Ligustilide improves aging-induced memory deficit by regulating mitochondrial related inflammation in SAMP8 mice

Results

Ligustilide improves learning and memory in SAMP8 mice

In Morris water maze test, the time for the mice to find the hidden platform decreased gradually during the five consecutive days, and the SAMP8 group spent longer time to find the platform than the SAMR1 group (Figure 1B and 1C). Compared with the SAMP8 group, the ligustilide-administered group had a relatively short time to find the platform, and the high-dose group was shorter than the low-dose group. The crossing time of the platform and the time spent in the target quadrant showed the similar tendency (Figure 1D and 1E). The swimming speed of each group was not changed (Figure 1F). In object recognition task (Figure 2A and 2B), the time to explore new objects in SAMP8 mice was less than that in SAMR1 mice. The ligustilide group spent more time to explore new objects, when compared with SAMP8 mice. In the open field test and elevated plus maze (Figure 2C–2F), SAMP8 group showed a significant anxiety-like behavior. Ligustilide relieved this symptom.

Figure 1. Ligustilide improves aging-induced cognitive dysfunction in SAMP8 mice. (A) The chemical structure of ligustilide. (B) Escape latency from five consecutive days of tests. (C) The swimming paths of each respective group on the sixth day. (D) Crossing times of the target platform in the probe trial. (E) Time spent in the target quadrant in the probe trial. (F) The swimming speed in the probe trial. Ligustilide 10 (10 mg/kg/d); Ligustilide 20 (20 mg/kg/d). Data represent mean ± SD (n = 20 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1; *p < 0.05, **p < 0.01, ***p < 0.001 versus SAMP8.

Figure 2. Ligustilide improves aging-induced cognitive dysfunction in SAMP8 mice. (A) Trajectory map for each respective group in the new object recognition. (B) Times by each respective group exploring a reference object and a new object. (C) Trajectory map for each respective group in the field test. (D) Time spent by each respective group in the field test. (E) Trajectory map for each respective group in the elevated plus maze. (F) Time spent by each respective group in the elevated plus maze. Ligustilide 10 (10 mg/kg/d); Ligustilide 20 (20 mg/kg/d). Data represent mean ± SD (n = 20 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1; *p < 0.05, **p < 0.01, ***p < 0.001 versus SAMP8.

Ligustilide ameliorates neurodegeneration in SAMP8 mice

As shown in Figure 3, the protein expressions of neuron-related factors, such as PSD95, PSD93 and BDNF in SAMP8 were significantly decreased. While after the treatment of ligustilide, the proteins of the high-dose group elevated to the normal level, the proteins of the low-dose group were slightly below normal. These changes indicated that ligustilide could ameliorate aging-related neurodegeneration in SAMP8 mice.

Figure 3. Ligustilide ameliorates neurodegeneration in SAMP8 mice. (A) Western blot analysis of (B) postsynapticdensity 93 (PSD93), (C) postsynapticdensity 95 (PSD95) and (D) BDNF. Ligustilide 10 (10 mg/kg/d); Ligustilide 20 (20 mg/kg/d). Data represent mean ± SD (n = 20 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1; *p < 0.05, **p < 0.01, ***p < 0.001 versus SAMP8.

Ligustilide improves mitochondrial function in SAMP8 mice

Mitochondrial dysfunction is often occurred in the aging-related disease. As shown in Figure 4, the protein expression of P-Drp1 was sharply increased, while Mfn1 and Mfn2 were decreased in SAMP8. After Ligustilide treatment, the levels of Mfn1 and Mfn2 increased and P-Drp1 decreased. Ligustilide treatment relieved the fusion and fission dysregulation of mitochondria in SAMP8. Ligustilide treatment also increased the protein level of P-AMPK and the content of ATP in SAMP8 mice.

Figure 4. Ligustilide ameliorates mitochondrial morphology in SAMP8 mice. (A) The levels of (B) Mfn1, (C) Mfn2, (D) P-DRP1, DRP1, (E) P-AMPK and AMPK were detected in the hippocampus. (F) The expression of ATP was detected in the hippocampus. Ligustilide 10 (10 mg/kg/d); Ligustilide 20 (20 mg/kg/d). Data represent mean ± SD (n = 20 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1; *p < 0.05, **p < 0.01, ***p < 0.001 versus SAMP8.

Ligustilide attenuates H₂O₂-induced and Rotenone-induced mitochondrial impairment in HT22 cells

To determine whether ligustilide improves mitochondrial dysfunction, we employed two cell models, H₂O₂-exposed and Rotenone-exposed HT22 cells, to study. The H₂O₂ and rotenone treatment significantly reduced the intracellular Mfn1, Mfn2, and P-AMPK levels in HT22 cells, while a strongly increase in ligustilide pretreatment in HT22 cells exposed to H₂O₂ and rotenone (Figure 5A and 5B). In addition, as shown in Figure 5A and 5B, the intracellular level of P-Drp1, which are a major factor of maintaining mitochondrial dynamic balance, was greatly ameliorated by the pretreatment with ligustilide compared to the H₂O₂ only group. In conclusion, these results were consistent with in vivo data, suggesting that ligustilide pretreatment protects HT22 cells by increasing the levels of Mfn1, Mfn2, and P-AMPK and down-regulating the levels of P-Drp1.

Figure 5. Ligustilide attenuates H2O2-induced and Rotenone-induced mitochondrial impairment in HT22 cells. (A, B) Western blotting was used to detect mitochondrial morphology-related proteins (C, G) Mfn1. (D, H) Mfn2. (E, L) P-Drp1. (F, I) P-AMPK.. Ligustilide 10 (10 mg/kg/d); Ligustilide 20 (20 mg/kg/d). Data represent mean ± SD (n = 20 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1; *p < 0.05, **p < 0.01, ***p < 0.001 versus SAMP8.

Ligustilide decreases oxidative stress and neuroinflammation in SAMP8 mice

As shown in Figure 6, the lipid peroxidation product MDA, a marker of oxidative stress, was increased in SAMP8 mice. Ligustilide significantly reduced the level of MDA. The anti-oxidative enzymes, SOD and GSH-PX, were also detected. Ligustilide increased the activity of SOD and GSH-PX. As shown in Figure 7, the level of neuroinflammation (NLRP3, IL-1β and NF-κB) was increased in SAMP8 mice. After ligustilide treatment, these proteins were decreased almost to normal level. These results indicated that ligustilide could confrontation AD-like neuropathology in SAMP8 mice.

Figure 6. Ligustilide reduces oxidative stress in the brains of SAMP8 mice. The supernatant from the hippocampus homogenate was used to assay (A) the MDA level, (B) activities of SOD and (C) the activities of Glutathione peroxidase (GSH-PX). Ligustilide 10 (10 mg/kg/d); Ligustilide 20 (20 mg/kg/d). Data represent mean ± SD (n = 20 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1; *p < 0.05, **p < 0.01, ***p < 0.001 versus SAMP8.

Figure 7. Ligustilide reduces neuroinflammation levels. (A) The levels of (B) NLRP3, (C) IL-1β and (D) NF-κB were detected in the hippocampus. Ligustilide 10 (10 mg/kg/d); Ligustilide 20 (20 mg/kg/d). Data represent mean ± SD (n = 20 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1; *p < 0.05, **p < 0.01, ***p < 0.001 versus SAMP8.

Ligustilide decreases neuronal apoptosis in SAMP8 mice

The expression of apoptotic markers, Bax, Bcl-2 and caspase-3, was detected to assess the apoptotic processes that occurred in all groups of tissues. As shown in Figure 8, the expression of apoptosis-related proteins Bax and cleaved Caspase-3 increased and Bcl-2 decreased in SAMP8. Ligustilide increased the Bcl-2 expression and decreased the Bax and cleaved Caspase-3 expressions.

Figure 8. Ligustilide protects against neuronal apoptosis in hippocampus. (A) The levels of (B) Bax, (C) Bcl-2 and (D) Cleaved caspase-3 were detected in the hippocampus. Ligustilide 10 (10 mg/kg/d); Ligustilide 20 (20 mg/kg/d). Data represent mean ± SD (n = 20 per group). #p < 0.05, ##p < 0.01, ###p < 0.001 versus SAMR1; *p < 0.05, **p < 0.01, ***p < 0.001 versus SAMP8.

Discussion

In this study, we demonstrate that ligustilide is capable of mitigating age-associated cognitive decline in SAMP8 mice. An eight-week-administration of ligustilide protected the learning and memory, improved mitochondrial dysfunction, reduced inflammation and apoptosis (Figure 9).

Figure 9. Schematic representation of ligustilide improves aging-induced memory deficit by regulating mitochondrial-related dysfunction in SAMP8 mice. Ligustilide improves aging-induced cognitive dysfunction in SAMP8 mice. Ligustilide ameliorates mitochondrial dysfunction in hippocampal neurons, reduces levels of oxidative stress and inflammation, reduces apoptosis in hippocampal neurons.

Alzheimer’s disease is a neurodegenerative disease with a variety of pathogenesis, including Cholinergic hypothesis, Amyloid beta (Aβ) hypothesis, Tau hypothesis, Excitotoxic hypothesis, Oxidative stress hypothesis, Apolipoprotein E (ApoE) hypothesis. These mechanisms eventually lead to neuronal damage or inflammation [16]. The rapid aging model mouse SAMP8 is currently recognized as an effective animal model for studying AD [17]. In this study, compared with the 10 -month-old SAMR1 mice, AD-like pathology represented by mitochondrial dysfunction, neurobehavioral deficits, oxidative stress, and inflammation were observed in the age-matched SAMP8 mice. Its behavioral and pathological features are very similar to the clinical symptoms of human AD and its complex neurological pathological changes (Xu et al. 2014). Therefore, we used SAMP8 mice as an experimental model to investigate whether Ligustilide could alleviate the pathology of AD in this study.

With the advent of aging society, the prevention and treatment of AD has become a hot topic. However, there is still no effective drug for AD treatment [18]. Recently, ligustilide has attracted widespread attention in the field of Alzheimer’s disease, owing to its capacity to reduce the levels of p-Tau, mutant APP, Aβ1-42 oligomers and monomers, ameliorate oxidative stress induced neuronal apoptosis, provide an effective neuroprotection [19, 20]. Learning and memory is an advanced function of the brain. It is an extremely complex process under the control of the central nervous system [21, 22]. In this study, we further investigated the neuroprotective effect of ligustilide on aging-related dementia.

Mitochondria are an important organelle that is ubiquitous in eukaryotic cells. It is the main site for ATP production in cells and the most important source of energy in cells. Most of the tissue cells in the human body rely on the oxidative phosphorylation of mitochondria to obtain the energy needed to maintain their own metabolism [23]. The role of mitochondria in aging was first proposed by Denham Harman in 1965. He proposed the free radical theory of aging, suggesting that with increasing age, oxygen species can attack cells and tissues and cause damage to the body. Mitochondria is one of the most important sources of reactive oxygen species [24, 25]. Since then, more and more evidence has shown that mitochondrial dysfunction causes age-related degenerative diseases, and the occurrence of mitochondrial dysfunction will increase with age, including accumulation of somatic mtDNA mutations, enhanced oxidative damage, decreased abundance and quality of mitochondria, as well as dysregulation of mitochondrial dynamics [26]. Mitochondrial dynamics means that mitochondria change their own morphology through continuous fusion and division to adapt to various stress conditions, which can meet the energy metabolism and other biological needs of cells [27]. Mitochondria are highly dynamic, their structure and function are regulated by a series of proteins involved in fission and fusion, Drp1 is involved in mitochondrial division, Mfn1 and Mfn2 are involved in mitochondrial fusion, precise regulation of mitochondrial fusion-split equilibrium helps the mitochondria to perform its normal function and ATP generation to maintain cell homeostasis [28, 29]. In the complement of Mfn1 and Mfn2, AMPK is also an energy-related signaling pathway. Due to ATP deficiency, activation of AMPK promotes mitochondrial respiration, thereby increasing mitochondrial potential [30]. In addition, AMPK activation reverses the expression of antioxidants and thus counteracts cellular oxidative stress [31]. Mitochondrial dysfunction was observed in SAMP8 mice. Since ligustilide treatment can reduce levels of Drp1 and increase levels of AMPK, Mfn1 and Mfn2 in the brain of SAMP8 mice. These results suggest that ligusrilide can protect against the mitochondrial dysfunction.

In order to further confirm the mechanisms by which ligustilide improves mitochondrial dysfunction of HT22 cells induced by H₂O₂ or ROT, we examined the expression of Mfn1, Mfn2, P-Drp1 and P-AMPK. In this study, H₂O₂ was used to induce the injury model of nerve cells, the combination of ROT and α-syn fibrils resulted in mitochondrial dynamic imbalance and leads to mitochondrial dysfunction [32]. We found that Ligustilide increase Mfn1, Mfn2 and P-AMPK, and reduce P-Drp1 in H₂O₂-exposed and Rotenone-exposed HT22 cells. These results again demonstrate that ligustilide can protect against the mitochondrial dysfunction.

There is increasing evidence that neuronal loss, mitochondrial mitigation and mitochondrial dysfunction occur during AD, leading to the progressive cognitive decline in AD, which may be associated with increased oxidative damage. Excessive oxidative stress can not only directly destroy cell membranes, RNA and protein, but also promote neuroinflammation and neuronal apoptosis [33]. Mitochondria are thought to be an important part of the production of reactive oxygen species in cells [34]. Oxygen is consumed in the mitochondria. When mitochondria are damaged, the body’s oxidative stress response is activated, which in turn causes apoptosis and inflammation, and the cells appear dysfunctional [35]. In this study, we find elevated levels of MDA and a decrease in GSH level and the activity of SOD in the SAMP8 mice under the treatment of ligustilide. Elevated oxidative stress was occurred in SAMP8 mice. Ligustilide effectively relieved this changes.

AD is a complex neurodegenerative disease. A variety of inflammatory factors such as IL-1β, NLRP3 and NF-κB are thought to play an important role in the pathogenesis of AD, leading to nerve damage, activity of astroglia and microglia and apoptosis [36, 37]. Glial cell hyperplasia, inflammatory cells and cytokine activation further promote or accelerate neuronal apoptosis. The apoptotic nerve cells reappear with new stimulation factors, which induce AD inflammatory reaction to occur many times and accelerate the deterioration of the disease [37]. Recent study has been demonstrated that ligustilide significantly ameliorated cognitive impairment and brain damage through antioxidant and anti-apoptotic mechanisms [38]. In our study, the level of NLRP3, IL-1β and NF-κB were decreased under the treatment of ligustilide. These results suggest that Ligustilide can protect against the inflammation.

In apoptosis, mitochondria are the center of regulation of apoptosis. Persistent mitochondrial dysfunction contributes to apoptosis due to the release of cytochrome c into the cytoplasm with subsequent activation of caspases, triggering a cascade, which initiate and carry out apoptosis [39]. The anti-apoptotic protein Bcl-2 can prevent the release of cytochrome C from mitochondria. In contrast, the pro-apoptotic protein Bax induces mitochondrial injury leading to cell death [40]. The present study showed that the apoptotic indexes Bax/Bcl-2 and cleaved Caspase-3 expressions significantly decreased after the treatment of ligustilide. These results indicated that ligustilide effectively inhibited mitochondrion-mediated apoptosis.

In conclusion, ligustilide prophylactic administration significantly improved the ability of learning and memory and brain tissue mitochondrial function in SAMP8 aging mice. Ligustilide is expected to be an effective drug for the treatment of AD in the clinic.

Author Contributions

Shi-Jie Zhang designed the study. Wen-Li Zhu, Jia-Yi Zheng, Wei-Wu Cai, Zhao Dai, Ben-Yue Li, Ting-Ting Xu, Hao-Fei Liu, Xiao-Qi Liu, Su-Fen Wei, Yi Luo, Hong Wang and Hua-Feng Pan conducted the experiment. Wen-Li Zhu and Shi-Jie Zhang contributed to initial data analysis and interpretation, drafted the initial manuscript. Qi Wang and Shi-Jie Zhang supervised all aspects of the study, critically reviewed and revised the manuscript, and approved the final manuscript as submitted.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Funding

This work was supported by the National Natural Science Foundation of China (No.81673627), Guangzhou Science Technology and Innovation Commission Technology Research Projects (201805010005).

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