD patients.⁴¹ It is suggested that the function of UCP4 may be associated with preventing oxidative injury in AD. The current study demonstrated that the down-regulation of UCP4 in MC mice might aggravate neuron damage by inducing oxidative stress. The oxidative stress further induced the aggregation of Aβ, and led to neuronal death. In the HL group, the expression of UCP4 was remarkably up-regulated, which could decrease ROS production, and protect neurons against oxidative injury in the hippocampus of APP/PS1 mice. HL-treatment may be responsible for enhancing the expression of UCP4 and therefore it alleviates neuronal oxidative damage. However, Donepezil doesn't have an obvious effect on the expressional level of UCP4. Hence, Donepezil doesn't protect against AD via the regulation of UCP4.

Superoxide dismutase (SOD₁) is one of the antioxidant enzymes, converting harmful superoxide radicals to hydrogen peroxide and molecular oxygen.⁴² Catalase (CAT) is another antioxidant enzyme that catalyzes the breakdown of hydrogen peroxide to oxygen and water.⁴³ Both SOD₁ and CAT are antioxidant defense systems against oxidative stress in vivo. The expression of SOD1 is decreased in the AD patients rather than in individuals without AD.⁴⁴ Furthermore, a decrease in CAT level is seen in the cognitive deficits of rat models.⁴⁵ In the present study, we found that the expressions of SOD₁ and CAT in the hippocampus of MC mice were significantly decreased compared to NC mice, suggesting an impaired antioxidant capacity in AD mice. However, the expressions of SOD₁ and CAT were significantly increased following HL-treatment and Donepezil-treatment. In particular, the expression of CAT in HL mice was significantly higher than that in Donepezil mice. These results indicated that HL may be more remarkable than Donepezil in relieving the oxidative stress in APP/PS1 mice.

Mitogen-Activated Protein Kinase (MAPK) signaling includes JNK and P38 signal pathways. Each one contributes to the physiologic function of cells, including the cell cycle, cell differentiation and cell survival.^46,47 A neuro-protective agent has been observed to affect neurodegenerative rats by the activation of JNK.⁴⁸ Antioxidant agents protect PC12 cells against tunicamycin mediated damage via enhancing JNK and P38 signal pathways.⁴⁹ The reported results are similar to the results in our present study. The expression levels of p-JNK and p-P38 in the MC group were significantly lower compared with the NC group. HL-treatment significantly enhanced the activation of MAPK signaling, including JNK and P38 pathways. The results suggest that the activation of MAPK signaling helped neurons combat oxidative stress injury. Therefore, HL may have potential therapeutic effects in AD, which are associated with the activation of P38 and JNK signaling. However, we observed that the activation of P38 wasn't dramatically changed after Donepezil-treatment. Hence, Donepezil might protect against AD through only the JNK signal pathway, but not the P38 pathway. Additionally, we found the lower activation of JNK and P38 signal pathways accompanied by lower expression of UCP4 in APP/PS1 mice. Meanwhile, there is significantly enhanced activation of JNK and P38 signal pathways and the significant up-regulation of UCP4 after HL-treatment. These results suggest that JNK and P38 signal pathways may be involved in the regulation of UCP4 expression following HL-treatment. However, the relationship among UCP4, JNK, and P38 signaling in AD needs to be further confirmed.

According to our recently on-line published data, HL is a mixture rich in antioxidant proteins, including catalase proteins, AhpC/TSA family protein, antibacterial peptide and so on.¹⁹ Though the above results in this paper showed the antioxidant protection of HL in AD, it is not known which protein is the exact one that works. In our next work, we plan to use the identified pure anti-oxidative protein of HL, such as the antibacterial peptide, AhpC/TSA family protein, etc. to test their protective activities in AD individually.

5. Conclusion

In summary, HL has a protective effect on AD. The molecular mechanism may be the up-regulation of UCP4, SOD1, and CAT, and the down-regulation of CyclinD1 via the activation of P38 and JNK MAPK signal pathways, and a reduction of oxidative injury (Fig. 6). Hence, HL might become a promising food adjuvant for the treatment of AD and further investigation is warranted.

Fig. 6 Potential neuro-protective mechanism of HL against oxidative damage in APP/PS1 mice derived from the above results.

Conflicts of interest

The authors declared no conflicts of interest.

Acknowledgements

The present study was supported by grants from the National Natural Science Foundation of China (81401150 and 81403129). The authors thank Associate Professor Shailendra Anoopkumar-Dukie, who provided guidance and critical help in manuscript drafts.

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Footnote

† These authors contributed equally to this work.

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