JAK2/STAT3 Signaling Pathway Plays a Key Role in Nicotine Mediated Neuroinammation Suppression in an Ischemic Rat Model

To explore the mechanism of nicotine mediated improvement of cognitive impairment in an established ischemic rat model. Endothelin-1 (ET-1) was injected into the left thalamic region in adult male Sprague-Dawley (SD) rats to establish ischemia model. 6 groups of rats (6 rats in each group) were then treated with nicotine, nicotine+DHβE, DHβE, AG490, nicotine +AG490 and saline respectively via intraperitoneal injection for 9 days. Another sham operation group was treated with saline as above. Morris Water Maze (MWM) test was performed for 6 consecutive days starting on the 4th day after operation to detect the cognitive function of rats in each group. 2-[ 18 F]-A-85380 microPET imaging was performed on day 10 to evaluate the changes of α 4 β 2 nAChRs in different brain regions of rats. Real-time PCR and Western blot were used to detect the amount of α 4 β 2 nAChRs, JAK2, STAT3 and inammatory factors in thalamus of rats in each group. imaging showed more uptake of 2-[ 18 F]-A-85380 the nicotine+AG490 and sham operation While the expression of p-JAK2, p-STAT3 and inammatory factors was not signicantly different in all the other groups (p>0.05). The study suggests nicotine inhibits the expression of inammatory factors by activating α 4 β 2 nAChRs through the activation of JAK2-STAT3 signaling pathway to improve cognitive impairment in ischemic rats. were fractionated by SDS–PAGE electrophoresis apparatus, transferred to PVDF membranes. Immunoblotting was carried out with antibodies against α 4 -nAChRs(1:500), β 2− nAChRs(1:500), IL-1β(1:2000), IL-6(1:2000), JAK2(1:1000), STAT3(1:1000), p-JAK2(1:1000), p-STAT3(1:2000), β-actin (1:1000) at 4 ℃ for 24h. The membranes were incubated with corresponding secondary antibodies at room temperature for 1h. Western blots were developed by enhanced chemiluminescence detection system. Image Lab software was used for gel electrophoresis image analysis. The gray value of the panel was used for quantitative analysis, and the histogram was obtained to show the protein expression of each group in each index. Quantitative analysis was performed basing on three individual samples preparation following with western bloting. Real-time PCR was performed to determine the relative expression of receptor subunits α 4 and β 2 nAChR in the left thalamus of rats. Three samples in each group were and three technical replications each sample were performed. The average CT values were obtained by amplifying β-actin and α 4 -, β 2 -nAChR respectively. The results showed that the expression of α 4 nAChR and β 2 nAChR mRNA were lower in ischemic rats given saline (ischemia group) than in the sham operation group (0.51±0.04, 0.68±0.04). The expression of α 4 nAChR and β 2 nAChR mRNA in the left thalamus of rats in nicotine group was signicantly higher than rats in ischemia group (0.75±0.16, p<0.05; 0.86±0.11, p<0.05). The expression levels of α 4 nAChR and β 2 nAChR mRNA in left thalamus of rats in nicotine+AG490 group were still signicantly higher than ischemic rats given saline (0.74±0.07, p<0.05; 0.82±0.03, p<0.05), and there was no signicant difference between nicotine+AG490 and nicotine groups, suggesting that nicotine could increase the number of α 4 nAChR and β 2 nAChR , while AG490 could not block this function. The expression of α 4 nAChR and β 2 nAChR mRNA in left thalamus of nicotine+DHβE group, DHβE group and AG490 group was signicantly lower than nicotine and nicotine+AG490 groups, but there was no signicant difference compared with ischemia group. signaling pathway


Introduction
Vascular cognitive impairment (VCI) is a kind of syndrome mainly caused by cerebral vascular disease, ranging from mild cognitive impairment to dementia. It is the second leading cause of dementia after Alzheimer's disease [1]. Studies showed that nicotine can play an important role in the protection of cognitive function [2][3][4][5]. Nicotine is an agonist of neuronal nicotinic acetylcholine receptors (nAChRs), which are composed of ve subunits arranged around a water-lled pore. The most abundant nAChR subtypes in the mammalian brain are heteromeric α 4 β 2 nAChRs and homomeric α 7 nAChRs, and α 4 β 2 is the most abundant subtype in the CNS and with high a nity for nicotine [6][7][8]. Histopathological studies have shown that under ischemic conditions, in ammation caused by microglia overactivation can destruct the blood-brain barrier, cause brain damage and prompt the occurrence and development of VCI [9][10][11][12]. Our prior studies have con rmed that the chronic ischemic cognitive dysfunction was correlated with the decrease of α 4 β 2 nAChRs [13], and nicotine played a role in inhibiting the in ammatory factors, which contributes to improving cognitive impairment by activating α 4 β 2 nAChRs in ischemic rats [14]. How α 4 β 2 nAChRs activated by nicotine acts on in ammatory factors remains uncertain.
The JAK-STAT signalling pathway is known to be a chain of interactions between proteins in a cell, and is involved in processes such as immunity, cell division, cell death and tumor formation. Abnormal activation of JAK2-STAT3 signaling pathway is often closely related to the occurrence, development and prognosis of in ammatory diseases, tumors, autoimmune diseases, etc. [15][16][17]. Studies have shown that JAK2 and STAT3 can regulate the plasticity of hippocampal synapses and are closely related to learning and memory [18]. In this study, we aimed to explore the relationship between JAK2-STAT3 signaling pathway and α 4 β 2 nAChRs activation induced by nicotine in ischemic rats. Nicotine, DHβE (the most potent competitive antagonist of α 4 β 2 nAChRs [19,20]) and AG490 (a speci c JAK2/STAT3 blocker [21,22]) were adopted for intervention treatment, and 2-[ 18 F]-A-85380 micro-PET imaging was performed for detecting α 4 β 2 nAChRs [23] in ischemic rats in this study.

Materials And Methods
All animal experiments were approved by the Ethics Committee of The First Hospital of China Medical University, and all experiments were performed in accordance with relevant guidelines and regulations.

Laboratory Animals and Groups
Forty-seven male Sprague-Dawley (SD) rats, about 8 weeks old, weighing 250 ± 20 g, were ordered (China Medical University animal laboratory, SYXK (Liao)2008-0005, Shenyang, China) to SPF animal laboratory. Before the experiment, the rats were fed freely and given 12h-12h light-dark cycle.
The other 3 SD rats did not receive any intervention neither operation nor drugs and they were used as a control group for PCR test. Another two rats were sacri ced 24 hours after surgery only for histological analysis with HE staining to make sure the success of ischemic model (Fig. 1), one from the sham operation group, the other from ischemia group.

Morris Water maze experiment
On the fourth day of drug intervention, Morris water maze (MWM) (endocrinology laboratory, China Medical University) test was performed to test the spatial learning and memory ability of rats. Rats of different groups were subjected to six consecutive days of experiments, which were divided into the following two parts: Directional navigation experiment: the experiment lasted for 5 days. On the rst day, a directional navigation program was set up.
Rats were put into the pool from any two quadrants to familiarize themselves with the water maze environment. Platform was placed in the center of a quadrant of the pool. The training tests were conducted at the same time every day for the next four days.
The software automatically recorded the swimming trajectory and swimming time of each rat in the pool.
The space exploration experiment: on the sixth day of the experiment, after setting up the space exploration program, the platform was removed, and the rats were put into any quadrant (except the target quadrant where the original platform was located). The software automatically recorded and analyzed the times that the rats crossed the target quadrant and the activity time in target quadrant in 120 seconds.
Quantitative analysis: Drawing ROI was based on standard rat brain atlas by Paxinos [25]. The cerebellum was used as the reference area, and the ratio of the average SUV (SUV ave ) in each area to the SUV ave of the cerebellum was calculated. The distribution of 2-[ 18 F]-A-85380 in the left thalamus and the whole brain of each group was quantitatively analyzed.

Real-Time PCR Detection of Receptor Subunits of nAChRs
The RNA extraction was performed rstly, followed by the reverse transcription synthesis of cDNA. The mixture was homogenized and the sample was placed on a reverse transcription instrument at 37℃, 15 min; 85℃, 5 s; 4℃, 10 min. Real-time PCR ampli cation curve and dissolution curve were con rmed after the reaction, and the CT value of the sample was calculated automatically by software. RNAiso plus (NO.9108/9109), SYBR Premix Ex Taq II (RR820A), Prime Script RT Reagent Kit (#RR037A) were purchased from TaKaRa company, Japan. Primers used for the study were as follows:

Western blot
The left thalamic tissue was taken and stored in an Eppendorf tube in a refrigerator at − 80℃. The tissues of each group were added with 1 ml RIPA buffer and PMSF mixture (RIPA: PMSF = 100:1). After homogenizing on ice by tissue homogenizer, the tissues were placed on ice for 30 minutes, centrifuged, 4℃, 15,000 rpm for 15 min. The supernatant was then taken and stored at − 80℃. According to BCA protein quantitative standard curve, different samples were adjusted to the same concentration with lysis buffer (3-5µg/µl for each sample). All the samples were denatured by heating at 95℃ for 10 mins, and stored at -20℃.

Statistical Analysis
All statistical analyses were performed using ANOVA of IBM SPSS Statistics. Variance analysis was applied for inter-group comparison and Tukey's method was used for in Multiple Comparison Correction. Shapiro-Wilk test was used to check the normal distribution. At least three individual trials were performed for each experiment and data was represented as mean ± SD. p < 0.05 was considered statistically signi cant. Speci c p values were indicated in notes of tables and gures.

MWM test indicated nicotine improving cognition but blocked by DHβE and AG490
Normality was checked using the Shapiro-Wilk test, which showed that the data t a normal distribution. We assumed normal distribution since the sample size was too small to test the normality assumption properly. Using variance analysis of repeated measurement design data to analyze the latency in directional navigation experiment, the escape latency of rats in each group decreased with the increase of experimental days, but the interaction between time and groups had no statistical signi cance (F=0.058, p=0.99> 0.01), indicating that the time factors did not vary with the grouping. There were signi cant differences in escape latency between different groups (F=4.942, p=0.01< 0.05) ( Table 1). The escape latency of rats in the nicotine group and the sham operation group was shorter than ischemic rats treated with saline only, and the difference was statistically signi cant (p<0.05) (Table 1, Fig. 2). There was no signi cant difference in escape latency between rats in the nicotine+DHβE group, DHβE group, nicotine+AG490 group and AG490 group compared with ischemic rats given saline (p>0.05).
The results of space exploration experiment: the number of times that the rats crossed the target quadrant and the activity time in the target quadrant area were listed in Table 1. There were signi cant differences in the number of times crossing the target quadrant and the activity time in target quadrant in the nicotine group and sham operation group compared with the ischemia group (p<0.05). There was no signi cant difference in the nicotine+DHβE group, DHβE group, nicotine+AG490 group and AG490 group compared with the ischemia group (p>0.05), indicating that nicotine improved the cognitive function of ischemic rats, but the effect was blocked by DHβE and AG490.
Micro-PET imaging showed nicotine up-regulating α 4 β 2 nAChRs was blocked by DHβE not AG490 The uptake of 2-[ 18 F]-A-85380 in left thalamus and whole brain of rats in the nicotine group, nicotine+AG490 group and sham operation group was signi cantly higher than ischemic rats treated with saline only (p<0.05) (Fig. 3, Table 2). There was no signi cant difference between the nicotine+DHβE group, the DHβE group and the AG490 group compared with the ischemia group (p>0.05).

Real-time PCR indicated nicotine upregulating α 4 β 2 nAChRs blocked by DHβE not AG490
Real-time PCR was performed to determine the relative expression of receptor subunits α 4 and β 2 nAChR in the left thalamus of rats. Three samples in each group were detected, and three technical replications for each sample were performed. The average CT values were obtained by amplifying β-actin and α 4 -, β 2 -nAChR respectively. The results ( Table 3, Fig 4) showed that the expression of α 4 nAChR and β 2 nAChR mRNA were lower in ischemic rats given saline (ischemia group) than in the sham operation group (0.51±0.04, 0.68±0.04). The expression of α 4 nAChR and β 2 nAChR mRNA in the left thalamus of rats in nicotine group was signi cantly higher than rats in ischemia group (0.75±0.16, p<0.05; 0.86±0.11, p<0.05). The expression levels of α 4 nAChR and β 2 nAChR mRNA in left thalamus of rats in nicotine+AG490 group were still signi cantly higher than ischemic rats given saline (0.74±0.07, p<0.05; 0.82±0.03, p<0.05), and there was no signi cant difference between nicotine+AG490 and nicotine groups, suggesting that nicotine could increase the number of α 4 nAChR and β 2 nAChR , while AG490 could not block this function. The expression of α 4 nAChR and β 2 nAChR mRNA in left thalamus of nicotine+DHβE group, DHβE group and AG490 group was signi cantly lower than nicotine and nicotine+AG490 groups, but there was no signi cant difference compared with ischemia group.
Western blot showed nicotine inhibiting in ammation by α 4 β 2 nAChRs upregulation and activation of JAK2-STAT3 signaling pathway Western blot was performed to detect the relative expression of protein α 4 and β 2 nAChR, IL-1β, IL-6, JAK2, STAT3, p-JAK2 and p-STAT3 in the left thalamus of rats. The internal reference was β-actin. Three samples were collected from each group, and the expression of each group was obtained by gray level detection. Western blot results (Table 4, Fig 5) showed that the expression of α 4 nAChR and β 2 nAChR protein in nicotine and nicotine+AG490 groups was signi cantly higher than ischemic rats treated with saline only (1.91±0.18, p<0.05; 2.05±0.12, p<0.05;1.88±0.12, p<0.05;1.91±0.03, p<0.05). The expression levels of p-JAK2 and p-STAT3 protein in nicotine group were signi cantly higher than ischemic rats given saline (0.95±0.03, p<0.05;1.12±0.02, p<0.05). The expression of p-JAK2 and p-STAT3 protein in nicotine+DHβE group, nicotine+AG490 group and AG490 group was not signi cantly different from rats in ischemia group. The expression of IL-1β and IL-6 protein in left thalamus of rats in nicotine group was lower than ischemia group (0.67±0.02, p<0.05;1.17±0.03, p<0.05), while the relative expression of in ammatory factors in nicotine+DHβE group, nicotine+AG490 group and AG490 group was not signi cantly different from ischemia group (p>0.05). These results suggested that the in ammatory response in the brain was enhanced after thalamic ischemia in rats. After nicotine intervention, α 4 β 2 nAChRs in the brain was up-regulated, and JAK2-STAT3 signaling pathway was then activated by α 4 β 2 nAChRs, thus reducing the expression of in ammatory factors in the brain.

Discussion
Our previous studies [13,14] showed that the cognitive impairment in ischemic rats induced by ET-1 was correlated with the impairment of α 4 β 2 nAChRs. Appropriate dose of nicotine could improve the learning and memory ability of ischemic rats. This improvement of cognition was related to the increased density of α 4 β 2 nAChRs in the thalamus and whole brain after nicotine intervention [14]. When thalamic ischemia happened, the in ammatory response in rat brain was elevated. Nicotine further inhibited the expression of in ammatory factors by activating the α 4 β 2 nAChRs, thereby improving cognitive function [14]. In ammatory reaction after cerebral ischemia injury is mainly manifested by in ltration of peripheral blood leukocytes in brain parenchyma and up-regulation of the intrinsic glial cells activation (microglia and astrocytes) [26]. In the early stage of cerebral infarction, it can trigger a series of in ammatory cascade reactions and prompt the excessive secretion of local in ammatory cytokines and chemokines, such as selectin, TNF-α, IL-1, IL-6, IL-1β, nitric oxide (NO), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), etc. [26]. The leukocytes were attracted to vascular endothelium and aggregated in ischemic area, thus activating the secondary in ammatory reaction downstream and forming a vicious circle [27,28]. At the same time, 2-4 hours after the early stage of cerebral ischemia, microglia were activated by in ammatory cytokines, which further aggravated brain injury [29].
Nowadays, studies have shown that neuroin ammation is closely related to cognitive impairment [30][31][32], especially the activation of microglia is the key point leading to neuroin ammation. Anti-in ammatory therapy has also been used to treat cognitive impairment. Guan Y.Z. [33] et al. found that nicotine could inhibit the release of in ammatory factors by inhibiting the increment of microglia after nicotine intervention.
JAK-STAT signaling pathway is an important target of a variety of human diseases. In the central nervous system, this signaling pathway is closely related to brain in ammation and the survival and development of neurons/glial cells [34]. It has been found that JAK2 and STAT3 can regulate synaptic plasticity in hippocampus, which is closely related to learning and memory. Therefore, more and more attention has been paid to the role of JAK2-STAT3 signaling pathway in central nervous system diseases, including Alzheimer's disease, depression and anxiety disorders. AG490 is a speci c antagonist of JAK2 and one of the PTK inhibitors [21]. As a speci c blocker of JAK2-STAT3 signaling pathway, AG490 inhibits the phosphorylation of JAK2 and STAT3 downstream of JAK2, thereby blocking cell signal transduction [35,36].
In this study, we established a rat model of ischemic cognitive impairment induced by ET-1, and explored the relationship between neuroprotective mechanism of nicotine, α 4 β 2 nAChRs, in ammation and JAK2-STAT3 signaling pathway. MWM test showed that the learning and memory ability was signi cantly improved by nicotine intervention, but no signi cant improvement was observed when nicotine was given with DHβE together, or with AG490 together. 2-[ 18 F]-A-85380 PET imaging showed that α 4 β 2 nAChRs increased signi cantly in rats brain when nicotine was given alone, or with AG490 together. The study demonstrated that nicotine intervention increased the density of α 4 β 2 nAChRs and improved cognitive impairment, but this effect would be blocked by AG490, while receptors were still upregulated. Essentially, when JAK2-STAT3 signaling pathway was blocked, nicotine could only upregulate the expression of α 4 β 2 nAChRs, but not improve the cognitive function. The results were further con rmed by PCR and Western blot analysis. Our ndings suggest that nicotine can improve ischemic cognitive impairment by up-regulating α 4 β 2 nAChRs, thereby activating JAK2-STAT3 signaling pathway to reduce in ammatory factors. Therefore, α 4 β 2 nAChRs-JAK2/STAT3 signaling pathway plays an important role in inhibiting neuroin ammation in ischemic rats.
One of the limitations in this study is that immuno uorescence co-staining assay was not performed to provide more direct evidence to con rm if nicotine acts via α 4 β 2 nAChRs in immune cells (microglia, astrocyte) or in neurons, for some study demonstrated the overexpression of α 4 β 2 nAChRs in both microglia and astrocytes from days 7-28 after experimental ischemic stroke [37]. The reaction of JAK2-STAT3 in immune cells taken from the ischemic region also needs to be investigated further. Another area needs to be further explored is that this study could not con rm the effect of JAK2-STAT3 activating by α 4 β 2 nAChRs was direct or indirect, for example, where nicotine affects the expression of another protein that then activates the JAK2/STAT3 pathway.

Conclusion
In an established ischemic rats model, nicotine could activate JAK2-STAT3 signaling pathway by up-regulating α 4 β 2 nAChRs to inhibit the expression of in ammatory factors, thereby improving rats cognitive function.    sham operation group 1.00±0.12* 0.98±0.18* Note: Relative expression means the mRNA expression of each index in different group relative to the normal rats which was the control group in RT-PCR. Each group was compared in pairs, only the nicotine, nicotine+AG490 and sham operation groups were statistically signi cant compared with the ischemia group. *: p<0.05 versus the ischemia group. Note: Relative expression was the protein expression of each index relative to β-actin. Each group was compared in pairs, only the nicotine group and nicotine+AG490 group were statistically signi cant compared with the ischemia group. *: p<0.05 versus the