Inhibition of cerebral ischemia/reperfusion injuryinduced apoptosis: nicotiflorin and JAK2/STAT3 pathway

Guang-qiang Hu, Xi Du, Yong-jie Li, Xiao-qing Gao, Bi-qiong Chen, Lu Yu,

1 Department of Anatomy, Southwest Medical University, Luzhou, Sichuan Province, China

2 Department of Chemistry, Southwest Medical University, Luzhou, Sichuan Province, China

3 Drug Discovery Research Center, Southwest Medical University, Luzhou, Sichuan Province, China

4 Department of Anatomy and Neurobiology, Southwest Medical University, Luzhou, Sichuan Province, China

Inhibition of cerebral ischemia/reperfusion injuryinduced apoptosis: nicotiflorin and JAK2/STAT3 pathway

Guang-qiang Hu1,＊, Xi Du2, Yong-jie Li3, Xiao-qing Gao4, Bi-qiong Chen2, Lu Yu2,＊

1 Department of Anatomy, Southwest Medical University, Luzhou, Sichuan Province, China

2 Department of Chemistry, Southwest Medical University, Luzhou, Sichuan Province, China

3 Drug Discovery Research Center, Southwest Medical University, Luzhou, Sichuan Province, China

4 Department of Anatomy and Neurobiology, Southwest Medical University, Luzhou, Sichuan Province, China

How to cite this article:Hu GQ, Du X, Li YJ, Gao XQ, Chen BQ, Yu L (2017) Inhibition of cerebral ischemia/reperfusion injury-induced apoptosis: nicoti fl orin and JAK2/STAT3 pathway. Neural Regen Res 12(1):96-102.

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Graphical Abstract

Nicoti fl orin protects against cerebral ischemia/reperfusion injury-induced apoptosisviathe JAK2/SA3 pathway

orcid: 0000-0002-8873-9138 (Lu Yu)

Nicoti fl orin is a fl avonoid extracted from Carthamus tinctorius. Previous studies have shown its cerebral protective e ff ect, but the mecha‐nism is unde fi ned. In this study, we aimed to determine whether nicoti fl orin protects against cerebral ischemia/reperfusion injury‐induced apoptosis through the JAK2/STAT3 pathway.e cerebral ischemia/reperfusion injury model was established by middle cerebral artery occlusion/reperfusion. Nicoti fl orin (10 mg/kg) was administered by tail vein injection. Cell apoptosis in the ischemic cerebral cortex was examined by hematoxylin‐eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Bcl‐2 and Bax expres‐sion levels in ischemic cerebral cortex were examined by immunohistochemial staining. Additionally, p‐JAK2, p‐STAT3, Bcl‐2, Bax, and caspase‐3 levels in ischemic cerebral cortex were examined by western blot assay. Nicoti fl orin altered the shape and structure of injured neurons, decreased the number of apoptotic cells, down‐regulates expression of p‐JAK2, p‐STAT3, caspase‐3, and Bax, decreased Bax immunoredactivity, and increased Bcl‐2 protein expression and immunoreactivity.ese results suggest that nicoti fl orin protects against cerebral ischemia/reperfusion injury‐induced apoptosisviathe JAK2/STAT3 pathway.

nerve regeneration; brain injury; nicoti fl orin; ischemic stroke; cerebral ischemia/reperfusion injury; treatment; cell apoptosis; terminal deoxynucleotidyl transferase dUTP nick end labeling; JAK2/STAT3 pathway; Bcl-2; Bax; caspase-3; neural regeneration

Introduction

Stroke is a serious leading cause of death that causes fi nan‐cial burden, especially in low‐income and middle‐income countries (Feigin et al., 2014; Levine et al., 2015; Banerjee and Das, 2016). Ischemic stroke accounts for 75% of all stroke patients (Zevallos et al., 2015). Within a certain time window, thrombolysis is thought to be the most effective treatment method, but many people cannot arrive at hospital within 4.5—6.0 hours, therefore systemic recombinant tissue plasminogen activator is limited (Zaidat et al., 2012; Akbik et al., 2016). Due to developments in pathophysiological stroke research, di ff erent mechanisms provide varied treatment op‐portunities (Lo et al., 2003; Hachinski et al., 2010; Liu et al., 2014).

In recent decades, studies have shown that significant blood fl ow reductions within the ischemic core accompany irreversible nerve cell necrosis, while programmed cell death (namely apoptosis) appears within the ischemic penumbra, and is reversible until a few hours aer cerebral ischemia (Xu and Zhang, 2011; Ghosh et al., 2012; Kalogeris et al., 2012). Hence, saving apoptotic cells is an important strategy in stroke treatment.

Cerebral ischemia/reperfusion (I/R) injury triggers mul‐tiple cell apoptotic pathways (Li et al., 1997; Polster and Fiskum, 2004; Wang et al., 2013; Feng et al., 2016). Indeed, there is overwhelming evidence that ischemia‐induced oxi‐dative stress and in fl ammation are principally responsible for subsequent cell death by necrotic or apoptotic mechanisms (Nita et al., 2001; Jin et al., 2013). Reactive oxygen species regulate cell survival/death by activating various cell signal‐ing pathways, such as p38, c‐Jun N‐terminal kinases, nuclear factor‐kappa B, and Janus kinase/signal transducers and acti‐vators of transcription JAK/STAT (Nakka et al., 2008; Wang et al., 2014; Hou et al., 2016). Studies show that JAK2/STAT3 activation contributes to cell apoptosis following transient focal cerebral ischemia (Satriotomo et al., 2006; Xie et al., 2007).

Traditional Chinese medicines are e ff ective and have less clinical side‐e ff ects for cerebral ischemia patients, but their mechanisms and targets need further investigation (Murphy, 2003; Sun et al., 2015). Nicoti fl orin, kaempferol‐3‐O‐rutino‐side, a fl avonoid glycoside extracted from Carthamus tinc‐torius, has shown protective effects against brain injury in a multi‐infarct dementia model (Xie et al., 2007). Similarly, other studies have shown that nicoti fl orin improves ischemic brain damage after transient focal cerebral ischemia (Li et al., 2006; Huang et al., 2007). Nicoti fl orin is neuroprotective against hypoxia‐, glutamate‐ or oxidative stress‐induced ret‐inal ganglion cell death (Nakayama et al., 2011). In addition, we have previously shown protective effects of nicotiflorin in brain injury and neuroin fl ammation by inhibiting STAT3 activation (Yu et al., 2013). As already noted, JAK2/STAT3 activation contributes to cell apoptosis following transient focal cerebral ischemia. However, it remains poorly under‐stood whether nicoti fl orin protects against cerebral I/R‐in‐duced cell apoptosis through the JAK2/STAT3 pathway. Ac‐cordingly, this is the focus of our present study, and indeed has not previously been investiagted.us, we examined the anti‐apoptopic effect and underlying nicotiflorin signaling pathway in rats following transient ischemia induced by I/R.

Materials and Methods

Animals

Twenty‐four male specific‐pathogen‐free Sprague‐Dawley rats weighing 260—310 g and aged 13—15 weeks were provid‐ed by the Experimental Animal Center, Southwest Medical University, China (license No. SCXK(Chuan)2013‐17).e rats were equally and randomly allocated to three groups: sham, I/R, and nicotiflorin. Rats were maintained under standardized temperature and humidity with a 12‐hour light/dark cycle, and free access to food and water. All pro‐cedures were performed in accordance with China Animal Welfare Legislation. Protocols were approved by the Ethics Committee of Southwest Medical University, China.

Surgical operation

Transient middle cerebral artery occlusion (MCAO) was performed on the right side using a nylon fi lament, as pre‐viously described (Longa et al., 1989). Briefly, rats were anesthetized with 10% chloral hydrate (350 mg/kg, intraper‐itoneally). The internal carotid artery and external carotid artery were carefully detached and a prepared segment of 4‐0 monofilament fiber was inserted from the external carotid artery into the internal carotid artery to block the origin of the middle cerebral artery. The sham operation involved a similar surgical procedure except for MCAO. Aer 2 hours of ischemia, the fiber was gently removed to enable reper‐fusion of the middle cerebral artery. Body temperature was maintained at 37°C throughout the operation. Rats with the following symptoms were assumed to be successful models: failure to fully extend left forepaw, circling to the left, or falling to the le. Aer 24 hours of reperfusion, rats were ex‐ecuted.

Drug administration

Nicotiflorin (Shanghai Winherb Medical Technology Co., Ltd., Shanghai, China) was dissolved in 25% polyethylene glycol 400. Nicotiflorin (10.0 mg/kg) was administered by tail vein injection to the nicoti fl orin group at the beginning of reperfusion. Vehicle was administered to the I/R and sham groups in the same manner. Rats were executed aer 24 hours of nicoti fl orin treatment.

Brain tissue extraction

Brain tissue was removed after reperfusion for 24 hours, then fi xed, embedded in para ffi n blocks, and cut into 5 μm‐thick coronal sections (0.26—0.51 mm anterior to bregma) for conventional hematoxylin‐eosin staining, terminal de‐oxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and immunohistochemical staining.

Hematoxylin-eosin staining

Figure 1 Representative hematoxylin-eosin (HE) stained micrographs of the cortical ischemic penumbra.

Figure 2 E ff ect of nicoti fl orin on cell apoptosis in the I/R model.

Figure 3 Nicoti fl orin e ff ect on expression of JAK2 and SA3 phosphorylation in the ischemic cerebral cortex of I/R rats.

Figure 4 Nicoti fl orin e ff ect on Bcl-2 and Bax immunreactivity in the ischemic cerebral cortex of I/R rats (immunohistochemical staining).

Figure 5 Nicoti fl orin e ff ect on Bcl-2, Bax, and caspase-3 expression in the ischemic cerebral cortex of I/R rats (western blot assay).

Briefly, slices were deparaffinized, hydrated in water, then stained with hematoxylin for 15 minutes, and washed in running tap water for 20 minutes. Next, slices were counter‐stained with eosin for 2 minutes, dehydrated in 95% absolute alcohol until excess eosin was removed, permeabilized in xylene, and mounted. Pathological changes were observed under a light microscope.

TUNEL staining was used to detect fragmented nuclear DNA during apoptosis (ArunaDevi et al., 2010; Bahmani et al., 2011), according to standard protocols for the TUNEL assay kit (Boster, Wuhan, China). Brie fl y, slices were depa‐raffinized, rehydrated, and then incubated in proteinase K for 15 minutes to digest DNA. Aer washing in a Tris bu ff er, sections were incubated in labeling buffer mixed with TdT and digoxigenin (DIG)‐d‐UTP at 37°C for 2 hours. Block‐ing reagent was added at room temperature for 30 minutes. Next, sections were incubated in anti‐DIG‐biotin for 30 min‐utes, with Strept Avidin Biotin Complex (SABC)‐FITC used for final detection. Stained cells were counted from three di ff erent views per section using a fl uorescence microscope (AMG EVos FL Microscopy, Seattle, WA, USA).e average count number was used for quanti fi cation and comparison between groups.

Immunohistochemical staining

Immunohistochemistry of coronal sections was performed as described previously (Yu et al., 2013). Rabbit anti‐B‐cell lymphoma 2 (Bcl‐2) associated X (Bax) polyclonal antibody (Boster; 1:200), and rabbit anti‐Bcl‐2 polyclonal antibody (Boster; 1:100) were used. Briefly, sections were dewaxed, rinsed with 3% H2O2for 20 minutes, and incubated in 5% bovine serum albumin for 30 minutes to block nonspeci fi c binding. Sections were then separately incubated in Bax and Bcl‐2 antibodies overnight at 4°C. Next, ready‐to‐use goat anti‐rabbit IgG secondary antibody (Boster) was incubated at 37°C for 1 hour. Sections were then stained with 3,3′‐di‐aminobenzidine and counterstained with hematoxylin. For each antibody staining, immunoreactive cells per 1 mm2of the cortex were counted using an optical microscope (Leica DM750, Solms, Germany) (Chu et al., 2006). The average count number was used for quanti fi cation and comparison between groups.

Western blot assay

Statistical analysis

All data are presented as the mean ± SEM, and were ana‐lyzed using SPSS 16.0 software (SPSS, Chicago, IL, USA). Di ff erences among groups were analyzed by one‐way analy‐sis of variance followed bypost hocTukey test. In all analyses, values ofP＜ 0.05 were considered statistically signi fi cant.

Results

Nicoti fl orin inhibited neuronal pathological changes

Hematoxylin‐eosin staining showed that normal neurons in the sham group exhibited regulatory round and bright blue nuclei (Figure 1). While in the I/R group, neurons showed apparent disorder, and part of them presented apoptosis fea‐tures: nuclear chromatin pyknosis, side scatter, or fracture. Nicotiflorin reduced pathological neuronal injury and the number of dead neurons (necrosis and apoptosis) induced by cerebral I/R.

Nicoti fl orin decreased cerebral I/R-induced cell apoptosis

In the sham group, there were only a few TUNEL‐positive cells (bright green fl uorescence) in the cerebral cortex (Figure 2A). Further, in the I/R group, the TUNEL‐positive cell number markedly increased after 2 hours of MCAO and 24 hours of reperfusion. In contrast, compared with the I/R group, TUNEL‐positive cells signi fi cantly decreased aer nicoti fl orin treatment (P＜ 0.01; Figure 2B).

Nicoti fl orin inhibited JAK2 and SA3 phosphorylation

Phosphorylation of JAK2 and STAT3 is thought to play an important role in apoptosis regulation.erefore, both pro‐teins were assessed by western blot assay. Activited pJAK2 and pSTAT3 both increased aer ischemic injury in the I/R group compared with the sham group (P＜ 0.01). However, compared with the I/R group, nicoti fl orin inhibited pJAK2 and pSTAT3 activation (Figure 3A, B).

Nicoti fl orin altered caspase-3, Bcl-2, and Bax immunoreactivity

Bcl‐2 family members play important regulatory roles in cerebral I/R injury‐induced apoptosis (Hardwick et al., 2012; Kalogeris et al., 2012; Kvansakul and Hinds, 2013; Troy and Jean, 2015). Therefore, we examined changes in Bcl‐2 and Bax immunoreactivity. Immunohistochemical staining showed increased Bax immunoreactivity and lower Bcl‐2 immunoreactivity in the cerebral cortex of the I/R group (Figure 4A). In contrast, nicotiflorin significantly reduced Bax immunoreactivity, but increased Bcl‐2 immunoreactivi‐ty (P＜ 0.05 orP＜ 0.01; Figure 4B).

To further examine this anti‐apoptotic e ff ect of nicoti fl o‐rin, caspase‐3, Bax, and Bcl‐2 expression was examined by western blot assay in brain tissue aer ischemia. Bax and Bcl‐2 con fi rmed the changes observed by immunohistochemis‐try. Following transient MCAO and 24 hours of reperfusion in the I/R group, increased Bax and caspase‐3 expression, and relatively less Bcl‐2 expression was detected. However, in the nicoti fl orin group, decreased Bax and caspase‐3, and increased Bcl‐2 expression was observed (P＜ 0.05 orP＜ 0.01,n= 3; Figure 5).

Discussion

Neuronal damage includes both apoptosis and necrosis (Charriaut‐Marlangue et al., 1996; Sugawara et al., 2004; Poon et al., 2010). Cerebral ischemia leads to irreversible neuronal injury in the ischemic core area within minutes of onset (Lavrik et al., 2005; Uyttenboogaart et al., 2009; Murray et al., 2010; Jiang et al., 2016). While in the infarct penumbra area, reversible apoptosis is the main cell death mechanism, and is closely related to the final infarct area (Olsen et al., 1983; Dirnagl et al., 1999; Lo, 2008; Popp et al., 2009; Deng et al., 2016). Promisingly, reversible apoptosis provides multiple opportunities for therapeutic intervention in ischemic stroke (Nakka et al., 2008; Broughton et al., 2009). Consequently, we examined the e ff ect of nicoti fl orin on neuronal apoptosis in I/R rats. TUNEL staining was chosen as an indicator of apoptotic neurons. Our results show significantly decreased apoptosis in the nicotiflorin group. In addition, hematoxy‐lin‐eosin staining showed that nicoti fl orin reduces pathologi‐cal neuronal injury and the number of dead neurons induced by cerebral I/R.is is the fi rst time the e ff ect of nicoti fl orin on neuronal apoptosis has been studied.

Apoptotic signal transduction mechanisms play a vital role (Chong et al., 2010; Wang et al., 2015).e JAK/STAT pathway is a critical human disease target (Ghoreschi et al., 2009; Babon et al., 2014; Tao et al., 2015; Chai et al., 2016). Within this fam‐ily, JAK2/STAT3 is closely associated with ischemia‐induced neuronal apoptosis and cancer cell apoptosis (Satriotomo et al., 2006; Xie et al., 2007; Du et al., 2012; Zhang et al., 2015). AG490, a potent JAK2 phosphorylation inhibitor, blocks post‐ischemic JAK2 and STAT3 phosphorylation, and signi fi‐cantly decreases cerebral infarction, cell apoptosis, and neu‐rological dysfunction (Satriotomo et al., 2006). We previously found that nicoti fl orin is protective against brain injury and neuroin fl ammation by inhibiting STAT3 activation (Yu et al., 2013). Whether nicoti fl orin prevents apoptosis and in fl uences ischemic outcomeviathe JAK2/STAT3 pathway is not known. In the present study, we examined JAK2 and STAT3 phos‐phorylation in post‐ischemic rat brain by western blot assay, in accordance with previous studies (Gorina et al., 2005; Satri‐otomo et al., 2006). Consistent with previous AG490 fi ndings, we found nicoti fl orin blocked post‐ischemic JAK2 and STAT3 phosphorylation.erefore, our results suggest that the protec‐tive e ff ect of nicoti fl orin against brain damage is related to the JAK2/STAT3 pathway.

To further understand the downstream anti‐apoptotic mechanism of nicotiflorin in cerebral I/R injury, we also examined Bax, Bcl‐2, and caspase‐3 expression. Undoubt‐edly, both extrinsic and intrinsic pathways are responsible for activation of apoptosis (Gorina et al., 2005; Venderova and Park, 2012). Internally, cerebral ischemia increases in‐tracellular calcium levels, activates calpains, and mediates cleavage of Bid to truncated Bid. Truncated Bid interacts with apoptotic proteins, such as Bax, which generally maintains balance with the anti‐apoptotic protein, Bcl‐2 (Hassan et al., 2014). Mitochondrial transition pores areopened and release cytochrome c or apoptosis‐inducing factor to activate caspase‐9, and subsequently caspase‐3, which leads to nuclear DNA damage and apoptosis (Mattson and Kroemer, 2003; Mishra and Kumar, 2005; Taylor et al., 2008; Broughton et al., 2009). Our results show signi fi cantly increased expression of the main pro‐apoptotic proteins, Bax and caspase‐3, in the ipsilateral cortical penumbra. Encour‐agingly, nicoti fl orin strongly inhibits their expression. Alter‐natively, the anti‐apoptotic protein, Bcl‐2, was signi fi cantly increased in the nicotiflorin group compared with the I/R group. These findings provide improved understanding of the cerebral protective mechanism of nicoti fl orin.

In conclusion, nicotiflorin ameliorates cortical neuronal shape and structure, and also decreases the number of apop‐totic neurons. Further, this protective mechanism involves the JAK2/STAT3 signaling pathway. Our fi ndings o ff er fur‐ther theoretical evidence for nicoti fl orin as a potential ther‐apeutic drug for ischemic stroke. Of course, other possible mechanisms need to be further studied.

Acknowledgments:We would like to thank Drug Discovery Research Center of Southwest Medical University of China for providing technical platform.

Author contributions:LY designed the experiments and wrote the paper. GQH performed the experiments. XD and BQC were responsible for data analysis. YJL and XQG provided technical support. 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, Hindle A, Wang J, Qiu Y, Li CH, Song LP, Zhao M

10.4103/1673-5374.198992

Accepted: 2016-10-22

＊Correspondence to: Guang-qiang Hu or Lu Yu, hgq863@sina.com or yulu863@sina.com.