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. 2020 Feb 14;40(8):1271–1281. doi: 10.1007/s10571-020-00812-7

Hydroxysafflor Yellow A Confers Neuroprotection from Focal Cerebral Ischemia by Modulating the Crosstalk Between JAK2/STAT3 and SOCS3 Signaling Pathways

Lu Yu 1,#, Zhili Liu 2,#, Wendi He 2,#, Huifen Chen 3, Zelin Lai 2, Yanhong Duan 2, Xiaohua Cao 2, Jie Tao 1, Chuan Xu 4, Qiujuan Zhang 4,✉, Zheng Zhao 2,✉, Jun Zhang 3,✉
PMCID: PMC11448784  PMID: 32060857

Abstract

Natural bioactive compounds have increasingly proved to be promising in evidence- or target-directed treatment or modification of a spectrum of diseases including cerebral ischemic stroke. Hydroxysafflor yellow A (HSYA), a major active component of the safflower plant, has drawn more interests in recent year for its multiple pharmacological actions in the treatment of cerebrovascular and cardiovascular diseases. Although the Janus kinase signaling, such as JAK2/STAT3 pathway, has been implicated in the modulation of the disease, the inhibition or activation of the pathway that contributed to the neuronal prevention from ischemic damages remains controversial. In this study, a series of experiments were performed to examine the dose- and therapeutic time window-related pharmacological efficacies of HSYA with emphasis on the HSYA-modulated interaction of JAK2/STAT3 and SOCS3 signaling in the MCAO rats. We found that HSYA treatment significantly rescued the neurological and functional deficits in a dose-dependent manner in the MCAO rats within 3 h after ischemia. HSYA treatment with a dosage of 8 mg/kg or higher markedly downregulated the expression of the JAK2-mediated signaling that was activated in response to ischemic insult, while it also promoted the expression of SOCS3 coordinately. In the subsequent experiments with the use of the JAK2 inhibitor WP1066, we found that the treatment of WP1066 alone or combination of WP1066/HSYA all exhibited inhibitory effects on JAK2-mediated signaling, while there was no influence on the SOCS3 activity of corresponding efficacious data in the MCAO rats, suggesting that excessive activation of JAK2/STAT3 might be necessary for HSYA to provoke SOCS3-negative feedback signaling. Taking together, our study demonstrates that HSYA might modulate the crosstalk between JAK2/STAT3 and SOCS3 signaling pathways that eventually contributed to its therapeutic roles against cerebral ischemic stroke.

Keywords: Hydroxysafflor yellow A, Cerebral ischemia, Negative feedback signaling, JAK2/STAT3 pathway, SOCS3

Introduction

Cerebral ischemic stroke is one of the leading causes of death and disability, and approximately 15 million people worldwide suffer from minor strokes each year (Chen et al. 2012). The disease is basically caused by the lack of blood supply to the brain that leads to selective nerve damage (Bacigaluppi et al. 2010) and triggers a cascade of pathophysiological events including neuronal excitotoxicity, oxidative and nitrative stress, inflammation, and apoptosis (Li et al. 2017a). No efficient therapeutics for the disease are currently available mainly due to the narrow therapeutic window (Christophe et al. 2017), though the recombinant tissue-type plasminogen activators (rtPAs) such as the reteplase are nowadays the standard therapeutics in acute ischemic stroke within certain symptomatic time window (Demers et al. 2012). Even the tPA treatment, however, has been associated frequently with an increased mortality in the patients in the first week after the onset of stroke symptoms (Wardlaw et al. 2012), presumably due to the tPA-induced blood–brain barrier damage, hemorrhagic transformation, and neurotoxicity (Chen et al. 2015). These suggest an urgent requirement for further understanding of neuropathological mechanisms underlying therapeutic targets that would be propitious to seek new therapeutic alternatives for the disease. To this regard, an excellent effort is the use of natural compounds that have proved to be promising in evidence- or target-based treatment or modification of a wide spectrum of diseases including cancer (Redondoblanco et al. 2017) and ischemic stroke (Chen et al. 2015; Chao et al. 2017). The flower of the safflower plant, Carthamus tinctorius L., has been widely used in traditional Chinese medicine for the treatment of cerebrovascular and cardiovascular diseases. It is described in the Compendium of Materia Medica as being able "to invigorate the circulation of blood" with potential benefits for homeostasis of the circulation system (Feng et al. 2013). Hydroxysafflor yellow A (HSYA) is the major active component of the plant that is structurally identified to be a quinochalcone C-glycoside (Jin et al. 2008; Jiang et al. 2010). Recent studies showed that HSYA possesses multiple neuroprotective activities and exhibits beneficial functions in reducing the neurological deficit score and the percentage of infarction volume in cerebral ischemic animal models (Wei et al. 2005; Tian et al. 2008; Shan et al. 2010). These neuroprotective efficacies of HSYA were found to be contributed by its roles in reducing malondialdehyde (MDA, the major decomposition product of peroxides derived from polyunsaturated fatty acids) content and increasing SOD activity (Wei et al. 2005), scavenging peroxynitrite and inhibiting iNOS production (Li et al. 2013), or mediating innate immune toll-like receptor 4 signaling pathway (Lv et al. 2015).

Evidence is mounting that the Janus kinase 2/signal transducer and activator of transcription three (JAK2/STAT3) signaling pathway play critical roles in the pathophysiology of cerebral ischemic stroke (Satriotomo et al. 2006; Wang et al. 2010, 2017; Li et al. 2017b). This pathway represents a fundamental signaling cascade in the brain that appears mostly to be inactivated under basal conditions, but turns into activated in response to the release of cytokines triggered by extracellular stimuli such as ischemic injury, by which cells control gene transcriptions that regulate cell proliferation, differentiation, and survival (Baggiolini, 1995; Kubo et al. 2003; O’Shea et al. 2015). Nonetheless, abnormal and/or extended JAK2/STAT3 signaling events is detrimental and could impair neuronal growth and function (Kershaw et al. 2013). The activity of the Janus kinase thus acquires to be properly regulated by its own pseudokinase domain and the action of phosphatases, or via interactions with other molecular and cellular signaling cascades (Wunderlich et al. 2013). As an essential modulating mechanism among others, the suppressor of cytokine signaling protein three (SOCS3) has been found to function essentially in the negative feedback regulation of JAK2/STAT3 signaling through binding to Janus kinase, cytokine receptors, and certain signaling molecules (Krebs and Hilton 2000; Kubo et al. 2003; Tamiya et al. 2011; Kwon et al. 2016). Despite the accumulated evidence that indicated a potent pharmacological interaction of HSYA with JAK2/STAT3 pathway under brain pathological condition (Zhang et al. 2014), challenges still remain as to which one, activation (Shyu et al. 2008) or inhibition (Wang et al. 2017) of JAK2/STAT3, is responsible for beneficial outcome after experimental brain ischemia. Meanwhile, the effect of SOCS3 feedback modulation on the activity of JAK2/STAT3 pathway following HSYA treatment is unclear. This study was therefore designed to investigate the efficacies of HSYA upon varied dosages and therapeutic time window on the functional and neurological outcomes,and then to explore if such therapeutic responses could be attributed to the pharmacological actions of HSYA on JAK2/STAT3 and SOCS3 signaling pathways in a middle cerebral artery occlusion (MCAO) rat model.

Materials and Methods

Chemicals and Reagents

HSYA (> 98% in HPLC purity) was provided by Shanghai Yuanye Pharmaceutical Co. Ltd. (Shanghai, China). The antibodies for the phosphorylated janus kinase 2 (p-JAK2), the phosphorylated signal transducers and activators of transcription three (p-STAT3), the suppressors of cytokine signaling three (SOCS3), and the reference β-actin were obtained from Cell Signaling Technology (Boston, USA). The JAK2/STAT3 inhibitor WP1066 was purchased from Selleck Chemicals (Houston, USA). 2,3,5-Triphenyltetrazolium chloride (TTC) was obtained from Xinong BioTechnologies (Beijing, China).

Animals

Sprague–Dawley male rats at 9–11 weeks old (250–290 g) were obtained from Shanghai Laboratory Animal Center of Chinese Academy of Sciences (SLACCAS). The rats were housed under the conditions of controlled temperature (22–26 °C) and humidity (40–70%) with a 12/12 h light/dark cycle. The rats had ad libitum access to food and water. All experiments in this study were performed in accordance with NIH Animal Welfare Act guidance and were approved by the Institutional Animal Care and Use Committee (IACUC approval ID #M07016) of the East China Normal University.

Focal Cerebral Ischemia/Reperfusion

Focal cerebral ischemia/reperfusion injury was induced by middle cerebral artery occlusion (MCAO) on rat according to the method described by Longa et al. (1989). The rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (400 mg/kg; Sinopharm Group Chemical Reagent Co., Ltd., Shanghai, China). The right common carotid artery (CCA), external carotid artery (ECA), and the internal carotid artery (ICA) were isolated via blunt dissection with a midline incision of the neck. The branches of the ECA were cut off, and the whole of the ECA was ligated. A monofilament nylon suture with a polylysine-coated tip (0.26 mm; Beijing Xinong BioTechnologies Co. Ltd., China) was inserted from the CCA into the right side of ICA in the depth of 19 ± 0.5 mm, to occlude the origin of the middle cerebral artery (MCA). The suture was fixed and the incision was closed. Occlusion was done for a period of 1.5 h. After 1.5 h occlusion, reperfusion was achieved by withdrawing the suture to restore blood supply to the MCA territory. After waiting for another 1.5 h, 4.5 h, and 7.5 h, the incision of rat was reopened and HSYA (at a dose of 4 mg/kg, 8 mg/kg, and 16 mg/kg dissolved in 0.9% saline) was injected into the unilateral CCA at each time point with a constant speed (0.05 mL/min). The rats in the normal group did not undergo surgery. The sham-operated rats underwent the identical surgery, but without the suture inserted. Body temperature was maintained 37 ± 0.5 °C throughout the surgery by means of a heating blanket and a lamp.

Rat Groups and Treatment

The experiment conducted on MCAO rats was divided into three parts, including the effect of HSYA on neurological function, the effect of HSYA on JAK2/STAT3/SOCS3 signaling pathway, and the effect of WP1066 or co-treatment of WP1066 and HSYA on JAK2/STAT3 and SOCS3 signaling. In the first part of the experiment, rats were randomly divided into five groups: sham-operated rats, MCAO (vehicle-treated) group, 4 mg/kg HSYA group, 8 mg/kg HSYA group, and 16 mg/kg HSYA group. And each group was further subdivided into three groups according to the therapeutic time point: 3, 6, and 9 h after ischemia. Thus, there were totally fifteen groups in the first part. In the second part, rats were randomly divided into five groups according to the therapeutic time point (3, 6, and 9 h after ischemia): sham-operated rats, MCAO group, 4 mg/kg HSYA group, 8 mg/kg HSYA group, and 16 mg/kg HSYA group. In the third part, rats were randomly divided into five groups: sham-operated rats, MCAO group, 4 mg/kg HSYA group, WP1066 group, and 4 mg/kg HSYA + WP1066 group. Drug treatment was performed at 3 h after ischemia.

In the first and second parts of the experiment, HSYA was dissolved in 0.9% saline (4, 8 or 16 mg/kg) and injected via the unilateral common carotid artery of rat at the specified time window point as shown in Fig. 1a. In the third part, WP1066 was dissolved in 1% DMSO. HSYA (4 mg/kg) and WP1066 (40 mM) were injected via the unilateral common carotid artery of rat at 3 h after ischemia as shown in Fig. 1b. In addition, total volume for treatments injected via unilateral common carotid artery is 0.5 ml. The measurements of brain infarct volume, edema, and neurological deficit scores were performed at 24 h after treatment. For protein assay, the animals were sacrificed at 24 h following treatment.

Fig. 1.

Fig. 1

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Diagram of the experimental procedure. a HSYA (0, 4, 8, or 16 mg/kg) treatment at 3, 6, or 9 h after ischemia. b HSYA (4 mg/kg) and/or WP1066 (40 mM) treatment at 3 h after ischemia

Measurement of Infarct Volume

Triphenyltetrazolium chloride staining was used to measure the infarct volumes 24 h after treatment. Rats (n = 6) were killed under deep anesthesia using 10% chloral hydrate and brains were rapidly removed and cut into 2-mm-thick coronal sections using the brain matrix (Beijing Xinong BioTechnologies, China). The fresh slices were incubated with light avoidance in 2% of 2,3,5-triphenyltetrazolium chloride solution at 37 ºC for 30 min to visualize the infarctions. Normal and damaged tissue was stained in red and white, respectively. The brain slices were photographed with a digital camera and the size of the infarct area (unstained) was assessed by Image J software. The percentage of the infarct volume was calculated according to the formula: [contralateral volume − (ipsilateral volume − infarct volume)/contralateral volume]×100%.

Measurement of Brain Water Content

Brain water content 24 h after treatment was measured by the standard wet/dry weight method (Hatashita et al. 1988). Brains (n = 6) were removed quickly under ice and weighted on an electronic balance to obtain the wet weight and then were dried in an oven at 100 ± 2 °C for 24 h to determine their dry weight. Brain water content percentage was calculated using the formula: [(wet weight − dry weight)/wet weight]×100%.

Assessment of Neurological Deficits

24 h after treatment, the neurological deficits of the rats (n = 10) were assessed using the Garcia test with an 18-point scale (Garcia et al. 1995). The examiners were blind to the procedures that the rat had undergone. The neurobehavioral study items included spontaneous activity, symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch.

Western Blot

Brain tissues were harvested and proteins were extracted from the ipsilateral cortices and processed as described previously (Gertz et al. 2012). The brain tissue was defrosted and immersed in ice-cold lysis buffer for 30 min. After centrifugation at 12,000 rcf for 15 min, the protein concentrations of the extracts in cleared lysate were measured by BCA assay. The proteins (40 μg) were resolved by SDS-PAGE and transferred onto a PVDF membrane (Millipore, Temecula, CA), then membrane was blocked 2 h by 5% nonfat dry milk. The following antibodies were used at the indicated concentrations: rabbit anti-p-JAK2 (Tyr1007/1008) (1:5000) (3776S, Cell Signaling Technology, Boston, USA), rabbit anti-p-STAT3 (Tyr705) (1:5000) (9145S, Cell Signaling Technology, Boston, USA), rabbit anti-SOCS3 (1:5000) (52113S, Cell Signaling Technology, Boston, USA), rabbit anti-β-actin (1:2000) (4970S, Cell Signaling Technology, Boston, USA), Anti-rabbit IgG (1:2000) (7074S, Cell Signaling Technology, Boston, USA). Immunoreativity was detected using an ECL-plus kit (Bio-Rad. American). β-actin was used as an internal control for all Western blot assays.

Data Analysis

The experimental data were expressed as mean ± SEM, and SPSS 18.0 software package was used for data processing. One-way analysis of variance (ANOVA) was used to compare the mean values of different groups. Comparisons between two groups were conducted by t-test. P value < 0.05 was considered as statistically significant.

Results

HSYA Promotes Neurological and Functional Recovery in the MCAO Rats

Dose (4, 8, and 16 mg/kg)- and therapeutic time window (3, 6 and 9 h after ischemia)-related efficacies of HSYA in the MCAO rats were firstly examined. Both 8 and 16 mg/kg, but not 4 mg/kg of HSYA significantly promoted recovery of neurological functions (Fig. 2a), inhibited development of cerebral edema (Fig. 2b), and reduced the infarct volume (Fig. 2c) in a dose-dependent manner in the rats with 3 h, but not 6 and 9 h ischemic reperfusion injury, suggesting that 8 mg/kg or more HSYA administrated through the carotid artery within 3 h after ischemia could achieve a satisfactory therapeutic outcome of the MCAO rats.

Fig. 2.

Fig. 2

Fig. 2

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Time window- and dosage-related effects of HSYA on the neurological score, the brain water content, and the infarct volume in the ischemic rats. a The neurological score (n = 10 for each group). b The brain water content (n = 6 for each group). c The infarct volume (n = 6 for each group). *P < 0.05, **P < 0.01, ***P < 0.001

HSYA Suppresses JAK2/STAT3 Activation in the MCAO Rats

The dose- and the time window-related changes of JAK2/STAT3 signaling pathway were examined. The levels of both p-JAK2 and p-STAT3 were increased in the MCAO rats without HSYA treatment as compared with that of rats in the sham group (Fig. 3). HSYA treatment inhibited p-JAK2 and p-STAT3 significantly in a dose-related way in the rats with 3 h (Fig. 3a, d), but not 6 or 9 h ischemic injury (Fig. 3b, c, e f), as compared with that of the MCAO rats without HSYA treatment.

Fig. 3.

Fig. 3

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Time window-related effects of HSYA on JAK2/STAT3 pathway in the ischemic brain (n = 4 for each group). a p-JAK2 at 3 h after ischemia. b p-JAK2 at 6 h after ischemia. c p-JAK2 at 9 h after ischemia. d p-STAT3 at 3 h after ischemia. e p-STAT3 at 6 h after ischemia. f p-STAT3 at 9 h after ischemia. **P < 0.01, ***P < 0.001

Activation of SOCS3 After HSYA Treatment in the MCAO Rats

The dose- and time window-related changes of SOCS3 were measured to assess its interacting relationship with JAK2/STAT3. The expression level of SOCS3 had no change in the model rats as compared with that in the sham group (Fig. 4). In line with the changes of JAK2-mediated signaling post HSYA treatment, SOCS3 expression was significantly upregulated in a dose-dependent manner in the rats with 3 h (Fig. 4a), but not 6 or 9 h ischemic injury compared to the MCAO control rats (Fig. 4b, c).

Fig. 4.

Fig. 4

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Time window- and dosage-related effects of HSYA on SOCS3 in the ischemic brain (n = 4 for each group). a 3 h after ischemia. b 6 h after ischemia. c 9 h after ischemia. ***P < 0.001

Modulation of SOCS3 Inhibitory Feedback Signaling on JAK2/STAT3 Excessive Activation After HSYA Treatment in the MCAO Rats

WP1066, a novel inhibitor of JAK2/STAT3, was used instead of HSYA or co-administrated with HSYA to evaluate their pharmacological effects on JAK2/STAT3 and SOCS3 signaling. The results illustrated that either WP1066 alone or co-treatment of WP1066 and HSYA led to the inhibition of p-JAK2/p-STAT3 (approximately by 36% for WP1066 vs 73% for WP1066/HSYA of p-JAK2, and 76% for WP1066 vs 88% for WP1066/HSYA of p-STAT3; Fig. 5b, c) with an obvious neuroprotective effect, while it had no influence on SOCS3 expression (Fig. 5d) in the model rats with 3 h MCAO insult.

Fig. 5.

Fig. 5

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Effects of WP1066 (40 mM), HSYA (4 mg/kg), and WP1066/HSYA (40 mM/4 mg/kg) on JAK2/STAT3 signaling and SOCS3 activity (n = 3 for each group). **P < 0.01, ***P < 0.001

Discussion

Convincing evidence is accumulated that natural compounds could be looked upon to be promising drug candidates for prevention and treatment of a wide spectrum of diseases including cerebral ischemic stroke. HSYA, an active component isolated from the flower of Carthamus tinctorius L., has drawn more interests in recent years for its multiple pharmacological effects in treating both myocardial and cerebral ischemic diseases (Wei et al. 2005; Tian et al. 2008; Shan et al. 2010; Li et al. 2013). Despite these advances in HSYA as a therapeutic candidate for ischemic stroke, challenge still lies ahead with regard to the understanding of molecular mechanisms that would be pharmacologically necessary to explain therapeutic potential of HSYA for cerebral ischemia.

In this study, we initially assessed the dose dependency and the time window of efficacy of HSYA in the MCAO rats. The results showed that HSYA treatment promoted recovery of neurological function (Fig. 2a), attenuated cerebral edema (Fig. 2b), and reduced cerebral infarct volume (Fig. 2c) in a dose-dependent manner in the rats with 3 h, but not 6 or 9 h, MCAO injury as compared with those of the sham rats. The tPA, which is until now the only FDA-approved thrombolytic drug for ischemic stroke, has proved to have improved outcome of ischemic stroke, but with a limited therapeutic window (within 4.5 h) (Chen et al. 2015). In this study, HSYA administrated through the carotid artery was originally supposed to be able to avoid the compound decomposition and promote rapid and even more direct distribution of the compound to the brain lesion site so that it could extend the time window comparable to tPA, but was unsuccessful still with a restrictive time window within 3 h post ischemia (Fig. 2). Nevertheless, the bench data of this study may not be simply translated to bedside, but may yield insights about pharmacodynamics and potentiality of HSYA for use bedside.

JAK/STAT pathway is held to be a well-conserved and fundamental paradigm for intracellular signaling cascade that starts from JAK activation on membrane by extracellular stimuli such as cytokines that then promotes the phosphorylation, nuclear translocation, and DNA binding of STAT, and hereby eventually controls transcriptional responses (Villarino et al. 2017). As an important member of JAK/STAT family, the JAK2-mediated signaling has been increasingly recognized to play crucial roles in the pathophysiology of ischemic stroke (Satriotomo et al. 2006; Wang et al. 2010, 2017; Dong et al. 2016). There are still critical concerns, however, as to what are the exact contributions, i.e., activated or inhibited, of JAK2-mediated signaling pathway to ischemic stroke. Indeed, several lines of evidence indicated that this pathway was activated (Shyu et al. 2008; Wang et al. 2010; Zhu et al. 2013; Liu et al. 2014; Dong et al. 2016; Li et al. 2017b), whereas inhibited in other studies, in in vivo or in vitro models of stroke (Satriotomo et al. 2006; Guo et al. 2015; Jia et al. 2017; Wang et al. 2017). To this point, the results of the present study provide evidence that the expression levels of both p-JAK2 and p-STAT3 were increased significantly in the MCAO model rats (Fig. 3), indicating that the ischemic insult would indeed lead to the activation of JAK2/STAT3 pathway. Furthermore, in match with the efficacy data (Fig. 2), we observed that HSYA treatment inhibited p-JAK2/p-STAT3 significantly in a dose-related way in the rats with 3 h ischemic reperfusion injury as compared with that of the MCAO rats without HSYA treatment (Fig. 3a, d). The results are supportive of the study by Satriotomo et al., in which the JAK2 phosphorylation inhibitor AG490 or STAT3 siRNA would reduce the infarct volume, number of apoptotic cells, and neurological deficits in rats with transient focal cerebral ischemia (Satriotomo et al. 2006), but inconsistent with the results from others, in which the activation of JAK2/STAT3 could exert neuroprotective effect against the cerebral ischemia (Shyu et al. 2008; Wang et al. 2010; Zhu et al. 2013; Liu et al. 2014; Dong et al. 2016; Li et al. 2017b). The discrepancies between the different responses of JAK2-mediated signaling pathway to cerebral ischemic stroke would be explained by differences in experimental settings such as the pathological model, therapeutic agent or intervention means, and analysis of the dose–effect correlation and the time window–effect relationship applied in our study and other studies (Satriotomo et al. 2006; Shyu et al. 2008). On the other hand, from the biochemical and pathophysiological views of the janus kinase signaling, which is mostly dormant under physiological conditions, and could be generally regarded as a cellular homeostatic mechanism of nuclear transcriptional control in response to extracellular insults such as ischemic/reperfusion injury (Shyu et al. 2008), the activation of JAK2-mediated pathway found in our experiments might be regarded as a defense or adaption response of the neuronal cells to the ischemic insult; and however, such an intrinsic defense response might be readily overloaded and be in turn detrimental to cell survival and require assistance of extrinsic force to meet ischemic challenges. Although the HSYA treatment in this study was obviously playing such a role by suppressing JAK2-mediated signaling (Fig. 3a, d), the further doubt to this stage still remains as to why the three doses of HSYA (4, 8, and 16 mg/kg) used in the study could all significantly inhibit the MCAO-induced activation of JAK2/STAT3 signaling, but only the treatments with two higher dosages of HSYA (8 and 16 mg/kg, rather than 4 mg/kg) would pharmacodynamically result in significant neurological and functional recoveries found in the MCAO rats (Fig. 2), and if there are other signaling pathways responsible for HSYA treatment, such as feedback signals, that could even more critically regulate and control the janus kinase signaling. Given that either over activated or excessively inhibited JAK2-mediated signaling would be detrimental, this pathway thus possesses “double-edged sword” property in nature and requires to be precisely regulated. Indeed, proper modulations of JAK2/STAT3 signaling pathway have been implicated in cell multiplication, cell cycle transformation, and angiogenesis (Darnell et al. 1994; Suganami et al. 2004; D'Ippolito et al. 2012; Raible et al. 2014). Growing evidence also demonstrated that the regulation of JAK2/STAT3 could exhibit its neurogenesis and neuroprotective roles through interaction with other molecular pathways, for instance, via stimulating VEGF production to promote neurovascular remodeling (Zhu et al. 2013; Dong et al. 2016; Li et al. 2017b).

In order to molecularly sort out the doubt as described above as to if there is coordinating signaling that would be engaged in the modulation of JAK2-mediated signaling in response to the cerebral insult as well as HSYA treatment, we then examined the changes of SOCS3, which is well identified to be a negative regulator upon the activation of JAK2/STAT3 pathway (HEINRICH et al. 2003; Kubo et al. 2003; Kwon et al. 2016). In addition to the findings that SOCS3 expression remained unchanged in the MCAO rats with no or 4 mg/kg HSYA treatment (Fig. 4a), the levels of SOCS3 were found to be increased significantly in the rats with 3 h ischemic injury following 8 or 16 mg/kg HSYA treatment (Fig. 4a), which appeared to be in line not only with the HSYA-mediated inhibitory changes of JAK2/STAT3 signaling (Fig. 3a, d), but also the neuroprotective efficacies of HSYA in the ischemic rats (Fig. 2). These results suggest that HSYA, at a dosage of 8 mg/kg or higher, could pharmacologically function on both molecular pathways, i.e., inhibition of JAK2-mediated signaling and promotion of SOCS3-provoked negative feedback signaling, to coordinately counteract the detrimental effects of ischemia-provoked JAK2/STAT3 activation. Similar observations with controversies were also documented previously showing that the phosphorylation of JAK2 and STAT3 were increased while SOCS3 was not upregulated after ischemic stroke (Wang et al. 2017), and that HSYA could rescue Aβ1–42-induced inhibition of JAK2/STAT3 phosphorylation (Zhang et al. 2014). Nevertheless, our results imply that MCAO ischemic insult could merely lead to cellular intrinsic adaptive activation of JAK2/STAT3 that seemed to be detrimental and incapable of provoking the negative feedback signal of SOCS3, and HSYA treatment might trigger signals simultaneously targeting JAK2/STAT3 and SOCS3, which all would eventually lead to the neuroprotective downregulation of JAK2-mediated signaling. This was confirmed further by the subsequent experiments, in which the JAK2/STAT3 inhibitor WP1066 was used instead of HSYA or co-administrated with HSYA to examine the differences in their pharmacological effects on JAK2/STAT3 and SOCS3 signaling. The results illustrated that, although both treatment protocols, i.e., WP1066 and WP1066/HSYA, had downregulating effects on JAK2-mediated signaling that linked to the alleviation of ischemic insults (Fig. 5b, c), there were differences in the downregulating extents of p-JAK2/p-STAT3 between the two protocols (p-JAK, 36% for WP1066 vs 73% for WP1066/HSYA; p-STAT3, 76% for WP1066 vs 88% for WP1066/HSYA compared to those of the MCAO model rats), indicating an overlying inhibitory effect of WP1066 and HSYA on the JAK2-mediated signaling (Fig. 5b, c). Importantly, neither WP1066/HSYA nor WP1066 alone exhibited activating effects on SOCS3 (Fig. 5d), suggesting that under the circumstance of readily inhibited JAK2/STAT3 by WP1066 or/and HSYA, there was no further need for HSYA to activate SOCS3. Collectively, these data may thus imply that, firstly, under the experimental transient focal cerebral ischemia that may functionally impair the janus kinase activities, downregulation to proper levels of JAK2-mediated signaling may be essentially required for improved functional recovery and reduced cell death; secondly, there were obvious differences between mechanisms of neuroprotective action of WP1066 and HSYA. The former appeared to impose inactivation of JAK2-mediated signaling predominantly by WP1006-mediated blockage of the JAK2/STAT3 interaction and the subsequent phosphorylation of STAT3, while the latter seemed to provoke SOCS3 inhibitory feedback role on JAK2-mediated signaling by further activation of JAK2/STAT3 and/or direct activation of SOCS3 under cerebral ischemia circumstance that eventually led to attenuation of JAK2/STAT3 signaling pathway; and thirdly, in the context of our study HSYA looks more like a pharmacological sensor, which may not impose its actions on JAK2/STAT3/SOCS3 pathway when cellular environments are physiologically normal, whereas it acts to maintain cellular homeostasis through coordinating multiple signaling pathways, such as JAK2-mediated signaling and the negative feedback role of SOCS3, when cells suffer cerebral ischemia. However, such a pharmacological action of HSYA would not be considered to be exclusive, and in fact, similar findings, for example, HSYA could apply neuroprotection against ischemic stroke by co-targeting BDNF and NMDARs (Mengya et al. 2016) or GSK3β and PI3K/Akt (Chen et al. 2013) signaling pathways, were also documented.

Conclusion

In summary, the present study reveals that HSYA treatment efficaciously rescued the functional and neuronal damages in brain of the rats with transient focal cerebral ischemia. The cerebral ischemia/reperfusion injury lasting for 3 to 9 h significantly activated JAK2/STAT3 phosphorylation, while it had no effect on the expression of SOCS3. The HSYA treatment within 3 h time window of ischemic reperfusion led to a net outcome of inhibition of the JAK2-mediated signaling, presuming HSYA could further activate the expression of p-JAK2/p-STAT3 that subsequently stimulated downstream SOCS3 activation and thereby provoke a negative feedback signal of SOCS3 on p-JAK2/p-STAT3. There might be also possibility that HSYA could directly activate SOCS3 which in turn neutralized the harmful activation of JAK2/STAT3. Nevertheless, the results were well corresponding with the efficacy data of HSYA in the ischemic rats (Fig. 6). Thus, HSYA can confer neuroprotection from focal cerebral ischemia by modulating the crosstalk between JAK2/STAT3 and SOCS3 signaling pathways, although the definite molecular action of SOCS3 on HSYA-mediated JAK2/STAT3 signaling still needs further study. Given that the toxicity of HSYA is neglIGIBLE (Liu et al. 2004), our findings may shed light on possible clinical utilities of HSYA as a multi-target-direct adjunct therapy with tPA for cerebral stroke.

Fig. 6.

Fig. 6

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The phosphorylation of JAK2/STAT3 was activated by the cerebral ischemia/reperfusion injury (3 to 9 h), but it had no effect on the expression of SOCS3. The HSYA treatment within 3 h time window of ischemic reperfusion led to the inhibition of the JAK2-mediated signaling, whereas activation of SOCS3 was achieved by the treatment.

Acknowledgements

This work was supported in part by grants from the National Natural Science Foundation of China (81503370 to LY, 31171019 to ZZ and 81470829 to JZ), Shanghai Natural Science Foundation (19ZR1447900 to LY), and MOST China–Israel Cooperation (2016YFE0130500 to XHC), and an opening grant from Key Laboratory of Brain Functional Genomics (ECNU), Ministry of Education, ECNU (JZ).

Author Contributions

LY, ZLL, WDH, XHC, QJZ, ZZ, and JZ conceived the project, planned the experiments, and analyzed and interpreted the data with support from HFC, ZLL, YHD, JT, and CX. LY, ZLL, WDH, HFC, ZLL, JT, and CX performed all experiments. LY, ZLL, WDH, QJZ, ZZ, and JZ prepared and reviewed the manuscript. All authors contributed to and approved the final manuscript.

Compliance with Ethical Standards

Conflict of interest

All the authors declare that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving animals were in accordance with NIH Animal Welfare Act guidance and were approved by the Institutional Animal Care and Use Committee (IACUC approval ID #M07016) of the East China Normal University.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lu Yu, Zhili Liu and Wendi He have contributed equally to this work.

Contributor Information

Qiujuan Zhang, Email: qiujuanzhang1014@163.com.

Zheng Zhao, Email: zzhao@brain.ecnu.edu.cn.

Jun Zhang, Email: zhangjun_c@hotmail.com.

References

  1. Bacigaluppi M, Comi G, Hermann DM (2010) Animal models of ischemic stroke part two: modeling cerebral ischemia. Open Neurol J 4:34–38 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baggiolini M (1995) Guidebook to cytokines and their receptors. FEBS Lett 369:347–348 [Google Scholar]
  3. Chao J, Dai Y, Verpoorte R, Lam W, Cheng YC, Pao LH, Zhang W, Chen S (2017) Major achievements of evidence-based traditional Chinese medicine in treating major diseases. Biochem Pharmacol 139:94–104 [DOI] [PubMed] [Google Scholar]
  4. Chen YC, Wu JS, Yang ST, Huang CY, Chang C, Sun GY, Lin TN (2012) Stroke, angiogenesis and phytochemicals. Front Biosci 4:599–610 [DOI] [PubMed] [Google Scholar]
  5. Chen L, Xiang Y, Kong L, Zhang X, Sun B, Wei X, Liu H (2013) Hydroxysafflor yellow A protects against cerebral ischemia–reperfusion injury by anti-apoptotic effect through pi3k/akt/gsk3β pathway in rat. Neurochem Res 38:2268–2275 [DOI] [PubMed] [Google Scholar]
  6. Chen HS, Qi SH, Shen JG (2015) One-compound-multi-target: combination prospect of natural compounds with thrombolytic therapy in acute ischemic stroke. Curr Neuropharmacol 15:134–156 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Christophe BR, Mehta SH, Garton ALA, Sisti J (2017) Current and future perspectives on the treatment of cerebral ischemia. Expert Opin Pharmacother 18:573–580 [DOI] [PubMed] [Google Scholar]
  8. Darnell JE, Kerr IM, Stark GR (1994) Jak-STAT pathways and transcriptional activation in response to ifns and other extracellular signaling proteins. Science 264:1415–1421 [DOI] [PubMed] [Google Scholar]
  9. Demers G, Meurer WJ, Shih R, Rosenbaum S, Vilke GM (2012) Tissue plasminogen activator and stroke: review of the literature for the clinician. J Emerg Med 43:1149–1154 [DOI] [PubMed] [Google Scholar]
  10. D'Ippolito S, Marana R, Nicuolo FD, Castellani R, Veglia M, Stinson J, Scambia G, Simone ND (2012) Effect of low molecular weight heparins (LMWHs) on antiphospholipid antibodies (aPL)-mediated inhibition of endometrial angiogenesis. PLoS ONE 7(1):29660 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dong W, Xian Y, Yuan W, Huifeng Z, Tao W, Zhiqiang L, Shan F, Ya F, Hongli W, Jinghuan W (2016) Catalpol stimulates VEGF production via the JAK2/STAT3 pathway to improve angiogenesis in rats stroke model. J Ethnopharmacol 191:169–179 [DOI] [PubMed] [Google Scholar]
  12. Feng ZM, He J, Jiang JS, Chen Z, Yang YN, Zhang PC (2013) NMR solution structure study of the representative component hydroxysafflor yellow A and other quinochalcone c-glycosides from carthamus tinctorius. J Nat Prod 76:270–274 [DOI] [PubMed] [Google Scholar]
  13. Garcia JH, Wagner S, Liu KF, Hu XJ (1995) Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Stat Valid Stroke 26:627–634 [DOI] [PubMed] [Google Scholar]
  14. Gertz K, Kronenberg G, Kälin RE, Baldinger T, Werner C, Balkaya M, Eom GD, Hellmannregen J, Kröber J, Miller KR (2012) Essential role of interleukin-6 in post-stroke angiogenesis. Brain 135:1964–1980 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guo S, Li ZZ, Gong J, Xiang M, Zhang P, Zhao GN, Li M, Zheng A, Zhu X, Lei H (2015) Oncostatin m confers neuroprotection against ischemic stroke. J Neurosci 35:12047–12062 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hatashita S, Hoff JT, Salamat SM (1988) Ischemic brain edema and the osmotic gradient between blood and brain. J Cereb Blood Flow Metab 8:552–559 [DOI] [PubMed] [Google Scholar]
  17. Heinrich PC, Behrmann I, Haan S, Hermanns HM, Müllernewen G, Schaper F (2003) Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374:1–20 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jia L, Wang F, Gu X, Weng Y, Sheng M, Wang G, Li S, Du H, Yu W (2017) Propofol postconditioning attenuates hippocampus ischemia-reperfusion injury via modulating JAK2/STAT3 pathway in rats after autogenous orthotropic liver transplantation. Brain Res 1657:202–207 [DOI] [PubMed] [Google Scholar]
  19. Jiang JS, He J, Feng ZM, Zhang PC (2010) Two new quinochalcones from the florets of carthamus tinctorius. Org Lett 12:1196–1199 [DOI] [PubMed] [Google Scholar]
  20. Jin Y, Zhang XL, Shi H, Xiao YS, Ke YX, Xue XY, Zhang FF, Liang XM (2008) Characterization of C-glycosyl quinochalcones in carthamus tinctorius L. by ultraperformance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. Rapid Commun Mass Spectrom Rcm 22:1275–1287 [DOI] [PubMed] [Google Scholar]
  21. Kershaw NJ, Murphy JM, Lucet IS, Nicola NA, Babon JJ (2013) Regulation of janus kinases by SOCS proteins. Biochem Soc T 41:1042–1047 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Krebs DL, Hilton DJ (2000) SOCS: physiological suppressors of cytokine signaling. J Cell Sci 113(Pt 16):2813–2819 [DOI] [PubMed] [Google Scholar]
  23. Kubo M, Hanada T, Yoshimura A (2003) Suppressors of cytokine signaling and immunity. Nat Immunol 4:1169–1176 [DOI] [PubMed] [Google Scholar]
  24. Kwon O, Kim KW, Kim MS (2016) Leptin signalling pathways in hypothalamic neurons. Cell Mol Life Sci 73:1457–1477 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Li S, Li Y, Ying F, Jing H, Xu Y, Hao L, Yan C (2013) Capacity of HSYA to inhibit nitrotyrosine formation induced by focal ischemic brain injury. Nitric Oxide Biol Chem 35:144–151 [DOI] [PubMed] [Google Scholar]
  26. Li H, Wu J, Shen H, Yao X, Liu C, Pianta S, Han J, Borlongan CV, Chen G (2017a) Autophagy in hemorrhagic stroke: mechanisms and clinical implications. Prog Neurobiol 163–164:79–97 [DOI] [PubMed] [Google Scholar]
  27. Li Y, Zhang X, Cui L, Rong C, Ye Z, Cong Z, Zhu X, He T, Shen Z, Dong L (2017b) Salvianolic acids enhance cerebral angiogenesis and neurological recovery by activating JAK2/STAT3 signaling pathway after ischemic stroke in mice. J Neurochem 143:87–99 [DOI] [PubMed] [Google Scholar]
  28. Liu Z, Li C, Li M, Li D, Liu K (2004) The subchronic toxicity of hydroxysafflor yellow A of 90 days repeatedly intraperitoneal injections in rats. Toxicology 203:139–143 [DOI] [PubMed] [Google Scholar]
  29. Liu X, Zhang X, Zhang J, Kang N, Zhang N, Wang H, Xue J, Yu J, Yang Y, Cui H (2014) Diosmin protects against cerebral ischemia/reperfusion injury through activating JAK2/STAT3 signal pathway in mice. Neuroscience 268:318–327 [DOI] [PubMed] [Google Scholar]
  30. Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84–91 [DOI] [PubMed] [Google Scholar]
  31. Lv Y, Qian Y, Fu L, Chen X, Zhong H, Wei X (2015) Hydroxysafflor yellow A exerts neuroprotective effects in cerebral ischemia reperfusion-injured mice by suppressing the innate immune tlr4-inducing pathway. Eur J Pharmacol 769:324–332 [DOI] [PubMed] [Google Scholar]
  32. Mengya X, Qingna S, Yiyi W, Yan C, Nan Z (2016) Hydroxysafflor yellow A increases BDNF and NMDARs in the hippocampus in a vascular dementia rat model. Brain Res 1642:419–425 [DOI] [PubMed] [Google Scholar]
  33. O’Shea JJ, Schwartz DM, Villarino AV, Gadina M, McInnes IB, Laurence A (2015) The JAK-STAT pathway: impact on human disease and therapeutic intervention. Ann Rev Med 66:311–328 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Raible DJ, Frey LC, Brooks-Kayal AR (2014) Effects of JAK2-STAT3 signaling after cerebral insults. JAK-STAT 3:e29510 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Redondoblanco S, Fernández J, Gutiérrezdelrío I, Villar CJ, Lombó F (2017) New insights toward colorectal cancer chemotherapy using natural bioactive compounds. Front Pharmacol 8:109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Satriotomo I, Bowen KK, Vemuganti R (2006) JAK2 and STAT3 activation contributes to neuronal damage following transient focal cerebral ischemia. J Neurochem 98:1353–1368 [DOI] [PubMed] [Google Scholar]
  37. Shan LQ, Ma S, Qiu XC, Zhou Y, Zhang Y, Zheng LH, Ren PC, Wang YC, Fan QY, Ma BA (2010) Hydroxysafflor yellow A protects spinal cords from ischemia/reperfusion injury in rabbits. BMC Neuroscience 11:1–8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shyu WC, Lin SZ, Chiang MF, Chen DC, Su CY, Wang HJ, Liu RS, Tsai CH, Li H (2008) Secretoneurin promotes neuroprotection and neuronal plasticity via the Jak2/Stat3 pathway in murine models of stroke. J Clin Investig 118:133–148 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Suganami E, Takagi H, Ohashi H, Suzuma K, Suzuma I, Oh H, Watanabe D, Ojima T, Suganami T, Fujio Y (2004) Leptin stimulates ischemia-induced retinal neovascularization: possible role of vascular endothelial growth factor expressed in retinal endothelial cells. Diabetes 53:2443–2448 [DOI] [PubMed] [Google Scholar]
  40. Tamiya T, Kashiwagi I, Takahashi R, Yasukawa H, Yoshimura A (2011) Suppressors of cytokine signaling (SOCS) proteins and JAK/STAT pathways: regulation of T-cell inflammation by SOCS1 and SOCS3. Arterioscler Thromb Vasc Biol 31:980–985 [DOI] [PubMed] [Google Scholar]
  41. Tian J, Li G, Liu Z, Fu F (2008) Hydroxysafflor yellow A inhibits rat brain mitochondrial permeability transition pores by a free radical scavenging action. Pharmacology 82:121–126 [DOI] [PubMed] [Google Scholar]
  42. Villarino AV, Kanno Y, O'Shea JJ (2017) Mechanisms and consequences of JAK-STAT signaling in the immune system. Nat Immunol 18:374–384 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wang G, Zhou D, Wang C, Gao Y, Zhou Q, Qian G, Decoster MA (2010) Hypoxic preconditioning suppresses group III secreted phospholipase A2-induced apoptosis via JAK2-STAT3 activation in cortical neurons. J Neurochem 114:1039–1048 [DOI] [PubMed] [Google Scholar]
  44. Wang XL, Qiao CM, Liu JO, Li CY (2017) Inhibition of the SOCS1-JAK2-STAT3 signaling pathway confers neuroprotection in rats with ischemic stroke. Cell Physiol Biochem 44:85–98 [DOI] [PubMed] [Google Scholar]
  45. Wardlaw JM, Murray V, Berge E, Del ZG, Sandercock P, Lindley RL, Cohen G (2012) Recombinant tissue plasminogen activator for acute ischaemic stroke: an updated systematic review and meta-analysis. Lancet 379:2364–2372 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wei X, Liu H, Sun X, Fu F, Zhang X, Wang J, An J, Ding H (2005) Hydroxysafflor yellow A protects rat brains against ischemia-reperfusion injury by antioxidant action. Neurosci Lett 386:58–62 [DOI] [PubMed] [Google Scholar]
  47. Wunderlich CM, Hövelmeyer N, Wunderlich FT (2013) Mechanisms of chronic JAK-STAT3-SOCS3 signaling in obesity. JAK-STAT 2:e23878 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhang Z, Wu Z, Zhu X, Hui X, Pan J, Xu Y (2014) Hydroxy-safflor yellow a inhibits neuroinflammation mediated by Aβ1-42 in BV-2 cells. Neurosci Lett 562:39–44 [DOI] [PubMed] [Google Scholar]
  49. Zhu H, Zou L, Tian J, Du G, Gao Y (2013) SMND-309, a novel derivative of salvianolic acid B, protects rat brains ischemia and reperfusion injury by targeting the JAK2/STAT3 pathway. Eur J Pharmacol 714:23–31 [DOI] [PubMed] [Google Scholar]

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