JNK‐IN‐8, a c‐Jun N‐terminal kinase inhibitor, improves functional recovery through suppressing neuroinflammation in ischemic stroke

Abstract C‐Jun N‐terminal kinase (JNK) is a pivotal MAPK (mitogen‐activated protein kinase), which activated by ischemia brain injury and plays a fairly crucial function in cerebral ischemic injury. Emerging studies demonstrated that JNK‐IN‐8 (a JNK inhibitor with high specificity) regulates traumatic brain injury through controlling neuronal apoptosis and inflammation. However, the function of JNK‐IN‐8 in ischemic stroke and the mechanisms underlying of JNK‐IN‐8 about neuroprotection are not well understood. In this work, male rats were treated with JNK‐IN‐8 after transient middle cerebral artery occlusion, and then the modified improved neurological function score (mNSS), the foot‐fault test (FFT), interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor‐α (TNF‐α) levels were assessed. We found that JNK‐IN‐8‐treated rats with MCAO exerted an observable melioration in space learning as tested by the improved mNSS, and showed sensorimotor functional recovery as measured by the FFT. JNK‐IN‐8 also played anti‐inflammatory roles as indicated through decreased activation of microglia and decreased IL‐6, IL‐1β, and TNF‐α expression. Furthermore, JNK‐IN‐8 suppressed the activation of JNK and nuclear factor‐κB (NF‐κB) signaling as indicated by the decreased level of phosphorylated‐JNK and p65. All data demonstrate that JNK‐IN‐8 inhibits neuroinflammation and improved neurological function by inhibiting JNK/NF‐κB and is a promising agent for the prevention of ischemic brain injury.


| INTRODUCTION
Ischemic stroke remains the main types of stroke and leads to longterm disability and countless efforts have been made towards the therapeutic treatment on the disease. Despite current diagnosis and prognosis for ischemic stroke can be facilitated by genetic or transcriptomic biomarkers, it becomes seriously limited in terms of early stroke management (Amouyel, 2012). It has been demonstrated widely that neuroinflammation acts as a fairly crucial function on the pathophysiology of ischemic injury stroke in which local neuroinflammation can cause neuronal damage and degeneration (Jin, Liu, Zhang, Nanda, & Li, 2013). Clinical research indicated that prognoses of stroke can be significantly influenced by systemic inflammation (Emsley & Hopkins, 2008) while inhibition on inflammatory responses could decrease brain injury (Yilmaz & Granger, 2008). Therefore, a comprehensive understanding of regulation on inflammatory processes in response to the brain insult can be a precondition of developing an effective treatment for ischemic stroke.
C-Jun N-terminal kinase (JNK), a pivotal mitogen-activated protein kinase (MAPK), which relates to inflammatory processes in many diseases (Chen & Tan, 2000;Choudhury, Ghosh, Gupta, Mukherjee, & Chattopadhyay, 2015;Utsugi et al., 2003;Wardyn, Ponsford, & Sanderson, 2015). JNK is considered as a major stressresponsive kinase and JNK signaling is reportedly associated with neuroinflammation, blood-brain barrier disruption, and oligodendroglia apoptosis in brain injury (Manning & Davis, 2003). Studies on novel therapeutic targets neuroinflammation and neuropathic pain have considered JNK as a promising candidate due to its regulatory roles in inducing neuroinflammation in vivo and in vitro (Ramesh, 2014). A specific JNK inhibitor, JNK-IN-8, has been used to inquiry JNK precise effect in many pathways and diseases (Zhang et al., 2012). Previous research has shown that a dual NO-donating oxime and JNK inhibitor is reported to safeguard cells against cerebral ischemia-reperfusion injury, indicating JNK inhibitor use is accessible for investigation on functions of JNK in ischemia injury (Atochin et al., 2016). Specifically, JNK-IN-8 is reported to significantly suppress tumor growth in vitro and in vivo, directly proving JNK regulating breast cancer tumorigenesis (Xie et al., 2017). These studies exhibit the valuable aspect of JNK inhibitors in protecting against molecular occurrence of different diseases, however, the precise effect of JNK-IN-8 on neuroinflammation related to ischemic stroke remains unclear.
In this study, our purpose is to explore whether JNK-IN-8 can improve functional recovery through suppressing neuroinflammation in ischemic stroke. We used established transient middle cerebral artery occlusion (tMCAO) rat model by JNK-IN-8 treatment. We assessed the modified improved neurological function score (mNSS), the foot-fault test (FFT), the interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) level. Current data demonstrated that JNK-IN-8 exerted a neuroprotective and anti-inflammatory effect and suppressed JNK activation and nuclear factor-κB (NF-κB) signaling activation in ischemic brain injury, proposing a potent treatment for ischemic brain injury. The animals were subjected to MCAO according to the previous description (Hata et al., 1998;Yu et al., 2018). In Brief, each rat was anesthetized by choral hydrate (350 mg/kg) intraperitoneally. The right vessels, including the common carotid artery, external carotid artery (ECA), and internal carotid artery (ICA) were exposed by a midline cervical incision. The ECA was coagulated and inserted into the ICA through the ECA to occlude the MCA. And 2 hrs later, the suture was withdrawn to allow MCA perfusion. The blood flow of MCA was observed to verify the occurrence of ischemia by a Laser Doppler flowmetry (Oxford Optronix, UK). SD rats of the sham-operated group were dealt with the same procedures except for MCA occlusion. A heating pad (Malvern, UK) was used to keep the temperature at 37.0°C and the rats were kept on it until the closure of the skin incision.

| Cell culture
The murine BV2 microglial cells, collected from the ATCC (Manassas, VA), treated with Dulbeccoʼs modified Eagleʼs medium (DMEM) contained with 10% Fetal bovine serum and 1% penicillin as well as streptomycin (YBio, Shanghai, China), and were were incubated at 37°C in a 5% CO 2 incubator. The oxygen-glucose deprivation was carried out through exposure of BV2 cells to DMEM containing no glucose or serum in a specific environment (5% CO 2 and 95% nitrogen) for 6 hr.

| Behavioral tests
The mNSS test was carried out to assess neurological function according to the previous description (Chen et al., 2001). This experiment was conducted before the rats received the injury and at 1, 3, 7, and 14 days after MCAO. About the mNSS, neurological functions including motor (muscle status and abnormal movement), ZHENG ET AL.

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sensory systems (visual, tactile, and proprioceptive) and reflexes. The mNSS was graded on a scale of 0-18, and the higher score indicates a more severe injury.
The sensorimotor function was assessed by the FFT according to the previous description (Zhang et al., 2015). The rats were placed on an elevated grid to walk, and the number of foot-fault errors (the paw slips between the wires) was recorded.

| Quantitative real-time polymerase chain reaction
The total RNA was extracted from microglia with TRIzol reagent  Table S1.

| ELISA assay for inflammatory cytokines
The ELISA assay was performed using ELISA kits (Biosource International Inc) as instructions. Briefly, to quantify TNF-α, IL-1β, as well as IL-6 protein level in the tissue of the brain, 96-well plates coated with indicated antibodies, were treated with the addition of the supernatant of brain tissue homogenate (1:20 dilution). After the reaction between enzyme and substrate, the absorbances of the sample was assessed at 450 nm using a microplate reader. All the procedures were repeated for at least three times.

| Immunofluorescence staining
The immunofluorescence staining in brain tissues was carried out according to the previous description (Burton, Sparkman, & Johnson, 2011). Specific primary antibody against Iba-1 (1:1,500; ab178846) was used to mark the section at 4°C overnight. Anti-rat Alexa Fluor 488 (1:1,000; Invitrogen) as secondary antibody was added to incubate the section and then counterstained with 4′,6-diamidino-2-phenylindole (ATOM). The samples were observed and analyzed by the LEICA TCS SPE microscope (Leica, Germany) and LEICA software LAS AF, respectively. And the positive cells were statistically counted and plotted.

| Statistics
All data were expressed as mean ± standard deviation. Student's t test was used to assess differences between groups. Values less than .05 was defined as the criteria to determine significance.

| JNK-IN-8 inhibits microglia activation in vivo after stroke
To unveil the potential mechanism of JNK-IN-8 on treating MCAO rats, microglia activation was analyzed by immunofluorescence analysis using Iba-1 antibody. Figure 2a   Previous research has shown that JNK-IN-8 possesses the potential to inhibit NF-κB activation in breast carcinoma cells (Ebelt et al., 2017), and NF-κB is a pivotal upstream modulator for proinflammatory cytokines in microglia (Jiang et al., 2017;Simmons et al., 2016). Therefore, we further investigated whether the neuroinflammation-relieving effect of JNK-IN-8 in MCAO rats is related to NF-κB activation. Figure 3c-f showed that the p65 expression was increased and I-κBα level was reduced by ischemic injury, indicating that NF-κB signaling was activated after MCAO, and the change in NF-κB signaling was reversed by JNK-IN-8 treatment.  (Chen et al., 2018). JNK signaling is reportedly associated with microglia activation and neuroinflammation (Manning & Davis, 2003). Additionally, JNK mediates NF-κB signaling activation, which is a major upstream modulator for proinflammatory cytokines in microglia (Jiang et al., 2017;Simmons et al., 2016).

| JNK-IN-8 inhibits microglia activation and the production of proinflammatory cytokines in vitro
Therefore, studies on novel therapeutic targets neuroinflammation and neuropathic pain have considered JNK as a promising candidate.
Various JNK-related synthetic inhibitors have been reported in cerebral ischemia/reperfusion injury, such as micromolecules SP600125 and IQ-1S. Guan et al. (2006)

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

AUTHOR CONTRIBUTIONS
H. T., O. D., D. J., and S. L. were involved in designing research route and conducting related work, collecting and analyzing data, and writing the manuscript. J. T. and Y. L. helped to draft the manuscript. W. G. was involved in the idea and design of the study, and eventually approved the submitted version. All authors declare that there is no competing interest.

ETHICS STATEMENT
Animal experiments were approved by the Committee for Animal Experiments at Wenzhou Medical University.