Breviscapine reduces neuronal injury caused by traumatic brain injury insult: partly associated with suppression of interleukin-6 expression

Ling Jiang, Yue Hu, Xiang He, Qiang Lv, Ting-hua Wang, Qing-jie Xia

Institute of Neurological Disease, Department of Anesthesiology and Translation Neuroscience Center, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China

Breviscapine reduces neuronal injury caused by traumatic brain injury insult: partly associated with suppression of interleukin-6 expression

Ling Jiang#, Yue Hu#, Xiang He, Qiang Lv, Ting-hua Wang, Qing-jie Xia＊

Institute of Neurological Disease, Department of Anesthesiology and Translation Neuroscience Center, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China

How to cite this article:Jiang L, Hu Y, He X, Lv Q, Wang TH, Xia QJ (2017) Breviscapine reduces neuronal injury caused by traumatic brain injury insult: partly associated with suppression of interleukin-6 expression. Neural Regen Res 12(1):90-95.

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Graphical Abstract

Breviscapine improves neurobehovior and down-regulates interleukin-6 expression in rats with traumatic brain injury

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orcid: 0000-0001-7135-2925 (Ling Jiang)

Breviscapine, extracted from the herbErigeron breviscapus, is widely used for the treatment of cardiovascular diseases, cerebral infarct, and stroke, but its mechanism of action remains unclear.is study established a rat model of traumatic brain injury induced by con‐trolled cortical impact, and injected 75 μg breviscapineviathe right lateral ventricle. We found that breviscapine signi fi cantly improved neurobehavioral dysfunction at 6 and 9 days aer injection. Meanwhile, interleukin‐6 expression was markedly down‐regulated following breviscapine treatment. Our results suggest that breviscapine is e ff ective in promoting neurological behavior aer traumatic brain injury and the underlying molecular mechanism may be associated with the suppression of interleukin‐6.

nerve regeneration; breviscapine; traumatic brain injury; neuroprotective e ff ect; interleukin-6; neural regeneration

Introduction

Breviscapine is a flavonoid extracted from the herbErigeron breviscapus. Scutellarin is the main active ingredient of bre‐viscapine, and is characterized by the structural formula of 4,5,6‐trihydroxy fl avone‐7‐glucuronide (Lou et al., 2015). Clin‐ical trails and experimental studies have shown that breviscap‐ine dilates blood vessels, reduces vascular resistance, improves microcirculation, and suppresses platelet aggregation (Wang et al., 2010; Guo et al., 2014). Accordingly, breviscapine has long been used in clinical practice of patented Chinese medicines to treat cardiovascular and cerebrovascular diseases (Tian et al., 2014). Moreover, breviscapine is considered a routine adminis‐tration for craniocerebral injury patients.

Traumatic brain injury (TBI) is associated with high dis‐ability and mortality, and exhibits a gradually increasing trend with development of society (Zaninotto et al., 2016).Although many proposed efforts have shown a promising outcome in pre‐clinical practice, none have survived the di ff erent phases of clinical trials due to the complex patho‐physiological process: TBI leads to cerebral structural dam‐age and functional deficits due to instantaneous primary mechanical injury accompanied by delayed secondary injury. Indeed, secondary brain injury plays a crucial role in prognosis of TBI (Kinoshita, 2016), with a cascade of in fl ammatory processes involved in pathology of secondary damage as a consequence of mitochondrial dysfunction, cerebral hypoxia, and disordered calcium homeostasis (Niklas et al., 2006; Bramlett and Dietrich, 2007). A body of evidence indicates that inflammation plays a dual role in TBI outcome. Inflammation stimulates reparation and regenerationviaclearance of necrotic and apoptotic cells (Wieloch and Nikolich, 2006; Ziv et al., 2006), while also facilitating secondary injuryviathe release of various in‐fl ammatory cytokines, which in turn drives and accelerates additional inflammatory processes (Morganti‐Kossman et al., 1997; Zhang et al., 2014).ese in fl ammatory cascades exacerbate brain tissue damage and cause irreversible cen‐tral nervous system impairment.

Interleukin 6 (IL‐6) is an important pro‐inflammatory cytokine, and one of the most widely studied molecules in TBI. IL‐6 is primarily produced in the central nervous sys‐tem (Hans et al., 1999; Lau and Yu, 2001), and is markedly up‐regulated after injury (Hillman et al., 2007) and also shown to correlate with increased production of other cen‐tral in fl ammatory cytokines (Di Santo et al., 1996). Mean‐while, IL‐6 expression in cerebrospinal fluid (Singhal et al., 2002; Chiaretti et al., 2008), serum (Arand et al., 2001), and brain parenchyma (Winter et al., 2004) is strongly as‐sociated with TBI outcome.erefore, IL‐6 may be a major contributor to the inflammatory response following TBI (Kumar et al., 2015). In neural regeneration, down‐regula‐tion of IL‐6 ameliorates cell in fl ammation, apoptosis, and oxidative stress, and may further promote neuronal surviv‐al and regeneration (Poulsen et al., 2005; Xu et al., 2014).e relationship between breviscapine and IL‐6 expression in neurological repair of neurotrauma has not been re‐ported.us, in the current study, we used a rat model of controlled cortical impact to examine the molecular mech‐anism of the neuroprotective e ff ect of breviscapine on TBI insult.

Materials and Methods

Animals and group assignment

Sixty healthy specific‐pathogen‐free Sprague‐Dawley rats aged 6—8 weeks and weighing 200—240 g were provided by the Laboratory Animal Center of Kunming Medical Univer‐sity in China (license No. SYXK (Dian) K2015‐0004). The rats were randomly divided into: sham group, TBI group, and TBI + breviscapine group (able 1). Rats were housed in a 12‐hour light‐dark cycle and supplied with food and water. All procedures were performed according to the Guide to the Care and Use of Experimental Animals published by the National Institute of Health (NIH publication 85—23, revised 1985), with animal protocols approved by the Animal Ethics Committee of Sichuan University, West China Hospital, Chi‐na (approval No. ScUEC‐145306).

Model preparation and drug treatment

Rats were intraperitoneally anesthetized with 3.6% chloral hydrate (CCl3CH(OH)2) (10 mL/kg), and placed in the prone position. Following routine disinfection, a midline incision was made through the scalp. A controlled cortical impact model was used to produce TBI in the parietal lobe. A cra‐niectomy was performed on the leanterior frontal area: 2.5 mm from the sagittal suture and 1.5 mm from the coronal suture (Wang et al., 2015).e craniectomy was approximate‐ly 5 mm in diameter and was administered using an electric micro drill. Aer exposure of the dura, a contusion was made using a 3.0 mm convex tip attached to an electromagnetic impactor (Leica, Wetzlar, Germany) mounted to a digitally calibrated manipulator arm. The impact parameters were set at a contusion depth of 2 mm (from dura), constant velocity of 1.9 m/s, and sustained impact of 300 ms. Following controlled cortical impact injury, rats in the TBI + breviscapine group were implanted with a dose of 3 μL (25 μg/μL) breviscapine (batch number 20121203‐1; approval number Z20053907; speci fi cation 25 mg; Longjing Pharmaceutical Limited Compa‐ny, Kunming, China). Breviscapine (composed of yellow loose lumps and dissolved in pure water as a 25 μg/μL solution) was implanted into the right lateral ventricle. The scalp was su‐tured. Finally, rats were placed in a water‐heated incubation chamber at 37°C until they fully recovered from anesthesia. Rats in the sham group were treated with the same procedure but without the controlled cortical impact injury. To note, greater attention should be paid to the dura, as rats with dis‐rupted dura were withdrawn from the study.

Neurobehavioral assessment

Severity of neurological de fi cit was evaluated using the neu‐rological severity score (NSS) system (Chen et al., 2001). Neurobehavioral function is graded on a scale of 0—18 (0, normal score; 18, maximal deficit score). NSS scoring re‐fl ects motor, sense, re fl ex, and balance functions. For injury severity, one point re fl ects the inability to perform a task or lack of an assessed re fl ex: 13—18, severe injury; 7—12, mod‐erate injury; and 1—6, mild injury.us, more severe injury is reflected by a higher score. Recovery of neurological function was observed and all rats’ scores recorded at 1, 3, 6, 9, and 14 days aer injury.

Sample harvest

able 1 Animal number in each group for each test

able 1 Animal number in each group for each test

NSS: Neurological severity score; RT‐PCR: reverse transcription‐polymerase chain reaction; TBI: traumatic brain injury.

RT‐PCR/ western blot assay (6 days) Sham Sham‐operated surgery 10 5 5 TBI TBI‐operated surgery 10 5 5 TBI + breviscapine TBI‐operated surgery + breviscapine 10 5 5 Group Treatment NSS (1, 3, 6, 9, 14 days) Immunohistochemistry (6 days)

Total RNA was extracted from harvested cerebral cortex tis‐sue using Trizol Reagent (SuperfecTRITM, Shanghai, China) prior to cDNA synthesis. To generate cDNA, reverse tran‐scription was performed according to the instructions of the RevertAid First Strand cDNA Synthesis Kit (ermo Scien‐ti fi c, Waltham, MA, USA). Single‐strand cDNA was synthe‐sized by incubating template RNA (2.5 μg) with oligo‐(dT) 18 primer (1 μL), and nuclease‐free water (to 12 μL) at 65°C for 5 minutes. Next, Revert Aid M‐MuLV Reverse Transcrip‐tase (200 U/μL, 1 μL) with 5× reaction buffer (4 μL), Ribo Lock RNase Inhibitor (20 U/μL, 1 μL), and 10 mM dNTP Mix (2 μL) was added (to a fi nal volume of 20 μL), and incu‐bated for 60 minutes at 42°C. Reactions were terminated by heating at 70°C for 5 minutes. PCR was performed using the T100TMermal Cycler (BIORAD, Hercules, CA, USA). Five μL of fi ve‐fold diluted template cDNA was added in a fi nal volume of 25 μL.e primer sequences were: IL‐6, sense 5′‐GAG GAT ACC ACT CCC AAC AGA CC‐3′ and antisense 5′‐GAG GAT ACC ACT CCC AAC AGA CC‐3′; annealing temperature: 58°C; and β‐actin, sense 5′‐GTA AAG ACC TCT ATG CCA ACA‐3′ and antisense 5′‐GGA CTC ATC GTA CTC CTG CT‐3′; annealing temperature: 52.5°C. PCR ampli fi cation was performed as follows: initial denaturation at 94°C for 5 minutes, 35 cycles of denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, with elongation at 72°C for 1 minute, followed by elongation at 72°C for 10 min‐utes. β‐Actin was used as the internal control. Relative gene expression was calculated using the 2−ΔΔCtmethod, in which Ct indicates the cycle threshold, with fractional cycle number being the fl uorescent signal that reached detection threshold. Normalized ΔCt values for each sample were calculated using β‐actin as the endogenous control gene.

Western blot assay

Cortical tissue was harvested, lysed, and sonicated in radioim‐mune precipitation assay bu ff er (Beyotime, Shanghai, China) supplemented with protease inhibitors (Roche, Basel, Switzer‐land). Protein quantification was performed using the bicin‐choninic acid assay kit (Beyotime). Protein samples (100 μg) diluted in sodium dodecyl sulphate loading bu ff er (Biosharp, Hefei, Anhui Province, China) were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels, and transferred to polyvinylidene di fl uoride membranes. Membranes were blocked with Tris‐bu ff ered saline Tween‐20 (TBST) for 2 hours at room temperature, and then incubated overnight at 4°C with IL‐6 primary polyclonal antibody (rabbit; 1:200; USCN, Wuhan, Hubei Province, China). Afterwards, blots were washed three times in TBST for 5 minutes each time. Secondary antibody (goat anti‐rabbit IgG; ZSGB‐BIO, Beijing, China) was applied at 1:5,000 dilution in TBST, and incubated for 2 hours at room temperature. Finally, samples were developed with enhanced chemiluminescence and analyzed using Alpha Innotech (BIORAD). Optical density values were determined using Image J soware (National In‐stitutes of Health, Bethesda, MD, USA), and represented as IL‐6 to β‐actin ratio.

Immunohistochemistry

Statistical analysis

Experimental data were expressed as the mean ± SD, and an‐alyzed using SPSS 20.0 soware (IBM Corporation, Armonk, NY, USA). One‐way analysis of variance was performed to compare three data sets, with Student’st‐test performed for two sets. A value ofP＜ 0.05 was considered statistically signi fi cant.

Results

Breviscapine improved neurobehavior inBI rats

NSS scoring was performed at 1, 3, 6, 9, and 14 days aer injury (Figure 1). NSS score was significantly higher in the TBI group compared with the sham group at each time point (P＜ 0.01). However, at 6 and 9 days aer injury, NSS score was signi fi cantly decreased in the TBI + breviscapine group compared with the TBI group (P＜ 0.01,P＜ 0.05). No obvious change was observed at 1, 3, and 14 days aer injury.

Breviscapine reversed IL-6 expression in the injured cortex ofBI rats

RT‐PCR and western blot assay were used to examine IL‐6 in the injured cortex at 6 days after injury. IL‐6 gene and protein levels were dramatically up‐regulated in the TBI group compared with the sham group (P＜ 0.05; Figure 2A). However, breviscapine treatment reversed this trend of up‐regulated IL‐6 induced by TBI insult, with IL‐6 gene and protein expression signi fi cantly down‐regulated in the TBI + breviscapine group compared with the TBI group (P＜ 0.01,P＜ 0.05; Figure 2B).

Breviscapine e ff ect on IL-6 distribution in injured brain tissue ofBI rats

Immunohistochemical staining showed IL‐6 was mainly lo‐cated in the cell membrane, with some in the cytoplasm and extracellular matrix (Figure 3A). Moreover, IL‐6 expression was increased in the TBI group, but decreased in the cor‐tex following breviscapine treatment. Quantitative analysis con fi rmed a signi fi cantly increased IL‐6‐positive cell number in the TBI group compared with the sham group (P＜ 0.01). In contrast, breviscapine administration notably decreased IL‐6‐positive cell number compared with the TBI group (P＜0.05; Figure 3B).

Discussion

In this study, we successfully established a TBI rat model and then administered breviscapine treatment. We found breviscapine improved neurobehavioral function that had been impaired by TBI insult. Further, both IL‐6 mRNA and protein expressions were markedly decreased compared to TBI rats with no breviscapine treatment.ese results show that breviscapine plays a neuroprotective role in rats with TBI injury that may be associated with down‐regulation of IL‐6.

TBI caused by head trauma always results in cognitive and behavioral disabilities (Stoller, 2015; Barman et al., 2016). Here, we found significantly impaired NSS scores with a declining trend. This indicates notable impairment of neu‐robehavioral function in the early period of TBI, followed by self‐rehabilitation in the later stage.e pathological process of TBI involves primary and secondary damage. Secondary damage results from external force aer injury, and includes cerebral edema and intracranial hemorrhage (Dardiotis et al., 2014; Hochstadter et al., 2014), which induce signi fi cant up‐regulation of intracranial pressure in the early period of injury. In clinical practice, increased intracranial pressure is associated with a worsened outcome aer TBI insult (Kukreti et al., 2014).erefore, more severe neurobehavioral dysfunc‐tion in the early stage of TBI may be associated with increased intracranial pressure. Conversely, self‐rehabilitation in the subsequent period may be associated with decreased intracra‐nial pressure induced by absorption of edema and hemor‐rhage.

We used breviscapine to treat TBI insult for the fi rst time. In the early stage of TBI, we found breviscapine treatment did not improve any neurological deficits. Contrarily, one day aer breviscapine treatment, NSS scores were higher in the TBI + breviscapine group compared with the TBI group. Previous evidence has shown that breviscapine can expand blood vessels, improve articulation, and anti‐platelet and red blood cell aggregation, and establish collateral circulation (Zheng et al., 2015). Accordingly, aggravated neuropatholo‐gy was observed in TBI rats with breviscapine treatment in the early stages, which may be associated with blood vessel dilatation and increased intracranial pressure, which there‐by exacerbates neurological deficits. Meanwhile, we found breviscapine improved neurological dysfunction at 6 and 9 days aer injection, with no signi fi cant e ff ect observed at 14 days. Iadecola et al. (1995) found that breviscapine injection could treat patients with severe brain injury.e protective e ff ect of breviscapine may be associated with its mechanism of improved energy metabolism, free radical scavenging, inhibition of intracellular Ca2+overload, excitatory amino acid toxicity, inflammatory suppression, and regulation of brain blood vessel activity (Wang et al., 2010; Zheng et al., 2015). Thus, after development of the acute stage of TBI, breviscapine may protect from neurological dysfunction. In the later stage, self‐rehabilitation and drug metabolism may have resulted in no signi fi cant di ff erence in neurobehavioral assessment between rats with and without breviscapine ad‐ministration.

To examine the underlying molecular mechanism, we in‐vestigated expression of IL‐6, a critical cytokine controlling the transition from innate to acquired immunity. IL‐6 is up‐regulated when neuroinflammation is expected, such as following central nervous system infection or injury or central nervous system disease (Erta et al., 2012). Previous reports have shown significantly increased IL‐6 expression post‐TBI in cerebrospinal fl uid, serum (Arand et al., 2001), and brain parenchyma (Winter et al., 2004), which is strong‐ly associated with clinical outcome (Singhal et al., 2002; Chiaretti et al., 2008).ese reports con fi rm our observation that cortical IL‐6 mRNA and protein expression is strikingly up‐regulated by TBI insult. It has been demonstrated that IL‐6 over‐expression is associated with neurodegeneration, blood‐brain barrier permeability, astrogliosis, and produc‐tion of other pro‐in fl ammatory cytokines, such as IL‐1β and TNF‐α (Campbell et al., 1993; Brett et al., 1995; Di Santo et al., 1996; Penkowa et al., 1999). Therefore, suppression of IL‐6 can improve neuronal survival and ameliorate neurobe‐havioral dysfunction.

Here, we show that breviscapine markedly down‐regulates IL‐6 mRNA and protein levels, which were up‐regulated by TBI injury. Except for the ability to dilate blood vessels, inhib‐it platelet aggregation, and scavenge free oxygen radicals, bre‐viscapine promotes recovery of neurological function, which is associated with a reduction of brain and in fl ammatory re‐actions induced by cerebral hemorrhage (Wang et al., 2011; Li et al., 2014). More importantly, Zhang et al. (2007) found that breviscapine decreased IL‐6 release associated with inhibition of protein kinase C‐alpha mRNA transcription to inhibit the in fl ammatory cascade.ese results fi rmly support our fi nd‐ing that breviscapine plays a protective role in TBI recovery, which may involve down‐regulated IL‐6 expression.

Figure 2 E ff ect of breviscapine on IL-6 expression in the injured cortex ofBI rats at 6 days after injury.

Figure 3 E ff ect of breviscapine on IL-6 distribution in the injured brain tissue ofBI rats.

Figure 1 E ff ect of breviscapine on neurobehavior ofBI rats.

In summary, we show for the fi rst time that breviscapine can treat TBI, with its neuroprotective e ff ect partly associat‐ed with suppression of IL‐6 expression.

Our results provide experimental evidence for brevis‐capine application in the treatment of neurotrauma. None‐theless, our findings do not support a direct relationship between the therapeutic effect of breviscapine and IL‐6 expression. Future investigations should confirm the exact relationship between IL‐6 expression and breviscapine treat‐ment for TBI.

Acknowledgments:We are very grateful to the Laboratory Animal Center of Kunming Medical University of China for providing animals. Also, we sincerely thank professor Jia Liu from Department of Experimental Zoology, Kunming Medical University in China for making substantial contributions in the analysis and experimental technical guidance.

Author contributions:QJX conceived the study and participated in its design and coordination. LJ prepared the animal model, performed statistical analysis and draed the paper. YH carried out the immunohistochemistry. XH helped to prepare the animal model and RT-PCR. QL carried out RTPCR and corrected the paper. THW participated in study design and language correction. All authors approved the fi nal version of the paper.

Con fl icts of interest:None declared.

Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

Peer review:

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Copyedited by James R, Frenchman B, Yu J, Li CH, Qiu Y, Song LP, Zhao M

10.4103/1673-5374.198990

Accepted: 2016-11-26

＊Correspondence to: Qing-jie Xia, Ph.D., xiaqj2005@126.com.