Aerobic exercise combined with huwentoxin-I mitigates chronic cerebral ischemia injury

hai-feng Mao, Jun Xie, Jia-qin Chen, Chang-fa Tang wei Chen Bo-cun Zhou rui Chen hong-lin Qu, Chu-zu wu

1 Key Laboratory of Physical Fitness and Exercise Rehabilitation of Hunan Province, Hunan Normal University, Changsha, Hunan Province, China

2 College of Physical Education, Yichun University, Yichun, Jiangxi Province, China

Aerobic exercise combined with huwentoxin-I mitigates chronic cerebral ischemia injury

hai-feng Mao1,2, Jun Xie2, Jia-qin Chen1,*, Chang-fa Tang1, wei Chen1, Bo-cun Zhou1, rui Chen1, hong-lin Qu1,2, Chu-zu wu2

1 Key Laboratory of Physical Fitness and Exercise Rehabilitation of Hunan Province, Hunan Normal University, Changsha, Hunan Province, China

2 College of Physical Education, Yichun University, Yichun, Jiangxi Province, China

How to cite this article:Mao HF, Xie J, Chen JQ, Tang CF, Chen W, Zhou BC, Chen R, Qu HL, Wu CZ (2017) Aerobic exercise combined with huwentoxin-I mitigates chronic cerebral ischemia injury. Neural Regen Res 12(4):596-602.

Open access statement:This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Funding:This work was supported by a grant from the Science and Technology Plans of Jiangxi Province Education Department of China, No. GJJ14705; a grant from the Science and Technology Plans of Health and Family Planning Commission of Jiangxi Province of China, No. 20175563.

Graphical Abstract

Neuroprotective ef f ect of huwentoxin-I (HWTX-I) in combination with exercise on mouse models of chronic cerebral ischemia

Ca2+channel blockers have been shown to protect neurons from ischemia, and aerobic exercise has signif i cant protective ef f ects on a variety of chronic diseases.e present study injected huwentoxin-I (HWTX-I), a spider peptide toxin that blocks Ca2+channels, into the caudal vein of a chronic cerebral ischemia mouse model, once every 2 days, for a total of 15 injections. During this time, a subgroup of mice was subjected to treadmill exercise for 5 weeks. Results showed amelioration of cortical injury and improved neurological function in mice with chronic cerebral ischemia in the HWTX-I + aerobic exercise group.e combined ef f ects of HWTX-I and exercise were superior to HWTX-I or aerobic exercise alone. HWTX-I ef f ectively activated the Notch signal transduction pathway in brain tissue. Aerobic exercise up-regulated synaptophysin mRNA expression.ese results demonstrated that aerobic exercise, in combination with HWTX-I, ef f ectively relieved neuronal injury induced by chronic cerebral ischemiaviathe Notch signaling pathway and promoting synaptic regeneration.

nerve regeneration; chronic cerebral ischemia; aerobic exercise; huwentoxin-I; Notch signaling pathway; calcium overload; neural regeneration

Introduction

Chronic cerebral ischemia, a sustained modest reduction in cerebral blood fl ow, is associated with neuronal damage and cognitive decline (Cechetti et al., 2012). Previous studies have focused on the mechanisms involved in chronic cerebral ischemia (Antipenko et al., 2016; Edrissi et al., 2016). In recent years, the ischemic mechanism of calcium overload in brain injury has become a hot research topic (Kostic et al., 2015; Zhang et al., 2016; Skovsted et al., 2017; Wang et al., 2017a).e Notch signaling pathway plays an important role in the regulation of cell differentiation, proliferation, and apoptosis, as well as a series of physiological and pathological processes (Huang et al., 2016; Demitrack et al., 2017; Li et al., 2017; You et al., 2017). Previous studies have shown that the Notch signaling pathway af f ects neuronal regeneration in cerebral ischemia animal models (Wang et al., 2009; Liu et al., 2011; Tao et al., 2014). Intracellular calcium overload is a main factor in apoptosis with cerebral ischemic injury (Li et al., 2015). Calcium channel blockers also provide protective ef f ects on ischemic brain injury in animal models (Maniskas et al., 2016).

In the present study, the chronic cerebral ischemia model was established in mice to investigate the ef f ects of aerobic exercise and HWTX-I on chronic cerebral ischemia.

Materials and Methods

Animals

Forty healthy, male, Kunming mice, 5–7 weeks of age and weighing 28–32 g, were provided by Hunan Slack Jingda Experimental Animals Co., Ltd., China (license No. SCXK (Xiang) 2011-0003). The study protocol was approved by the Hunan Normal University Medical Ethics Committee (Approval number: 2015(17)).e experimental design followed the national guidelines for the Care and Use of Laboratory Animals, and“Consensus author guidelines on animal ethics and welfare”by the International Association for Veterinary Editors. The article was prepared in accordance with the “Animal Research: Reporting of In Vivo Experiments Guidelines.”

Establishment of chronic cerebral ischemia models

The mice were placed in a supine position and a 1.5-cm long skin incision was made in the upper part of the neck.e subcutaneous fat, fascia, and muscle were separated to the trachea.e right common carotid artery was separated and permanently ligated using 3-0 surgical line. The color of the right eye changed from bright-red to grayish-white following ligation of the right common carotid artery. Aer regaining consciousness, the right eye remained closed and became dark red, although it was determined that a low-f l ow blood supply was obtainedviathe Willis circle after right brain ischemia (Figure 1).ese symptoms suggested successful establishment of the chronic cerebral ischemia model (Yoshizaki et al., 2008; Zhao et al., 2014).

Drug administration

HWTX-I (200 μg/ampoule, batch number: 010620, purity: 99.5%; license number: 260085434; Sinobioway Biomedicine Co., Ltd., Xiamen, Fujian Province, China) was provided by Institute of Protein Chemistry and Molecular Biology Laboratory of Hunan Normal University, China. Four days aer group assignment, mice in the HWTX-I and HWTX-I + aerobic exercise groups were administered 0.15 mL toxin solution to the tail vein at a dose of 0.05 μg/g. Mice in the model and aerobic exercise groups were administered the same volume of physiological saline, once every 2 days, for a total of 15 times over 30 days.

Exercise training

Following model establishment, mice in the aerobic exercise and HWTX-I + aerobic exercise groups were subjected to treadmill exercise by gradually increasing the running speed and time (Figure 2) (Sheng et al., 2015) for 5 weeks as follows: a) on days 1–3 of week 1, the speed was 7 m/min for 30 minutes, with a slope of 0 degrees; b) on days 4–6 of week 1, the training time was increased by 10 minutes, and the running speed increased by 1 m/min for each training; c) on day 6, the training time was 60 minutes, and the running speed was 10 m/min; d) on day 7, the mice rested; 3) weeks 2–5, the speed was 10 m/min for 60 minutes, with a slope of 0 degrees, six times a week until the end of week 5.

Neurological function assessment

A 28-point neurological def i cit score fi rst developed by Clark et al. (1997) was used to assess neurological functions in the mice, which included focal and general functional impairment scores. Each mouse group was scored according to the Clark neurological function scale at immediately aer group assignment and at the fih weekend. A higher score represented greater damage.

Tissue extraction

Nissl staining

Tissues were embedded in paraffin wax. Tissue sections approximately 5 μm thick were prepared using a rotary microtome (Jinhua YIDI Medical Appliance Co., Ltd., Jinhua, Zhejiang Province, China).e sections were then subjected to Nissl stained using toluidine blue (ShangHai EKEAR Bio@Tech Co., Ltd., Shanghai, China).e staining was observed and photographed using a light microscope (Olympus, Tokyo, Japan).

Real-time polymerase chain reaction (RT-PCR)

Figure 1 Establishment of chronic cerebral ischemia models.

Table 1 Primers used for real time-polymerase chain reaction

Figure 2 Aerobic exercise.

Brain tissue and blood total RNA was extracted using Trizol reagent (TakaRa Biotechnology (Dalian) Co., Ltd., Dalian, Liaoning Province, China) and the Magnetic Total RNA Kit (code No. E31004; Shanghai GenePharma Co., Ltd., Shanghai, China).e absorbance value of the extracted total RNA was determined using ultraviolet spectrophotometry, with anA260/A280ratio ＞ 1.7. PCR primers were designed using mouse cDNA sequences from Gene Bank and Primer 5.0 soware, and were synthesized by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China (Table 1).

Genomic DNA was removed as follows: 2.0 μL 5 × g DNA Eraser Buf f er was mixed with 1.0 μL gDNA Eraser and 7.0 μL total RNA. The samples were incubated at 42°C for 2 minutes, and the reaction was terminated at 4°C. Reverse transcription: 10.0-μL reaction solution contained 1.0 μL PrimeScript RT Enzyme Mix I, 1.0 μL RT Primer Mix, and 4.0 μL 5× PrimeScript Buf f er 2. Rnase-free dH2O was added to a fi nal volume of 20 μL.e samples were incubated at 37°C for 15 minutes and 85°C for 5 seconds to obtain fi rst-strand cDNA.e reagents used in the reverse-transcription reaction were PrimeScriptTMRT reagent Kit with gDNA Eraser (TaKaRa Biotechnology (Dalian) Co., Ltd).

A SYBR®Premix Ex TaqTMII (Tli RNaseH Plus) kit (Ta-KaRa Biotechnology (Dalian) Co., Ltd.) was used for RTPCR. The total reaction volume was 20 μL and contained the following: 2 μL cDNA template, 10 μL SYBR®Premix Ex Taq II (Tli RNaseH Plus) (2×), 0.8 μL forward primer, 0.8 μL reverse primer, 0.4 μL ROX-Reference Dye II (50×), 6 μL nuclease-free water. Reactions were prepared on ice. PCR was performed using an ABI 7500 Fast Real-time PCR System (Foster, CA, USA) at 95°C for 30 seconds, 95°C for 3 seconds, and 60°C for 30 seconds for 40 cycles. Data were processed using an ABI 7500 Fast.

Statistical analysis

All data, expressed as the mean ± SD, were analyzed with SPSS 22.0 software (IBM, Armonk, NY, USA). The sample means were compared using one-way analysis of variance.P＜ 0.05 represented a signif i cant difference.

Results

Ef f ects of aerobic exercise and HWTX-I on neurological function in chronic cerebral ischemia mice

As shown in Figure 3, there was no signif i cant difference in neurological def i cits between the groups prior to any interventions. At the fifth weekend, compared with the model group, focal and general neurological deficit scores significantly decreased in the intervention groups (P＜ 0.01). Compared with the HWTX-I group, general neurological def i cit scores signif i cantly decreased in the HWTX-I + aerobic exercise group (P＜ 0.05).

Ef f ects of aerobic exercise and HWTX-I on morphological changes in the cerebral cortex of chronic cerebral ischemia mice

Figure 3 Ef f ects of aerobic exercise and HWTX-I on neurological function in mice with chronic cerebral ischemia.

Figure 4 Ef f ects of aerobic exercise and HWTX-I on morphological changes in the cerebral cortex of chronic cerebral ischemia mice (Nissl staining, × 200).

Figure 5 Ef f ects of aerobic exercise and HWTX-I on Notch1, Jagged1, NCX1, SYP, and CaBP-D28k mRNA expressions in the brain (A) and blood (B) of chronic cerebral ischemia mice (real-time polymerase chain reaction).

Ischemia results in cellular morphological changes (Kitabatake et al., 2015; Arteaga et al., 2016; Zhang et al., 2017). To determine the neuroprotective ef f ects of aerobic exercise and HWTX-1 on these morphological changes, we analyzed Nissl staining in the cerebral cortex. In the model group, neurons in the cerebral cortex were deeply stained, with the presence of pyknosis, obvious vacuoles, nuclear necrosis, and distinct neuronal loss. In the HWTX-I group, vacuoles were not obvious, part of the nuclei and Nissl bodies were visible in the cerebral cortex, but the neurons were darkly stained and exhibited pyknosis. In the aerobic exercise group, the cerebral cortex exhibited a distinct loss of neurons and vacuoles; the neurons were darkly stained, and some neurons exhibited pyknosis, although morphology was better than in the model group. In the HWTX-I + aerobic exercise group, there were less vacuoles in the cortical neurons; the nuclei were lightly stained and exhibited distinct nucleoli; although pyknosis was still detectible, there was less neuronal loss; the Nissl bodies were clear and gathered to the periphery in the cytoplasm (Figure 4).

Ef f ects of aerobic exercise and HWTX-I on Notch1, Jagged1, NCX1, synaptophysin (SYP), and CaBP-D28k mRNA expression in the blood and brains of chronic cerebral ischemia mice

As displayed in Figure 5A, mRNA expressions of Notch1, Jagged1, NCX1, SYP, and CaBP-D28k were highest in the HWTX-I + aerobic exercise group compared with the model, HWTX-1, and aerobic exercise groups. Compared withthe model group, mRNA expressions of Notch1, Jagged1, NCX1, and SYP were signif i cantly decreased in brain tissue of mice in the HWTX-I group (P＜ 0.05). Compared with the model group, SYP mRNA expression was significantly increased in brain tissue of mice in the aerobic exercise group (P＜ 0.05). Compared with the model group, mRNA expressions of Notch1, Jagged1, NCX1, SYP, and CaBP-D28k were significantly increased in brain tissues of mice in the HWTX-I + aerobic exercise group (P＜ 0.01 orP＜ 0.05).

As shown in Figure 5B, mRNA expressions of Notch1, Jagged1, NCX1, and CaBP-D28k in the HWTX-I + aerobic exercise group, and SYP in the HWTX-I group, were highest. Compared with the model group, mRNA expressions of NCX1, SYP, and CaBP-D28k were signif i cantly increased in the blood of mice in the HWTX-I group (P＜ 0.01 orP＜0.05). Compared with the model group, mRNA expressions of CaBP-D28k and SYP were significantly increased in the aerobic exercise group (P＜ 0.01 orP＜ 0.05). Compared with the model group, mRNA expressions of Notch1, NCX1, SYP, and CaBP-D28k were signif i cantly increased in the HWTX-I + aerobic exercise group (P＜ 0.01 orP＜ 0.05).

Discussion

Shamsaei et al. (2015) confirmed a sparse distribution of hippocampal CA1 neurons in rats with cerebral ischemia, with a signif i cantly decreased number of normal cells. Naderi et al. (2017) found that minocycline pretreatment signif i cantly attenuated ischemia-induced pyramidal cell death and microglial activation in the CA1 region, and provided neuroprotective ef f ects on cerebral ischemia-induced memory def i cits. Shamsaei et al. (2015) used Nissl staining to show that pre-ischemic exercise significantly reduced necrotic cell death in the hippocampal CA1 region and prevented memory def i cits aer cerebral ischemia. Wang et al. (2017b) described a series of 11 studies that showed the signif i cant effects of astragaloside IV on ameliorating neurological function scores following ischemic stroke.ese studies suggested that different interventions exert neuroprotective effects following cerebral ischemia/reperfusion injury through antioxidant, anti-inf l ammatory, or anti-apoptotic properties.

SYP is a 38-kDa integral membrane protein closely related to synaptic structure and function; it has been identified in almost all synaptic terminals of the central and peripheral nervous systems. Pinheiro et al. (2014) showed that SYP protein expression was strongly associated with synaptic formation and function, and SYP protein expression decreased aer ischemia. Hou et al. (2011) suggested that willed movement therapy promotes recovery of neurological functions in rats with cerebral ischemia, which could be related to increased SYP expression. He et al. (2017) showed that bone marrow stromal cell treatment signif i cantly improved neurobehavioral performance following ischemic brain injury by SYP and growth-associated protein 43 expression. Fonteles et al. (2016) showed that increased synaptogenic activity and anti-inflammatory action exert protective effects on memory in a permanent middle cerebral artery occlusion mouse model. Results from the present study suggested that the aerobic exercise-induced increase in SYP mRNA expression following cerebral ischemic injury is responsible for recovery of neurological function.

The calcium-binding protein CaBP-D28k is expressed in neurons of most brain regions, and CaBP-D28k-positive neurons have a direct relationship with ischemic brain injury (Yenari et al., 2001). Ca2+overload plays a role in ischemia-reperfusion injury in neurons. Neuronal injury can be delayed or avoided using voltage-gated calcium channel inhibitors to prevent Ca2+inf l ux. CaBP-D28k selectively binds with Ca2+and down-regulates intracellular-free calcium levels. Ouh et al. (2013) determined that ischemic damage reduces expression of calcium-binding proteins, thereby leading to cell death. Results from Lee et al. (2013) showed that longer maintenance of calcium-binding proteins may contribute to less and more delayed neuronal death/damage in young animals. Yenari et al. (2001) concluded that Calbindin D28K overexpression leads to neuroprotection in an animal model of central nervous system injury. Sodium-calcium exchangers (NCX) are ion transporters that are widely expressed in animal cell membranes. Ca2+concentration stability in cells is maintained by pumping Ca2+out from the cytoplasm using reverse transport. It is believed that NCX1 plays a protective role in neuronal injury (Boscia et al., 2012; Vinciguerra et al., 2014; Secondo et al., 2015). NCX1 and the calcium-binding protein Calretinin cooperate within the striatum to confer tolerance against cerebral ischemia (Boscia et al., 2016). Results from the Formisano et al. (2013) study demonstrate that the RE1-silencing transcription factor may represent a potential drug target for treating brain ischemia by regulating NCX1 expression.is resulted in delayed or reduced neuronal damage, andthe ef f ect was increased through aerobic exercise. Although the Ca2+overload took place during the time window prior to intervention, HWTX-I increased NCX1 expression and promoted Ca2+outf l ow, thereby reducing the Ca2+concentration and delaying neuronal damage. Circulating nucleic acid includes circulating DNA and RNA. Recently, the detection of nucleic acid molecules in peripheral blood circulation has become a hot research topic (Guo et al., 2017; Huang et al., 2017). Although circulating nucleic acid can serve as a sensitive and ef f ective tumor biomarker (Roth et al., 2011; Perkins et al., 2012; Greenberg et al., 2014), very little is known about the application of circulating RNA in cerebral ischemic injury. Our results showed that Notch1 and NCX1 mRNA expression in peripheral blood coincided with a low degree of injury, suggesting that these molecules could serve as peripheral blood markers for cerebral ischemia detection.

Results from this study suggested that HWTX-I and moderate-intensity treadmill exercise for 5 weeks alone and used as a combined intervention in a mouse model of chronic cerebral ischemia effectively reduced brain tissue damage. Aerobic exercise aer cerebral ischemia resulted in increased SYP mRNA expression, suggesting a role for SYP in the recovery of neurological function. Although both HWTX-1 and exercise reduced injury, the mechanisms of action were different. HWTX-I combined with aerobic exercise was superior to HWTX-I or aerobic exercise alone.

Acknowledgments:We are grateful to Dr. Zhong-hua Liu at the Department of Biochemistry, College of Life Sciences , Hunan Normal University of China for his useful suggestions in this study.

Author contributions:JQC and CFT conceived and designed the study. HFM, JX, WC, BCZ, HLQ, and CZW performed the experiments. HFM and RC wrote the paper. All authors approved the f i nal version of the paper.

Conf l icts of interest:None declared.

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

Peer review:This paper was double-blinded and stringently reviewed by international expert reviewers.

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Copyedited by Yu J, Li CH, Qiu Y, Song LP, Zhao M

*< class="emphasis_italic">Correspondence to: Jia-qin Chen, M.D., chenjiaqin2010@sina.com.

Jia-qin Chen, M.D., chenjiaqin2010@sina.com.

orcid: 0000-0002-2968-9193 (Jia-qin Chen)

10.4103/1673-5374.205099

Accepted: 2017-02-28