MicroRNA-146a protects against intracerebral hemorrhage by inhibiting inflammation and oxidative stress.

The present study aimed to investigate the role of microRNA-146a (miR-146a) in intracerebral hemorrhage (ICH), and to further assess its underlying mechanism. An ICH rat model was established in the current study and 1 h following ICH induction, rats were treated with or without an miR-146a mimic. A total of 3 days following ICH induction, rat neurological score, brain water content and neuronal apoptosis were measured via flow cytometry. Levels of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β were detected via ELISA and certain biomarkers of oxidative stress, including malondialdehyde, superoxide dismutase and glutathione peroxidase, were also determined in current study. The expression of genes and proteins were detected in current study via reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. MicroRNA.org software and a dual luciferase reporter assay were used to confirm the association between miR-146a and TRAF6. The results of the current study revealed that miR-146a was significantly downregulated in ICH rats, and its overexpression reduced neurological damage and brain edema, as evidenced by decreased neurological scores and brain water content. Results from further analyses demonstrated that the overexpression of miR-146a inhibited neuronal apoptosis, reduced pro-inflammatory cytokine production and prevented oxidative stress in ICH rats. In addition, it was revealed that the upregulation of miR-146a repressed the TRAF6/NF-κB pathway in the brain tissue of ICH rats. TRAF6 was also determined to be a target of miR-146a. In conclusion, these data indicated that miR-146a protects against ICH by inhibiting inflammation and oxidative stress. Copyright: © Qu et al.

Introduction

As a common clinical neurological disease, intracerebral hemorrhage (ICH) accounts for ~30% of acute cerebrovascular diseases in China alone, particularly in the elderly (1). ICH is one of the most destructive stroke sub-types with a poor prognosis and high mortality (2,3). Despite significant advances in the treatment of ischemic stroke over the past decade, little progress has been made in the treatment of ICH (4). Furthermore several novel treatment strategies, including the use of factor VIIa and certain neuroprotective agents, have revealed conflicting results (3,5). Currently, no medication or surgical intervention is satisfactory for the treatment of ICH (3). Therefore, it is necessary to assess the pathogenesis of ICH and identify effective novel treatment methods.

ICH induced neurological damage can be divided into primary and secondary brain injury (6). Physical injury, which is considered to be primary brain injury, was caused by the rapid formation of hematoma (7). This primary brain injury will then develop a variety of parallel pathological processes including neuronal apoptosis, oxidative stress, neuroinflammation and excitotoxicity, leading to secondary brain injury (8,9). Therefore, the underlying mechanism of secondary injury may provide a promising strategy for the treatment of ICH.

MicroRNAs (miRNAs) are a group of small non-coding single stranded RNAs that negatively regulate the expression of target genes by binding to the 3′untranslated region (UTR) of multiple target mRNAs (10). The dysregulation of miRNA expression is involved in a variety of pathophysiological processes, including organ development, cell growth and cell differentiation (11–13). The role and underlying mechanism of various miRNAs in the pathogenesis and development of ICH have become increasingly studied (14–17).

miR-146a, a member of the miR-146 family located on the long arm of chromosome 5, is involved in the occurrence and development of various diseases, including cancer (18), spinal cord injury (19), myocardial dysfunction (20) and allergic airway inflammation (21). Recently, miR-146a has been studied in several neurological disorders (22). For example, Chu et al (23) indicated that miR-146a served an important role in ischemia-reperfusion injury by regulating the expression of interleukin-1 receptor associated kinase 1. Furthermore, the abnormal expression of miR-146a in murine models of Down's Syndrome and Alzheimer's Disease has been revealed (24). Additionally, the altered production of miR-146a in astrocytes has been considered to be a contributing factor in astrocyte-mediated spinal muscular atrophy pathology (25). Levels of miRNA-146a were also positively associated with the level of inflammation in rats with status epilepticus (26). These reports therefore indicate the crucial role of miR-146a in neurological disorders. Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), a member of the TNF receptor associated factor family, mediates signaling from members of the TNF receptor super-family and the Toll/IL-1 family (27,28). A previous study has determined that TRAF6 is closely associated with neurological disorders (29). Interestingly, TRAF6 has been confirmed as a target of miR-146a (30,31) that was involved in the development of ICH (32,33). A previous study has also revealed a lower expression of serum miR-146a in patients with ICH (34). However, to the best of our knowledge, the functional role of miR-146a in ICH remains unclear. Thus the current study was performed.

The current study aimed to determine the role of miR-146a in the development of ICH and to further assess its underlying molecular mechanism.

Materials and methods

Animals

A total of 60 Adult male Sprague-Dawley rats (weight, ~320g; 10 weeks old) were purchased from the Vital River Laboratories Co., Ltd. (Beijing, China). All rats were housed under a standard environment (temperature, 22±2°C; humidity, 55±5%; 12 h light/dark cycle) with free access to food and water. All experimental procedures were performed according to the Recommended Guideline for the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research. The present study was approved by the Animal Ethics Committee of the Xuan Wu Hospital, Capital Medical University (Beijing, China).

ICH model establishment

The ICH model was constructed via the stereotaxic intrastriatal injection of collagenase type VII (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as previously described (35,36).

A total of 60 rats were randomly divided into four groups (n=15): A sham, an ICH, an ICH+mimic control and an ICH+miR-146a mimic group. A total of 1 h following ICH induction, the mimic control or miR-146a mimic, synthesized by GenePharma, Inc. (Sunnyvale, CA, USA), was delivered to rats via an intracerebroventricular injection as described previously (37).

Neurological scores evaluation

To evaluate the neurological abnormalities of treated rats, the modified neurological severity score method (38) was utilized at 1 h and 3 days following ICH induction or sham surgery. To assess neurological scores, various tests, including sensory tests, motor tests, beam balance tests and absent reflexes and abnormal movements were performed (38). Neurological function was rated on a scale of 0–18 (normal score=0; maximal deficit score=18).

Brain edema assessment

To assess brain edema, the brain water content of rat brain tissue was measured. A total of 5 rats were randomly selected from each group and anesthetized with intraperitoneal 5% chloral hydrate (400 mg/kg) and decapitated. Brain tissue was then sectioned into the contralateral and ipsilateral hemispheres, and the cerebellum. Tissue was then immediately weighed to obtain the wet weight. The dry weight was determined following tissue drying for 24 h at 160°C. Finally, brain water content was calculated using the following formula: Brain water content (%)=(wet weight-dry weight)/wet weight × 100%.

Detection of oxidative stress factors

The thiobarbituric acid reaction method was used to detect the level of malondialdehyde (MDA) in brain tissue as previously described (36). To determine superoxide dismutase (SOD) activity in brain tissue, a SOD assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) was used in accordance with the manufacturer's protocol. The activity of glutathione peroxidase (GSH-Px) was also detected in samples using a GSH-Px assay kit (Cayman Chemical Company) in line with the manufacturer's protocol.

ELISA

A total of 3 days following ICH induction, blood and cerebrospinal fluid (CSF) were collected from all rats by piercing the heart and foramen magnum prior to sacrifice. Blood samples were centrifuged for at 1,000 × g for 5 min at 4°C and CSF samples were immediately centrifuged at 12,000 × g for 30 min at 4°C. The supernatants were subsequently collected for the detection of TNF-α (cat no. PT516) and interleukin (IL)-1β (cat no. PT303) levels using their respective ELISA kits (Beyotime Institute of Biotechnology) following the manufacturers' protocol.

Flow cytometric analysis

An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining kit (cat no. 70-AP101-100; Hangzhou Multi Sciences (Lanke) Biotech Co., Ltd., Hangzhou, China) was used to detect brain cell apoptosis. Brain tissues were regionally dissected from rats and dissociated to a single-cell suspension via enzymatic degradation using a neural tissue dissociation kit (Miltenyi Biotec, Inc., Cambridge, MA, USA) according to the manufacturer's protocol. Cells were collected following centrifugation at 1,200 × g for 5 min at 4°C, re-suspended in Annexin V-FITC binding buffer and PI solution and incubated at 37°C for 10 min in dark. Apoptotic cells were analyzed immediately using a FACScan flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Early apoptosis and late apoptosis were calculated and presented in this study using WinMDI software (version 2.5; www.cyto.purdue.edu/flowcyt/software/Catalog.htm).

Western blot analysis

The protein levels of TRAF6, phosphorylated (p)-nuclear factor (NF)-κB (p-p65) and matrix metallopeptidase (MMP)-9 were detected via western blotting. Protein from brain tissue was extracted using radioimmunoprecipitation assay lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) according to the manufacturer's protocol. A bicinchoninic acid assay (Thermo Fisher Scientific, Inc.) was performed to detect protein concentrations. An equal quantity of protein (30 µg/lane) was separated using 12% SDS-PAGE. Samples were then transferred onto polyvinylidene difluoride membranes and blocked with 5% skim milk at room temperature for 2 h. Subsequently, the membranes were incubated with primary antibodies (all 1:1,000; all Cell Signaling Technology, Inc.) against TRAF6 (cat no. 8028), p-NF-κB (p-p65; cat no. 3033MMP-9 (cat no. 13667) and β-actin (cat no. 4970) overnight at 4°C. Samples were then incubated with anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibodies (cat no. 7074; 1:2,000; Cell Signaling Technology, Inc.) at room temperature for 2 h. At the end of the experiment, protein bands were visualized using an enhanced chemiluminescence detection system (Super Signal West Dura Extended Duration Substrate; Pierce; Thermo Fisher Scientific, Inc.) and quantified with Image J 1.38X software (National Institutes of Health, Bethesda, MD, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from brain tissue was extracted by using TRIzol regent (Invitrogen; Thermo Fisher Scientific, Inc.) as per the manufacturer's protocol. The PrimeScript reverse transcription reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) was utilized to synthesize cDNA according to the manufacturer's protocol. cDNA was then analyzed using a TaqMan Universal PCR Master Mix kit (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. All primer sequences used in qPCR were obtained as required: U6 forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′ and reverse, 5′-CGCTTCACGAATTTGCGTGTCAT-3′; GAPDH forward, 5′-CTTTGGTATCGTGGAAGGACTC-3′ and reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′; miR-146a forward, 5′-GCGAGGTCAAGTCACTAGTGGT-3′ and reverse, 5′-CGAGAAGCTTGCATCACCAGAGAACG-3′; MMP-9 forward, 5′-AGACCTGGGCAGATTCCAAAC3′ and reverse, 5′-CGGCAAGTCTTCCGAGTAGT-3′; TRAF6 forward, 5′-GAGTTTGACCCACCTCTGGA-3′ and reverse, 5′-TTTCATTGTCAACTGGGCACT-3′. GAPDH or U6 was used as the internal control. The amplification conditions for qPCR were as follows: 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/elongation at 60°C for 30 sec. The 2−ΔΔCq method (39) was used for relative gene expression quantification.

Cell culture

The human cerebrovascular endothelial cell line (hCMEC/D3) was obtained from Shanghai Yu Bo Biological Technology Co., Ltd. (Shanghai, China). hCMEC/D3 cells were cultured in 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). Cells were maintained in a 5% CO2 incubator at 37°C.

Dual luciferase reporter assay

A biological prediction website (http://www.microRNA.org) was used to predict the targets of miR-146a. The results revealed that TRAF6 was a potential target of miR-146a. To confirm this prediction, wild type (WT)-TRAF6 and mutant (MUT)-TRAF6 3′-UTRs were cloned into a pmiR-RB-ReportTM dual luciferase reporter gene plasmid vector (Guangzhou RiboBio Co., Ltd., Guangzhou, China). To point-mutate the miR-146a binding domain on the 3′UTR of TRAF6, a QuikChange Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc., Santa Clara, CA, USA) was applied and used in accordance with the manufacturer's protocol. A total of 48 h following co-transfection with WT-TRAF6 or MUT-TRAF6 and miR-146a or mimic control using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), the luciferase activity of hCMEC/D3 cells was measured using a dual-luciferase assay system (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Luciferase activity was normalized to that of renilla luciferase.

Statistical analysis

All data analyses were performed using SPSS Version 18.0 (SPSS Inc., Chicago, IL, USA) and were presented as the mean ± standard deviations. Variations between groups were statistically assessed using a Student's t-test or one-way analysis of variance followed by a post-hoc Student-Neuman-Keuls test. P<0.05 was considered to indicate a statistically significant result.

Results

miR-146a is downregulated in ICH rats

To assess the role of miR-146a in ICH, miR-146a levels in the brain tissue of ICH and sham rats were determined using RT-qPCR. The results revealed that compared with sham group, the level of miR-146a significantly decreased in ICH rats (Fig. 1A). To further elucidate the protective role of miR-146a in ICH, miR-146a mimics or mimic controls were transiently transfected into ICH rats via intraparenchymal injection. Transfection efficiency was then confirmed via RT-qPCR 48 h post cell transfection. The results demonstrated that when compared with ICH rats, miR-146a levels were significantly upregulated by miR-146a mimics in ICH rats (Fig. 1B).

Figure 1.

miR-146a was downregulated in ICH rats. The level of miR-146a in the brain tissues of rats was detected using RT-qPCR. (A) miR-146a levels in Sham and ICH rat brain tissue. (B) miR-146a mimic or mimic controls were transiently transfected into ICH rats via intraparenchymal injections. Following 48 h, miR-146a levels in brain tissue were assessed using RT-qPCR. Data are presented as the mean ± standard deviation. **P<0.01 vs. the Sham group; ##P<0.01 vs. the ICH group. miR, microRNA; ICH, intracerebral hemorrhage; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

miR-146a reduces neurological damage in ICH rats

The neurological severity scores of rats from different groups were determined in current study. All ICH rats exhibited varying degrees of neurological damage 1 h following ICH induction (Fig. 2A), revealing that the ICH model was successfully constructed. As presented in Fig. 2B, compared with the ICH group, the neurological score of the miR-146a mimic group was significantly reduced at day 3 following ICH injury, indicating that miR-146a administration relieved neurological damages in ICH.

Figure 2.

Effect of miR-146a on neurological damage. The neurological severity score of rats from different groups was determined using the modified neurological severity score method (A) 1 h or (B) 3 days following ICH or sham surgery. Data are presented as the mean ± standard deviation. **P<0.01 vs. the Sham group; ##P<0.01 vs. the ICH group. miR, microRNA; ICH, intracerebral hemorrhage.

miR-146a reduces brain edema of ICH rats

As brain edema serves an important role in ICH-associated brain injury (40), the current study determined rat brain water content using the wet/dry method. As presented in Fig. 3, compared with the sham group, at 3 days following ICH, the brain water content in the ICH group was significantly increased, which was subsequently inhibited following administration of the miR-146a mimic.

Figure 3.

Effect of miR-146a on the brain water content of ICH rats. A total of 3 days following ICH or sham surgery, brain water content was determined using the wet/dry method. Data are presented as the mean ± standard deviation. **P<0.01 vs. the Sham group; ##P<0.01 vs. the ICH group. miR, microRNA; ICH, intracerebral hemorrhage.

miR-146a inhibits neuronal apoptosis in ICH rats

ICH-induced neuronal apoptosis is a key factor that results in secondary brain injury (41). To assess whether miR-146a exhibits an inhibitory effect on neuronal apoptosis, flow cytometry was performed. The results revealed that compared with the sham group, ICH induced neuronal apoptosis (early+late apoptosis). This effect was then reduced following treatment with miR-146a mimics (Fig. 4).

Figure 4.

Effect of miR-146a on ICH rat neuronal apoptosis. A total of 3 days following ICH or sham surgery, neuronal apoptosis (early apoptosis + late apoptosis) was determined using flow cytometry. Early apoptosis + late apoptosis were calculated and presented. Data are presented as the mean ± standard deviation. **P<0.01 vs. the Sham group; ##P<0.01 vs. the ICH group. miR, microRNA; ICH, intracerebral hemorrhage; PI, propidium iodide; FITC, fluorescein isothiocyanate.

miR-146a inhibits the pro-inflammatory cytokine levels in ICH rats

To assess whether miR-146a suppresses inflammation following ICH, MMP-9, an indicator of inflammation, was detected in the brain tissue of ICH rats via western blotting and reverse transcription-quantitative polymerase chain reaction. The findings revealed that, compared with the sham group, the protein and mRNA levels of MMP-9 were significantly increased in the ICH group, while miR-146a mimic treatment reversed this effect (Fig. 5A and B). Furthermore, the levels of pro-inflammatory cytokines (TNF-α and IL-1β) in the blood and CSF of rats were detected using ELISA. The results demonstrated that compared with the sham group, levels of TNF-α and IL-1β were significantly enhanced in the serum and CSF of ICH rats. In addition, these increases were eliminated following treatment with miR-146a mimics (Fig. 5C-F).

Figure 5.

Effect of miR-146a on the inflammatory response of ICH rats. A total of 3 days following ICH induction, the (A) protein and (B) mRNA levels of MMP-9 were assessed using western blotting and reverse transcription-quantitative polymerase chain reaction. Levels of (C and D) TNF-α and (E and F) IL-1β in the blood and CSF of all rats were also detected by performing ELISA. Data were presented as the mean ± standard deviations. *P<0.05 and **P<0.01 vs. the Sham group; #P<0.05 and ##P<0.01 vs. the ICH group. miR, microRNA; ICH, intracerebral hemorrhage; MMP-9, matrix metallopeptidase-9; TNF-α, tumor necrosis factor-α; IL, interleukin; CSF, cerebrospinal fluid.

miR-146a inhibits oxidative stress in ICH rats

Abnormal high oxidative stress leads to ICH-induced brain injury (42). Therefore, the current study determined whether miR-146a exhibited an effect on oxidative stress in ICH by assessing the levels of certain biomarkers of this condition, including MDA, SOD and GSH-Px in the brain tissue of rats. The results indicated that compared with the sham group, levels of MDA significantly increased and the activity of SOD and GSH-Px significantly decreased in ICH rats. Furthermore, following miR-146a mimic treatment, these changes were reversed (Fig. 6).

Figure 6.

Effect of miR-146a on oxidative stress in ICH rats. A total of 3 days following ICH induction, the levels of oxidative stress biomarkers, including (A) MDA, (B) SOD and (C) GSH-Px were measured in the brain tissue of rats. Data were presented as the mean ± standard deviation. **P<0.01 vs. the Sham group; #P<0.05 and ##P<0.01 vs. the ICH group. miR, microRNA; ICH, intracerebral hemorrhage; MDA, malondialdehyde; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase.

TRAF6 is a target of miR-146a

A biological prediction website (http://www.microRNA.org) was used to predict the targets of miR-146a. The results revealed that TRAF6 is a potential target of miR-146a (Fig. 7A). To confirm whether miR-146a directly modulates TRAF6 expression by interacting with potential binding sites, a luciferase reporter assay was performed. As presented in Fig. 7B, compared with cells co-transfected with WT-TRAF6 and mimic control, luciferase activity was significantly reduced following co-transfection with WT-TRAF6 and miR-146a mimics. These data indicate that miR-146a directly targets TRAF6 in hCMEC/D3 cells.

Figure 7.

TRAF6 is a direct target of miR-146a. (A) Interactions between miR-146a and the 3′UTR of TRAF6 was predicted using microRNA target site prediction software (http://www.microRNA.org). (B) Luciferase activity results of a reporter containing WT-TRAF6 3′UTRs or MUT-TRAF6 3′UTRs. All data are presented as the mean ± standard deviation of three independent experiments. **P<0.01 vs. the mimic control. TRAF6, tumor necrosis factor receptor-associated factor 6; miR, microRNA; UTR, untranslated region; WT, wild-type; MUT, mutant.

miR-146a represses the TRAF6/NF-κB pathway in ICH rats

Previous studies have revealed the inhibitory effect of miR-146a on the TRAF6/NF-κB pathway (28,40) and its involvement in ICH (32,33). To determine the molecular mechanism underlying the protective role of miR-146a in ICH, the current study determined whether the TRAF6/NF-κB pathway is involved. As presented in Fig. 8, protein and mRNA levels of TRAF6 and p-NF-κB (P-65) were enhanced in the brain tissue of ICH rats when compared with rats of the sham group. Furthermore, miR-146a mimic treatment significantly decreased the protein and mRNA levels of TRAF6 and p-NF-κB (P-65) in the brain tissue of ICH rats.

Figure 8.

Effect of miR-146a on the TRAF6/NF-κB pathway in ICH rats. A total of 3 days following ICH induction, the (A) protein and mRNA levels of (B) TRAF6 and (C) P-65 were determined using western blotting and reverse transcription-quantitative polymerase chain reaction, respectively. Data are presented as the mean ± standard deviation. **P<0.01 vs. the Sham group; ##P<0.01 vs. the ICH group. miR, microRNA; TRAF6, tumor necrosis factor receptor-associated factor 6; ICH, intracerebral hemorrhage; P-65, nuclear factor-κB.

Discussion

The present study determined that miR-146a was downregulated in ICH rats and that miR-146a mimic treatment significantly relieved neurological injury and brain edema in ICH rats, as evidenced by reduced neurological scores and brain water content. Furthermore, the results revealed that the miR-146a mimic inhibited neuronal apoptosis, reduced pro-inflammatory cytokine production and reduced oxidative stress in ICH rats. Further assessment indicated that the miR-146a mimic repressed the TRAF6/NF-κB pathway in the brain tissue of ICH rats. These data indicated that miR-146a protects against intracerebral hemorrhage by inhibiting inflammation and oxidative stress. Therefore, miR-146a may be a novel and promising therapeutic target for the treatment of ICH.

ICH is an important public health problem associated with a high mortality and morbidity, with no effective available treatment (2,3). Therefore, determining the pathogenesis of ICH and identifying novel, effective treatment strategies have important clinical significance. Increasing evidences have indicated that miRNAs serve important roles in the pathogenesis and development of ICH (12–15). The study of miRNAs in ICH presents a novel direction for its diagnosis and treatment (43–47).

miR-146a has been revealed to be involved in the development and progression of several diseases. Tan et al (48) reported that miR-146a enhanced chemotherapeutic resistance in lung cancer cells by regulating the expression of DNA damage inducible transcript 3. Furthermore, Li et al (49) suggested that miR-146a was involved in systemic juvenile idiopathic arthritis via the modulation of macrophage polarization by targeting inhibin subunit beta A. miR-146a has also been studied in coronary heart disease (50) and is also involved in the progression of nervous system disease (51,52). A previous study has revealed that a lower expression of miR-146a is exhibited in the serum of patients with ICH (34). However, the functional role of miR-146a in ICH remains unclear. The present study was therefore performed to assess the role of miR-146a in the development of ICH in vivo, to provide more theoretical basis for the treatment of ICH.

The current study established an ICH rat model and subsequently assessed the level of miR-146a in ICH and sham rats via RT-qPCR. Consistent with a previous study (34), the results indicated that when compared with sham group, levels of miR-146a were significantly downregulated in ICH rats. The present study then assessed whether the miR-146a mimic exerted a protective effect on ICH rats, the results of which revealed that the miR-146a mimic reduced neurological damage and brain edema in ICH rats, as evidenced by decreased neurological scores and reduced brain water content. Neuronal apoptosis, the inflammatory response and oxidative stress are three crucial factors of secondary brain damage (53). Further analyses in the current study indicated that neuronal apoptosis and oxidative stress were significantly prevented following miR-146a mimic treatment. Previous studies have demonstrated that miR-146a represses the inflammatory response by inhibiting the release of several pro-inflammatory cytokines including IL-6 and TNF-α in neurological disorders (54,55). The results of the present study revealed that the overexpression of miR-146a significantly reduced pro-inflammatory cytokine (MMP-9, TNF-α and IL-1β) production in ICH rats. Previous studies have reported the inhibitory effect of miR-146a on the TRAF6/NF-κB pathway (30,43) and its involvement in ICH (32,33). Therefore, the current study determined whether the TRAF6/NF-κB pathway was involved in the molecular mechanism underlying the protective role of miR-146a in ICH rats. The results demonstrated that the miR-146a mimic significantly repressed the TRAF6/NF-κB pathway in the brain tissue of ICH rats. Furthermore, it was revealed that miR-146a directly targets TRAF6 in the human cerebrovascular endothelial cell line hCMEC/D3.

Taken together and to the best of our knowledge, this was the first study to reveal that miR-146a is downregulated in ICH rats, and that the miR-146a mimic serves a protective role in ICH development by inhibiting the inflammatory response and oxidative stress at least partly by regulating the TRAF6/NF-κB pathway. Therefore, miR-146a may be a novel and promising therapeutic target for the treatment of ICH. However, the current study has several limitations. The level of reactive oxygen species was not determined to assess oxidative stress in the present study, and immunohistochemistry was not performed to visually assess the condition of ICH in rats. The current preliminary study determined the role of miR-146a in ICH, but the results require further validation in future study. For example, the effect of miR-146a downregulation in ICH rats should be assessed. Furthermore, the correlation of miR-146a expression with the clinical characteristics and prognosis of patients with ICH are required to confirm the role of miR-146a in ICH.