What is the neuroprotective effect of astaxanthin in a neonatal rat model of hypoxic-ischemic brain injury?

 Neonatal ischemic-hypoxic injury (IHI) is a common disease in newborns[1] , which mainly refers to asphyxia during perinatal period, labor and delivery and postnatal period, resulting in hypoxic brain injury, which seriously affects the growth and development of newborns[2] . However, there is still a lack of effective measures for the treatment of neonatal ischemic-hypoxic brain injury [3-4], so in-depth study of the specific molecular mechanisms of ischemic-hypoxic injury and the development of safe and effective therapeutic drugs and methods are the hotspots and focuses of perinatal medicine research at home and abroad [5-8]. A large number of previous studies have found that inflammatory response and oxidative stress are important molecular mechanisms leading to the occurrence of ischemic-hypoxic injury in neonatal rats [9-11], and drugs that down-regulate the inflammatory response and oxidative stress can significantly improve the prognosis of neonatal ischemic-hypoxic brain injury in neonatal rats [12].

 

Astaxanthin, an oxygenated derivative of carotenoids, has been found to have significant anti-inflammatory and antioxidant effects [13-19], and is widely found in crustaceans, marine organisms, and a few plants [20-21], and can play a significant neuroprotective role in acute injuries of the central nervous system, such as subarachnoid hemorrhage [22-23], and its protective effects on ischemic-hypoxic injuries in neonatal rats were investigated. In this paper, we intend to investigate its protective effects on neonatal rats with ischemic-hypoxic injuries, in order to provide new ideas and methods for the treatment of neonatal ischemic-hypoxic diseases in clinical practice.

 

1 Materials and methods  

1.1 Molding animals and materials  

Ninety-eight neonatal clean-grade SD male suckling rats, aged 7 d, body mass 12-18 g, were provided by the Laboratory Animal Center of Fujian Medical University under the animal license No. SCXK(Min)2004-0002, and were kept in closed groups under 24 ℃ under 24 h of day and night light irradiation control and strict and standardized card registration management.

Astaxanthin powder was purchased from Sigma, USA, and dissolved in 10% by volume DMSO solvent before use.

 

1.2 Molding methods and interventions  

The experiments were completed from May 2013 to May 2014 at the Medical Laboratory Center of the Second Affiliated Hospital of Fujian Medical University.

Grouping and modeling method: 30 rats were randomly selected from 98 suckling rats as the sham group, and the rest of the rats were modeled as ischemic-hypoxic brain injury.

 

The model of ischemic-hypoxic brain injury in neonatal rats was made with reference to the previous literature [24]: after the suckling rats were anesthetized with ether, they were fixed on the surgical plate in the supine position, the skin of the neck was disinfected routinely, and a mid-cervical incision was made, the left common carotid artery was freed, the distal and proximal ends of the left common carotid artery were ligated with sterilized silk wire, and the incision was closed one by one and the incision and surrounding skin were disinfected, and then the suckling rats were placed in the recovery position on the rewarming blankets for 2 hours. After recovering for 2 h on a rewarming blanket, the mice were placed in a Plexiglas hypoxic chamber. Nitrogen was first introduced to make the oxygen volume fraction in the hypoxic chamber reach 8%, and then 92% by volume of special standard gas and 8% by volume of oxygen were introduced at a rate of 1.5-2.5 L/min to maintain the oxygen volume fraction of 8% in the chamber, and then the animals were taken out and released back to the original feeding environment and fed by the mothers for 2 h. The animals were then fed by the mothers.

 

Criteria for successful modeling: after ischemia and hypoxia, the mice showed decreased muscle strength, imbalance, impaired mobility, and behaviors such as difficulty or inability to roll over, tail pinching and left rotation. In the sham-operated group, only the neck skin was incised. Successful rats were randomly divided into cerebral ischemia-hypoxia group and astaxanthin-treated group, 30 rats each.

Astaxanthin treatment: the astaxanthin treatment group was injected 80 mg/kg of astaxanthin intraperitoneally immediately after the model was successfully made, and then again 12 h later [25].

 

1.3 Western blot assay  

Twelve hours after drug administration, rats were anesthetized, and 100 mL of saline was perfused through the left apical region of the heart, and the parietal cortex of the ischemic injury area was taken out and put into a refrigerator at -80 ℃ for storage and waiting for use. The total protein was extracted according to the instructions of the kit (Biyuntian), and the protein was quantified by the Bradford method. 35 μg of protein was added to each well at a ratio of 1:4 in 5×loading boiling water for 10 min, denatured, and then enclosed for 1 h after electrophoresis and membrane transfer. After adding β-actin, p-Akt, p-GSK-3β (Ser9), cleaved-caspase-3 and Bcl-2 (1:1,000; Cell signaling company, USA) primary antibody dilution, shaking the bed at 4 ℃ overnight, adding the secondary antibody, washing the membrane, dropping the ECL developer, and analyzing the grayscale analysis by Image J software (National Institutes of Health, USA).

 

1.4 TUNEL method detection  

The TUNEL assay was performed with reference to the instruction manual of the kit (Roche, USA) and previous literature [26]: 24 h after ischemia-reperfusion injury, the mouse brain was fixed with saline and paraformaldehyde via left apical perfusion, the brain was removed by severing the head, and then routinely embedded in paraffin wax, and then the brain tissue sections were routinely dewaxed and dehydrated, and then soaked in distilled water and washed in PBS with a droplet of H2 O2, and then placed in an oven at 37 °C for 40 min. Then the sections were placed in the oven at 37 ℃ for 40 min, removed and washed with PBS for 5 min each time for 3 times, and incubated with proteinase K for 10 min in the oven at 37 ℃, removed and washed with PBS for 5 min each time for 3 times.

 

Reagents A and B of the apoptosis detection kit were prepared into a TUNEL reaction mixture, mixed thoroughly and added onto the sections, and then placed in the oven at 37 ℃ for 60 min, washed with PBS after removal, and washed with POD after 30 min in the oven at 37 ℃, and then washed by using DAB color development, hematoxylin staining, dehydrated, transparent, and sealed. Two experienced pathologists observed the sections under 400× microscope and counted the number of apoptotic cells by double-blind method. 10 fields of view were taken from each section and the average value was calculated.

 

1.5 Main observational indicators  

Expression levels of β-actin, p-Akt, p-GSK-3β(Ser9), cleaved-caspase-3 and Bcl-2 proteins and neuronal apoptosis in the parietal cortex of neonatal rats with ischemic injury.

 

1.6 Statistical analysis  

SPSS 15.0 software (IBM, USA) was used for the processing of the results, data were expressed in ± s. Comparisons between multiple groups were performed by one-way ANOVA.

 

2 Results

2.1 Analysis of the number of laboratory animals  

There were no deaths in the modeling, but a total of 8 deaths after astaxanthin and solvent injections, including 5 deaths in the cerebral ischemia-hypoxia group and 3 deaths in the astaxanthin-treated group, with the difference in mortality between the two groups not being significant, and no deaths were observed in the neonatal group in the sham-operated group. Twenty rats in each group were used for immunoblotting and 10 rats in each group were used for TUNEL. The experimental procedure is shown in Figure 1.

 

2.2 Effects of astaxanthin on gross behavior in a neonatal rat model of hypoxic-ischemic brain injury  

The behavior of the neonatal rats in each group was normal before the experiment, but the neonatal rats became restless and rolled over about 10 min after entering the hypoxic environment after the ischemic surgery, and then gradually developed limb trembling, head trembling, inability to roll over, tail pinching and left swiveling, and even convulsions and lethargy, whereas the neonatal rats in the sham-operated group did not have the above mentioned abnormal behaviors.

 

2.3 Effects of astaxanthin on the p-Akt/GSK3β signaling pathway in the parietal cortex of the ischemic injury zone of the neonatal rat model of hypoxic-ischemic brain injury  

Compared with the sham-operated group, the expression levels of p-Akt and p-GSK3β in the parietal cortex of the ischemic injury area of the neonatal rats in the cerebral ischemia-hypoxia group were significantly increased (P < 0.05); astaxanthin significantly up-regulated the expression levels of p-Akt and p-GSK3β in the parietal cortex of the ischemic injury area of the neonatal rats in the cerebral ischemia-hypoxia group (P < 0.05), as shown in Fig. 2 and Table 1. Table 1.

 

2.4 Effects of astaxanthin on ischemic injury in a neonatal rat model of hypoxic-ischemic brain injury

The effects of cleaved-caspase-3 and Bcl-2 expression levels in the parietal cortex of the ischemic injury zone of neonatal rats were shown in Fig. 2 and Table 1. Compared with the sham-operated group, the expression levels of cleaved-caspase-3 in the parietal cortex of the ischemic injury zone of neonatal rats in the ischemic injury zone of the ischemic ischemic group were significantly increased (P < 0.05), while that of Bcl-2 was significantly decreased (P & lt; 0.05). 0.05); compared with the ischemia-hypoxia group, astaxanthin significantly inhibited the expression of cleaved-caspase-3 in the parietal cortex of the ischemic injury area of neonatal rats (P < 0.05), and significantly increased the expression of Bcl-2 (P < 0.05).

 

2.4 Effects of astaxanthin on apoptosis in brain tissue of a neonatal rat model of hypoxic-ischemic brain injury  

TUNEL staining showed that the number of apoptotic cells in the brain tissues of neonatal rats in the ischemic-hypoxic group was significantly increased compared with that of the sham-operated group (P < 0.05), and that astaxanthin treatment significantly reduced the number of apoptotic cells in the neonatal brain tissues of neonatal rats compared with that of the ischemic-hypoxic group (P < 0.05), as shown in Fig. 3 and Table 2.

 

3 Discussion

Akt is an important kinase in the phosphatidylinositol 3-kinase signaling pathway, also known as protein kinase B. After activation through Ser473 residue phosphorylation, Akt can further phosphorylate and activate the downstream proteins such as Bad, GSK3β, and Caspase9 [27]; in addition, activated Akt can bind to GSK-3β and phosphorylate its N-terminal Ser9 site, thereby leading to the inactivation of GSK-3β, and ultimately affecting the transcriptional activities of the downstream substrates NF-κB and c-Jun transcription factors, thus exerting significant anti-apoptotic effects [28]. In addition, activated Akt can also bind to GSK-3β and phosphorylate the Ser9 site at its N-terminus, leading to the inactivation of GSK-3β, which ultimately affects the transcriptional activity of the downstream substrates, NF-κB and c-Jun transcription factors, and thus exerts a significant anti-apoptotic effect[28-30] . Previous studies in transient ischemic brain injury and localized ischemic brain injury have found that the expression level of p-Akt is significantly increased after neurological injury[31] , and the use of selective inhibitors of PI3K/Akt can significantly aggravate the degree of brain injury[13] , whereas the use of drugs up-regulating the expression of p-Akt can inhibit the activity of GSK-3β[32] , the activity of apoptotic proteins such as cleaved-caspase3 and caspase3, as well as the activity of the apoptotic proteins such as NF-κB and c-Jun, and thus exert significant anti-apoptotic effects[28-30] . The use of drugs that up-regulate p-Akt expression can inhibit GSK-3β activity [32], inhibit the expression of the apoptotic protein cleaved-caspase3, and increase the expression of the anti-apoptotic protein Bcl-2, thereby significantly reducing neuronal apoptosis and improving neurological function [33-34]. In view of the fact that Akt can prevent apoptosis by regulating many substrates, targeting Akt for the treatment of cerebral ischemic-hypoxic injury has become a hot spot and focus of current research.

 

Numerous studies have shown that astaxanthin has the effects of tumor inhibition, anti-inflammation, antioxidant, immune enhancement, and prevention of cardiovascular and cerebrovascular diseases [35-39], suggesting that astaxanthin has a good neuroprotective effect in central nervous system injurious diseases [40-41], but there is a lack of research on its protective effect in hypoxic-ischemic brain injury in neonatal rats. The results of this experiment showed that astaxanthin significantly reduced the number of apoptotic neuronal cells in neonatal rats in the ischemic-hypoxic group, and further mechanistic studies revealed that astaxanthin significantly up-regulated the level of p-Akt in ischemic-hypoxic brain tissues, which is consistent with the neuroprotective effects previously observed in brain injury with subarachnoid hemorrhage[41-42] , suggesting that astaxanthin can be applied in the treatment of a variety of neurological injuries; in addition, studies have also found that astaxanthin has a protective effect in neonatal rats with hypoxic-ischemic brain injury[40-41] . In addition, astaxanthin was found to increase the expression of p-GS3β (Ser9), significantly inhibit the expression of cleaved-caspase-3, an apoptosis key execution protein, and increase the expression of Bcl-2, an anti-apoptotic protein, which may be one of the mechanisms for its neuroprotective effects.

 

In conclusion, astaxanthin plays a significant neuroprotective role in neonatal ischemic-hypoxic injury, and its mechanism may be related to the up-regulation of Akt/GSK3β signaling pathway, suggesting that astaxanthin may be a potential therapeutic agent for neonatal ischemic-hypoxic injury.

 

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