Neuroprotective effects of ebselen in traumatic brain injury model involving nitric oxide and P38 MAPK

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Neuroprotective effects of ebselen in traumatic brain injury model: involvement of nitric oxide and P38 MAPK signaling pathway

Abstract

Objective

This study focused on the neuroprotective effects of ebselen (Ebs) in the traumatic brain injury model.

Methods

90 SD rats were randomly grouped into five groups, including sham-operation group, injury model group, low Ebs group (3mg/kg), moderate Ebs group (10mg/kg) and high Ebs group (30mg/kg). The traumatic brain injury model was made according to modified Feeney’s model. Rats in Ebs groups received Ebs intragastrically while saline was intragastrically administrated to rats in the sham-operation group and the injury model group. The expression level of nitiric oxide (NO), iNOS, toll like receptor 4 (TLR4) and P38 mitogen-activated protein kinase (P38-MAPK) were examined 24h after Ebs administration.

Results

Ebs administration significantly decreased the neurological severity score and the percentage of brain water content in injury model rats. Ebs also significantly inhibited the expression of NO and iNOS, which is mainly mediated by the inhibition of TLR4 and p-P38-MAPK signaling pathway after traumatic brain injury.

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Key Words ebselen, traumatic brain injury, neuroprotection, inflammation

Introduction

Traumatic brain injury (TBI), leading to death of millions of individuals in the world, is one of the most severe diseases that affect the quality of human life [1,2]. TBI includes primary injuries and secondary injuries. Secondary injuries, which are mainly caused by mass lesion and extracranial damages, resulting in pathological and physiological changes, are the main aspect for clinical treatment of TBI. The pathological mechanism of the secondary injuries includes inflammation [3], glutamate excitotoxicity [4] and changes in the cerebral blood flow [5,6]. Inflammation responses, which affect the survival and functional recovery of neurons, play a crucial role in the secondary injuries after TBI [7]. TBI could cause hyperactivity of astrocytes and microglia in brain, resulting in increased inflammatory factors, oxygen radicals and cytotoxic substances[8]. Thus, inhibition of inflammation responses is an important step in the treatment of secondary injuries after TBI.

Ebselen (Ebs), 2-phenyl-1, 2-benzo-isoselenazole-3(2H)-ketone, which is a class of organic selenium compounds, has anti-inflammation, immune-regulation, and neuro-protective effects[9]. Previous investigations found that Ebs is able to treat neurodegenerative diseases caused by radical and acute total cerebral ischemia [10,11]. However, the effects of Ebs on TBI have not been reported.

Here in this paper, we studied the neuro-protective effects of Ebs on TBI rat model following a modified Feeney’s weight-drop method. We found that Ebs administration significantly decreased inflammatory responses and improved the performance of TBI model rats. Moreover, Ebs treatment significantly decreased the expression level of toll like receptor 4 (TLR4) and P38 mitogen-activated protein kinase (P38-MAPK), indicating TLR4 mediated P38-MAPK signaling is a potential molecular mechanism for the effects of Ebs on the secondary injuries of TBI.

Materials and Methods

Animals

Male Sprague-Dawley (SD) rats were provided by the Animal Center of Chinese Academy of Sciences with the weight of 250±20 gram. The rats were housed in a animal facility with the temperature of 22-25℃ and the humidity of 70%. The rats can get access to food and water ad libitum. All experiments were approved by the animal control committee.

Traumatic brain injury rat model

90 SD rats were randomly grouped into the sham operation group, injury model group, low Ebs (Sigma Co., USA) group (3 mg/kg), moderate Ebs group (10 mg/kg) and high Ebs group (30 mg/kg). Animals were anesthetized by 10% chloral hydrate (35 mg /kg). The head of rats were fixed. A craniotomy (posterior, 2mm, lateral, 2mm, diameter, 5mm) was performed with a drill. Following a modified Feeney’s weight-drop model[12], contusion was produced by letting a 20 g weight hammer drop on the drilling position from a height of 30 cm. In the sham operation group, a bone window was opened without traumatic injury. Rats were handled carefully after modeling to prevent infections. Ebs or saline were administrated to rats 4 h after modeling, respectively. All experiments were performed 24h after drug administration.

Neurological severity score (NSS)

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NSS of rats was assessed 24h after drug administration, including movement, sensation, balance and reflex [13,14]. The highest score of 18 represents the most severe neurological deficit, while the lowest score of 0 indicates normal neurological function. The rater is well-experienced and blind to experimental groups.

Analysis of brain water content

Rats in each group were anesthetized and decapitated. The hemisphere with injury was separated and weighed of wet weight (W) immediately. Then it was dried at 100℃, following the measurement of dry weight (D). The brain water content was calculated as (W - D) /W×100% [15,16].

Hematoxylin-Eosin (HE) Staining

Brain slices were stained by haemotoxylin stained for 10 min, followed by 20-30 s staining of eosin solution. Then the slices were mounted for pathological observations.

Measurement of NO

Brains tissues were collected and measured according to standard protocol (Jiancheng Bioengineering Institute, Nanjing, China). Briefly, NO was extracted and determined by reading the optical density at 550 nm.

Real-time polymerase chain reaction (PCR)

Total brain tissue RNA was extracted using Trizol (Invitrogen Co., USA) reagent. Total RNA was reverse transcribed using reverse transcription kit (Promega Co., USA). Primers (Shenggong Bioengineering Co., Shanghai, China) used for quantification measurement of iNOS gene were, forward, 5'-CAG CCC TCA GAG TAC AAC GAT -3', reverse, 5'-CAG CAG GCA CAC GCA ATG AT -3'. For glyceraldehydes-3-phosphate dehydrogenase (GAPDH), the primer sequence were, forward, 5'-GCT GGT CAT CAA CGG GAA A-3', reverse, 5'- ACG CCA GTA GAC TCC ACG ACA-3'. GAPDH was used as controls.

Western-blotting

The expression of TLR4 and p-P38-MAPK was examined by western blot experiment according to standard protocol. All antibodies used were bought from Cell Signal Co. (1:1000). Band intensities were measured using image analysis software and expressed as the ratio to β-actin.

Statistical analysis

The results were analyzed using one-way ANOVA test in SPSS13.0 software. P < 0.05 was considered as significant difference.

Results

Effects of Ebs on NSS

The neurologic functions were impaired in TBI model as there is severe deficit in the movement and reflex of limbs. Rats in the TBI model group exhibited significantly higher NSS scores compared to the sham operation group (P < 0.05). Moreover, Ebs treatment significantly decreased the NSS score of TBI rats in a dose-dependent manner (P < 0.05) (Table 1).

Effect of Ebs on brain water content in TBI rats

Brain water content was significantly increased in TBI model rats (P < 0.05). Compared to the injury model group, Ebs treatment significantly reduced the brain water content in a dose-dependent manner (P < 0.05) (Table 2).

HE Staining

The rat brain exhibited severe structural abnormality, vacuolized fibrous structure and chromatin pyknosis after TBI as compared to control (Figure 1A, B). Ebs treatment significantly decreased the histopathologic deficits in TBI rats (Figure 1C-E).

Effects of Ebs on NO level

The NO expression was significantly increased in TBI model rats (P < 0.05). Compared to the injury model group, Ebs treatment significantly reduced NO level in a dose-dependent manner (P < 0.05) (Table 3) (Figure 2).

Effects of Ebs on iNOS expression

The iNOs mRNA level was significantly increased in TBI model rats (P < 0.01). Compared to the injury model group, Ebs administration significantly reduced iNOS expression in a dose-dependent manner (P < 0.05) (Figure 3).

Effects of Ebs on the expression of TLR4 and p-P38-MAPK

The expression level of TLR4 and p-P38-MAPK was significantly increased in TBI model rats (P < 0.01). Compared to the injury model group, Ebs administration significantly reduced the expression of both TLR4 and p-P38-MAPK in a dose-dependent manner (P < 0.05), indicating Ebs can reduce TBI-induced brain damages through the inhibition of TLR4 and P38-MAPK signaling (Figure 4).

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Discussion

The pathological changes after TBI, including inflammatory responses, apoptosis, oxidative stress responses and encephaledema, are the main causes of secondary brain injuries [17,18]. So it is important to suppress these adverse factors for the treatment of TBI. Ebselen is a class of organic selenium compounds, which is reported to have anti-inflammation, immune-regulation, and neuro-protective effects. It has been demonstrated that Ebs can protect the nerves system through inhibition of oxidative damages, neuronal apoptosis and neurotoxicity of glutamic acid and NO [9,19,20]. Here we studied the mechanism of Ebs in neuro-protection by the examination of behavioral functions, brain water content, expression of NO and iNOS in TBI rat model, as well as the P38-MAPK signaling pathway under these conditions.

TBI increases the expression of inflammation factors, chemotactic factors and adhesion molecules in the neural system, resulting in the transformation from primary injuries to secondary injuries. In TBI condition, there are increased permeability of capillaries and assembled intracellular fluid, leading to encephaledema. Thus, reducing the content of brain water is one of aspects in the treatment of TBI. Here we found that Ebs treatment significantly reduced brain water content and severe brain damages after TBI.

NO is a class of toxic free radicals, which is closely related to inflammatory neurological diseases. NO can mediate oxidative stress and inflammatory response through the impairment of proteins and DNA, leading to apoptosis and necrosis of neurons. iNOS is a major rate-limiting factor in the process of NO production. So, inhibition of iNOS and NO production is one of the effective treatments for inflammation. Here we reported that the expression of iNOS and NO was significantly increased in the injured brain tissues, while Ebs administration significantly inhibited NO expression and iNOS mRNA level, indicating the neuro-protective effects of Ebs is mediate by suppression of NO expression.

TLR4 family, first found in drosophila, is one of the important receptors for pathogen recognization. Under TBI conditions, TLR4 is highly expressed by glia cells, activating several signaling pathways, including P38-MAPK, JNK and NF-κB, resulting in increased expression of inflammation factors, such as NO, cyclooxygenase-2 and interleukin-1β [21]. Therefore, TLR4 is one of the important molecular targets for immune responses. P38-MAPK, which is one of the subclasses in the downstream of MAPK signaling pathway mediated by TLR4, can promote the aggregation and activation of cells, involving in the regulation of inflammation responses [22,23,24]. In this study, we found that Ebs treatment significantly decreased the expression level of TLR4 and p-P38-MAPK in a dose-dependence manner. Thus, Ebs is involved in the neuroprotection of TBI rat model by regulation of P38-MAPK signaling pathway, indicating Ebs is a potential therapeutic drug for the prevention of secondary brain injuries.

References

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Table 1 Effects of Ebs on neurological severity score in TBI rats ("",n=18 )

Group

Sham

Model

Ebs groups

(3mg/kg)

(10mg/kg)

(30mg/kg)

NSS

1.87± 0.61

8.82± 1.13 *

7.36± 1.42

5.77±1.22 #

3.59±1.61 #

*, P < 0.05, significant difference between the injury model group and the sham operation group.

#, P < 0.05, significant difference between the injury model group and Ebs groups.

Table 2 Effects of Ebs on brain water content in TBI rats ("",n=18 )

Group

Sham

Model

Ebs groups

(3mg/kg)

(10mg/kg)

(30mg/kg)

brain water content (%)

77.63± 1.02

84.81± 1.65*

81.52± 0.76 #

79.61±0.89 # #

78.43±1.15 # #

*, P < 0.05, significant difference between the injury model group and the sham operation group.

#, P < 0.05, ##, P < 0.01, significant difference between the injury model group and Ebs groups.

Table 3 Effect of Ebs on NO production in TBI rats ("",n=18 )

Group

Sham

Modle

Ebs groups

(3mg/kg)

(10mg/kg)

(30mg/kg)

NO (μM)

38.07±2.31

50.74±4.05*

45.95±5.51 #

43.41±3.22 # #

39.93±3.77 # #

*, P < 0.05, significant difference between the injury model group and the sham operation group.

#, P < 0.05, ##, P < 0.01, significant difference between the injury model group and Ebs groups.

""

Figure 1 Effects of Ebs on the hisopathologic changes in TBI rats

  1. HE staining of rat cerebral cortex in the sham-operation group.
  2. HE staining of rat cerebral cortex in the injury model group.
  3. HE staining of rat cerebral cortex in the low Ebs treatment group (3mg/kg).
  4. HE staining of rat cerebral cortex in the moderate Ebs treatment group (10mg/kg).
  5. HE staining of rat cerebral cortex in the high Ebs treatment group (30mg/kg).

""

Figure 2 Effects of Ebs on NO production in TBI rats ("",n=18 )

*, P < 0.05, statistically significant difference between model group and sham group.

#, P < 0.05, # #, P < 0.01, statistically significant differences between model group and Ebs groups.

""

Figure 3 Effects of Ebs on iNOS mRNA level in TBI rats

Up, representative gel band showing iNOS and GAPDH expression.

Down, bar graph of iNOS mRNA level in each condition relative to GAPDH expression.

#, P < 0.05, statistically significant difference between model group and sham group.

*, P < 0.05, **, P < 0.01, statistically significant differences between model group and Ebs groups.

""

Fig.4 Effect of Ebs on TLR4 and p38-MAPK expression in TBI rats

  1. TLR4 expression in each condition (up, representative gel band showing TLR4 and β-actin expression, down, bar graph of TLR4 protein level in each condition relative to β-actin expression).
  2. pP38-MAPK expression in each condition (up, representative gel band showing p-P38-MAPK and P38-MAPK expression, down, bar graph of p-P38-MAPK protein level in each condition relative to P38-MAPK expression).

#, P < 0.05, statistically significant difference between model group and sham group.

*, P < 0.05, **, P < 0.01, statistically significant differences between model group and Ebs groups.