Introduction

Vascular dementia (VD) refers to acquired intelligence disorder syndrome, which is finally caused by the long-term exposure to various risk factors of cerebral vascular diseases such as cerebral ischemia and hypoxic damage [1]. The development of VD negatively impacts patient cognitive function, depression and anxiety, and working memory [2]. In recent years, the incidence of cerebrovascular diseases has been increasing with the acceleration of population aging, as well as the incidence of VD. Although the exact etiopathogenesis of VD remains unclear, numerous reports have shown that inflammatory response and oxidative stress may be the pathogenesis of cognitive dysfunction, resulting in brain structure abnormalities and dysfunction in VD mice [3, 4]. Therefore, it is urgent to identify potential therapeutic drugs for VD.

Astaxanthin (3,3′-dihydroxy-b, b′-carotene-4,4′-dione, AST), a natural carotenoid with high anti-oxidative activity, is widely distributed in algae, crab, shrimp, salmon, and crustaceans [Detected of superoxide dismutase (SOD) and malondialdehyde (MDA)

To further determine whether AST influences anti-oxidation, we analyzed SOD activity and MDA content in the hippocampus and prefrontal cortex of each group. All hippocampus tissue were obtained after normal perfusion (0.9% NaCl). A 10 mg hippocampus tissue was weighed and added to 100 ml SOD sample preparation fluid. The mixture was homogenized at 4 °C or ice bath. Then supernatants were obtained and used as a sample to be tested after centrifugation at 12,000g for 3–5 min at 4 °C. Bicinchoninic acid kit used to determine the protein concentration of each sample, and SOD and MDA tests were performed according to kits steps (Beyotime, Shanghai, China) [18].

Calculation of total SOD activity in samples: SOD enzyme activity units = percent inhibition/{1 − (A blank control 1 − A sample)/(A blank control 1 − A blank control 2) × 100%} units. Then convert the SOD activity units into U/g or U/mg protein [18, 19].

Determination of MDA content in samples: the initial concentration of MDA in the sample solution was determined by the protein concentration per unit weight to the tissue weight, such as μmol/mg of protein or tissue.

Statistical analysis

All data were statistically analyzed with SPSS 15.0 (International Business Machines Corporation, IBM, USA). For two-way analysis of variance (ANOVA), the procedure (sham) and the treatment (AST 50, 100, 200 mg/kg) were taken as between-group factors. One-way ANOVA with Tukey’s post hoc test was used for multiple comparisons to determine whether the means differed significantly between two groups. A value of P ≤ 0.05 was considered statistically significant, results were presented as mean ± SEM.

Result

Weight of mice

During the period of dministration, the average body weight of each group increased steadily for 30 days, except for slight fluctuations in the first ten days. But there was no significant difference between each group (P > 0.05) (Fig. 1), indicating that AST did not affect the body weight of mice.

Fig. 1
figure 1

The weight of each group mice in the 30 days, with 13 mice per group

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AST improves non-spatial cognitive function in VD mice

After LUCCAO, mice exhibited a significant reduction in discrimination ability when compared with mice in sham group (P < 0.0001) (Fig. 2). Whereas, the discrimination indices of mice treated with AST (50, 100 and 200 mg/kg) were dramatically higher than that in the model group (P < 0.001, P < 0.001 and P < 0.0001, respectively). Taken together, these data indicated that AST could improve the non-spatial cognitive function in mice with VD.

Fig. 2
figure 2

The discrimination index of different groups, with 13 mice per group. (****P < 0.0001: compared with sham group; ###P < 0.001, ####P < 0.0001: compared with LUCCAO group)

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AST ameliorates spatial cognition impairment in VD mice

As shown in Fig. 3a, b, LUCCAO-treated mice had longer escape latency and path length when compared with sham group, which were markedly shortened after administration of AST 200 mg/kg. LUCCAO induced a decrease in crossing platform time and the number of times crossing the platform quadrant, which were also reversed by treatment of AST 200 mg/kg [F (4, 60) = 22.28, P < 0.0001; F (4, 60) = 40.97, P < 0.0001] (Fig. 3c, d). These findings indicated that AST might ameliorate the impairment of spatial acquired function to a certain extent.

Fig. 3
figure 3

The results of morris water maze test, with 13 mice per group. a The average escape latency period of every group in the five days. b The path length of each group in the five days. c Time for mice crossing platform quadrant. d The number of times of mice cross the platform (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001: compared with sham group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001: compared with LUCCAO group)

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Effect of AST on hippocampus neuron in VD mice

Results showed that the hippocampal neuron structure in LUCCAO-treated mice was significantly damaged, and their nuclei was lost (Fig. 4). In particular, the neurons in the hippocampal CA1 and CA3 area of mice in model group contracted seriously and the adjacent gap were enlarged, with disordered and hyperchromatic arrangement of neurons. After AST treatment, the damage of neuron was alleviated, and the neurons in CA1 and CA3 areas were remarkably improved, with orderly arrangement and large and clear nucleus. These findings indicated that AST could alleviate the morphological impairment caused by VD in mice.

Fig. 4
figure 4

The hematoxylin and eosin staining results of each group, with 13 mice per group. Bar: 100 um

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AST regulates IL-1β and IL-4 expression in the hippocampus and prefrontal cortex of VD mice

IL-1β level in the LUCCAO-treated mice was significantly elevated compared to that in sham group in the hippocampus and cerebral cortex. However, AST dose-dependently abated IL-1β expression in the hippocampus (P < 0.05), and only AST 200 mg/kg lowered IL-1β expression in cerebral cortex (P < 0.0001) (Fig. 5a, b). Besides, AST elevated the expression of IL-4 in the hippocampus and prefrontal cortex in a dose-dependent manner when compared with that in LUCCAO group, and reached the highest at AST 200 mg/kg (P < 0.0001) (Fig. 5c, d) According to the above results, it was implied that AST could regulate the expression of inflammatory cytokines in VD mice.

Fig. 5
figure 5

The expression of IL-1β and IL-4 in the hippocampus and prefrontal cortex of each group mice, with 13 mice per group. a The expression of IL-1β in the hippocampus. b The expression of IL-1β in the prefrontal cortex. c The expression of IL-4 in the hippocampus. d The expression of IL-4 in the prefrontal cortex (***P < 0.001, ****P < 0.0001: compared with sham group; #P < 0.05, ###P < 0.001, ####P < 0.0001: compared with LUCCAO group)

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AST suppresses oxidative stress in the hippocampus and prefrontal cortex of VD mice

From Fig. 6a, b, we found that SOD activity was noticeably decreased in the hippocampus and prefrontal cortex of LUCCAO group (P < 0.001). However, mice treated with AST (50, 100, and 200 mg/kg) had higher SOD activity in the hippocampus and prefrontal cortex, and the maximal effect was achieved when AST dose was 200 mg/kg (P < 0.05). Compared with sham group, LUCCAO group showed a significant increase of MDA content in the hippocampus and prefrontal cortex (both P < 0.001), which was diminished after AST treatment (100 and 200 mg/kg) in a dose dependent manner (P < 0.01) (Fig. 6c, d). These findings clarified that AST could inhibit the level of oxidative stress.

Fig. 6
figure 6

SOD activity and MDA content in the hippocampus and prefrontal cortex of each group, with 13 mice per group. a The value of SOD activity in the hippocampus. b The value of SOD activity in the prefrontal cortex. c The level of MDA in the hippocampus. d The level of MDA in the prefrontal cortex (***P < 0.001, ****P < 0.0001: compared with sham group; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001: compared with LUCCAO group)

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Discussion

The most significant finding of this study was that AST ameliorated the cognitive function and hippocampal neuron in VD mice, which may be associated with the inhibition of inflammatory response and oxidative stress, revealing that the potential value of AST in the treatment of VD.

VD is recognized as the second most common type of dementia following Alzheimer’s disease, which is caused by a reduction in blood supply due to blockage in the vascular system, leading to a gradual decline in memory and cognitive function [20]. Currently, many VD animal models have been established, among which 2VO rat model is a classic VD model of whole cerebral ischemia. Numerous studies have shown that permanent hypoxia and hypoperfusion of brain induced by bilateral common carotid arteries (2VO) in rat model can lead to VD and oxidative stress of brain neurons, and ultimately cholinergic dysfunction and decreased learning and memory ability [21, 22]. However, 2VO is only limited to rats and is not suitable for mice, because rats have a complete circle of Willis. Mice lacking complete circle of Willis will suffer from severe ischemia and even death if perform 2VO [23]. Recently, UCCAO model is adopted for VD mice model, which is modified from 2VO model [24]. After 28 days of permanently ligated of a common carotid artery, cerebral perfusion in the ipsilateral hemisphere of mice declined by 35–55% [25]. In this study, we established VD mice model induced by LUCCAO.

Due to the complex pathogenesis of VD-related cognitive dysfunction, there is still no effective treatment. Therefore, it is necessary to find a specific and effective therapeutic drug. Recent studies have shown that AST has the effect of anti-inflammation, anti-apoptosis, anti-oxidation, anti-aging, anti-tumor, and boosting immunity [26, 27]. In the central nervous system (CNS), AST is regarded as a potential neuroprotective drug because of its powerful antioxidant property [28]. In this study, AST alleviated cognitive dysfunction and hippocampal structural damage to a certain extent, indicating that AST had therapeutic effects on VD cognitive dysfunction. In addition, many previous studies have shown that AST can be used as a protective agent in ischemia model owing to its antioxidant potential [29, 30], which is consistent with this study.

It has been demonstrated that inflammatory response and oxidative stress are involved in VD-related cognitive impairment [31, 32]. Overexpression of inflammatory factors can lead to neuron damage in the hippocampus, thus affecting the cognitive function of mice [33]. Miao et al. have confirmed that the levels of interleukin-6 (IL-6), tumor necrosis factor-a, and COX-2 are increased in the brain tissue of type 2 diabetes mellitus rats with cognitive dysfunction [34]. Research conducted by Paloma Bermejo et al. has found that inflammatory response may be an early factor in the development of Alzheimer’s disease [35]. The above studies demonstrate that inflammatory cytokines play a role in the pathogenesis of cognitive dysfunction. In the present study, we found that pro-inflammatory cytokine IL-1β level was increased, while anti-inflammatory IL-4 level was decreased in VD mice, which was consistent with the view that inflammatory cytokines were associated with the pathogenesis of VD-related cognitive dysfunction. However, AST treatment remarkably lowered the IL-1β expression and enhanced IL-4 expression in the hippocampus and prefrontal cortex, indicating that the alleviation of AST in cognitive impairment may correlated with suppression of inflammatory response.

Under normal and non-stress conditions, oxidation and antioxidants levels are relatively balanced, but are out of balance under harmful external stress, thereby leading to the mass production of reactive oxygen species and the relative shortage of antioxidants [36]. Subsequently, a large amount of reactive oxygen species in the organism results in the formation of MDA. MDA is a vital indicator of lipid peroxidation and reflects the severity of oxidative stress injury [37]. In addition, as a free radical scavenger, SOD can protect brain tissue from oxidative stress damage [38]. Mamun et al. have also confirmed that ATX can inhibit neuronal oxidative stress caused by aluminum chloride and ameliorate spatial memory impairment in mice [39]. In this current study, MDA production was elevated and SOD activity was diminished in the hippocampus and prefrontal cortex after LUCCAO, which were significantly reversed by AST treatment. Therefore, we hypothesized that ATX could play a neuroprotective role in cognitive dysfunction of VD mice through its antioxidant effect. Oxidative stress caused by VD can damage the structure of neurons. The results of this study presented damaged hippocampal neuron structure and less nucleus in VD mice, which were alleviated by ATX, indicating that ATX might play a neuroprotective role through anti-oxidative stress and reduced the hippocampal injury of VD mice.

There are some limitations of the study. AST may reduce the damage of neurons in hippocampus induced by VD through oxidative stress, but the apoptosis of neurons was not detected. Besides, we did not detect VD markers such as S100B, C-reactive protein and IL-6. Further studies on these issues were required to carried out.

In summary, this study proves the existence of behavioral impairment in VD mice. Additionally, AST has a significant protective effect on VD mice induced by LUCCAO, which can reduce oxidative stress and enhance the inflammatory levels in a dose-dependent manner. Furthermore, AST 200 mg/kg shows the best neuroprotection property among the three dosages.