Vanillin Attenuates Pro-Inammatory Factors in tMCAO Mice Model Via Inhibiting of TLR4/NF-kB Signal Pathway

Purpose Vanillin has been reported to reduce hippocampal neuronal death in rats of global cerebral ischemia. However, the immunoregulatory mechanism of vanillin in ischemic mice is still unclear. Hence, this study aims to investigate the role of vanillin in transient middle cerebral artery occlusion (tMCAO) mice. Transient cerebral ischemic stroke was induced by tMCAO surgery following by reperfusion in mice. After 24 hours of ischemia/reperfusion, Berderson scoring and TTC staining were used to evaluate neurological decit and infarct volume, respectively. Furthermore, the expression of cytokines in ipsilateral hemisphere were detected by qPCR, ELISA and immunouorescence. In vitro, LPS-stimulated primary and BV2 microglia cells were used to mimic neuroinammation after ischemic stroke. Similarly, the expression of cytokines was detected by qPCR and ELISA. In addition, Western blotting was performed to evaluate the expression of Toll-like receptor 4 (TLR4), nuclear factor-κ-gene binding p65 (NF-κB p65) and phosphorylated NF-κB p65. factor-κ-gene binding, rt-PA: recombinant tissue-type plasminogen activator, TLR4: Toll-like receptor 4.


Introduction
Stroke is the second leading cause of death in the world, with the characteristics of high incidence, high recurrence, high disability and high mortality. It is reported that 80% stroke are ischemic stroke [1], which is caused by vascular occlusion that blood cannot ow into brain and results in tissue damage. At present, recombinant tissue-type plasminogen activator (rt-PA) is the only recognized effective therapy for ischemic stroke [2]. However, due to technical requirements, many patients do not bene t from rt-PA treatment. In recent decades, drugs and methods for the treatment of ischemic stroke have mainly focused on neuroprotection. However, few drugs have been translated into clinical trials [3]. Therefore, there is an urgent need to investigate new targets and drugs for the treatment of ischemic stroke.
Neuroin ammation is considered to be an important factor affecting the prognosis of ischemic stroke, so intervention for in ammatory response has become research hotpot. In the acute phase of ischemic stroke, microglia play neuroprotective or neurotoxic effects [4]. After ischemic stroke, microglia migrate to the lesion site and aggravate tissue damage by producing in ammatory cytokines and cytotoxic substances. On the other hand, microglia also promote tissue repair and remodeling by removing cell debris and producing anti-in ammatory cytokines [5]. Hence, therapies that inhibit the activation of proin ammatory microglia while augment repair would offer great promise for ischemic stroke. Nevertheless, during acute phase, TLRs is considered to play a key role after ischemic stroke and thus is an important therapeutic target [6]. Above all TLRs, TLR4 is mainly expressed on microglia and participates in the regulation of microglia activation after ischemic stroke [7]. It was reported that TLR4 is one of the targets of neuronal injury and in ammation, and the regulation of TLR4/MyD88/NF-κB signal pathway is expected to improve the prognosis of ischemic stroke[8], [9].
Vanillin (4-oxy-3-methoxybenzaldehyde, C8H8O3) is a small molecular obtained from orchid pods. It was reported that vanillin has a variety of biological activities, including anti-mutagenesis, antibacterial, antitumor, anti-oxidation, anti-in ammation and neuroprotection. For example, vanillin has been shown to reduce hippocampal neuronal death caused by global cerebral ischemia [10]. Recently, it has been found that vanillin could pass through the blood-brain barrier (BBB), decreasing BBB damage and oxidative damage, and reducing infarct volume as well as brain edema after hypoxic-ischemic brain injury [11], which suggested that vanillin may be an effective drug for the treatment of ischemic stroke. Nevertheless, the role of vanillin in ischemic stroke has not been investigated to date. Here, our study illustrated the neuroprotective effect of vanillin on regulating the expression of in ammatory cytokines in tMCAO mice, and the suppression of TLR4/NF-κB signal pathway was involved in the anti-in ammatory mechanism of vanillin.

Cell Culture
The immortalized microglia BV2 cell line was purchased from China Center for Type Culture Collection and the test for mycoplasma contamination was negative. Cells were cultured in DMEM medium with 10% fetal bovine serum (FBS, Thermo Fisher Scienti c, Waltham, MA, USA, 10099141C) in a 5 % CO2 incubator at 37 °C. In vitro isolation and culture of primary microglia, cells were isolated from newborn Sprague-Dawley rat as previously described [12]. By utilizing anti-Iba1 antibodies (Millipore, MA, USA, MAB360, 1:300) for immuno uorescence staining, we veri ed the purity of the primary microglia cells was more than 95%. We used LPS-stimulated microglia cells to mimic the activated microglia after stroke. Vanillin was obtained from Sigma-Aldrich (St. Louis, MO, USA, V1104).
tMCAO Young adult C57BL/6 male mice (22g-26g) were purchased from Guangzhou University of Chinese Medicine, with free access to food and water on light cycle 12/12 h light/dark. All procedures were in accordance with the Animal Use and Care Committee for Research and The Fifth a liated Hospital of Sun Yat-sen University (No. 00081). All animals were randomly divided into groups according to their body weight before operation, and fasted for 12 hours. The mice were anesthetized with iso urane and transient cerebral ischemia was induced by inserting a nylon mono lament into middle cerebral artery.
Body temperature was maintained at 37°C through a feedback heating pad. tMCAO was induced by 90 min of reversible middle cerebral artery occlusion following by 24 hours reperfusion. Sham-operated animals only underwent the procedure of separating artery, but the nylon mono lament was not advanced into the artery. Animals' exclusion criteria: death, subarachnoid hemorrhage, Berderson scoring less than 1 point or more than 4 points. During the whole procedure, investigators were blinded with the treatment group. animals were given vanillin (56mg/kg) after 90 minutes of ischemia. Berderson scoring was performed after 24 hours reperfusion to evaluate neurological de cit as following: score 0: no obvious neurological de cit symptoms; 1: the left forelimb buckling to the chest wall, the right forelimbs extending to the ground when suspending; 2: failure to overcome the resistance; 3: rotating spontaneously towards the left; 4: no spontaneous walking; 5: animal death. The mice with a score of 1-3 were included in the experiment.
Quantitative evaluation of infarct volume 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, USA, T8877) staining was used to evaluate infarct volume. Following by 90 min ischemia and 24 hours reperfusion, mice were anesthetized deeply with iso urane. The brain was rapidly removed and frozen at -20°C for 25 min. Then brain tissues were sliced coronally into 2-mm thick sections. The sections were stained with 1.0% TTC at 37°C for 15 min and xed in a 4.0% paraformaldehyde solution at room temperature for 12 hours. Finally, the brain sections were photographed by a digital camera and analyzed using Adobe Photoshop CS6. The infarct volume was calculated as following: corrected infarct volume = contralateral brain volume -(ipsilateral brain volume -infarct volume).

Real-Time qPCR
Total RNA was obtained using Trizol kit (EZBioscience, USA, B0004D) according to the manufacturer's procedure. Then cDNA was generated with 1μg of total RNA. After that, SuperReal qPCR PreMix (SYBR Green) (Tiangen, Beijing, China, FP202-01) was used to perform the RT-qPCR reaction on a 7500 Fast Real-time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's procedure. Samples were subjected to 40 cycles of ampli cation at 95 °C for 15s and 60 °C for 60s. IL-1β: Cytokine Analysis by ELISA Secretion levels of IL-1β, TNF-α, IL-4 and IL-10 in each group were detected by ELISA (RayBio, Atlanta, USA). The brain tissues were grinded with 500 μ L PBS with 1% protease inhibitor. The tissue or cell samples were performed by according to the manufacturer's instructions. Three replicates of each sample were analyzed in each assay. Assays were repeated on three separate occasions.

Immuno uorescence
After 24 hours of ischemia/reperfusion, the brains were perfused with PBS, xed with paraformaldehyde overnight, and embedded with para n. Next, the brains were coronally sliced into 10 μ m thick sections. The slices were dipped into xylene and series of ethanol gradient in order to dewax and rehydrate. The hours, the membranes were incubated with corresponding secondary antibodies for 1 hour at room temperature. Finally, the blot images were detected with enhanced chemiluminescence reagent (Millipore, MA, USA, WBKLS0500) and captured using ChemiDocTM MP System (Bio-Rad, UK).

Statistical analysis
GraphPad Prism 6.0 and IBM SPSS Statistics 25 were used to analyze the experimental data. All data were given as the mean ± SD. Data were analyzed by one-way analysis of variance (ANOVA) followed by the post hoc comparisons LSD test for multiple comparisons. P-values less than 0.05 was considered as statistically signi cant.

Results
Vanillin reduced infarct volume and promoted motor function in tMCAO mice As showed in Fig. 1a, 1b and Additional le 1, compared with sham-operated mice, 90 min of ischemia following by 24 hours reperfusion resulted in 36.3±6.5 infract volume in whole brain. Low dose concentration of vanillin (7 mg / kg) did not reduce infarct volume in tMCAO mice. 28 mg / kg concentration of vanillin could decrease infarct volume but the result without statistical difference. Nevertheless, high dose concentration of vanillin (56 mg / kg) signi cantly decreased infarct volume (p = 0.017) to 23.3±9. In addition, compared with saline group, high dose concentration of vanillin ameliorated motor function de cit in tMCAO mice (Fig. 1c). Therefore, in following animal in vivo research, 56 mg / kg concentration of vanillin was used.
Vanillin reduced the expression of pro-in ammatory cytokines of IL-1β and TNF-α in ipsilateral hemisphere It was reported vanillin has multiple anti-in ammatory effect. To investigate whether vanillin regulated cytokines expression, qRT-PCR and ELISA were used to detect the expression level of pro-in ammatory cytokines (such as IL-1β and TNF-α) and anti-in ammatory cytokines (such as IL-4 and IL-10) in ipsilateral hemisphere. As shown in Fig. 2a-d, mRNA levels of IL-1β, TNF-α and IL-4 increased to 8.5±0.7, 3.2±0.5, 2.6±0.4 folds in ipsilateral hemisphere, respectively, compared with sham-operated mice. Furthermore, protein levels of IL-1β, TNF-α and IL-4 in ipsilateral hemisphere increased (Fig. 2e-h). Vanillin signi cantly reduced the mRNA level of pro-in ammatory cytokines, IL-1β and TNF α, to 5.9±0.6, 1.9±0.7 folds respectively. Similarly, vanillin signi cantly decreased the protein levels of pro-in ammatory cytokines, IL-1β and TNF-α, to 69.9±10.3 and 165.6±15.1 folds respectively. However, vanillin did not regulate the expression levels of anti-in ammatory cytokines IL-4 and IL-10. Furthermore, we verify the expression levels of cytokines using immuno uorescence. In accordance with the result of ELISA, vanillin reduced the expression of IL-1β and TNF-α ( Fig. 2i-j), and the expression of IL-4 and IL-10 did not be in uenced (see Additional le 2).
Vanillin inhibited expression of pro-in ammatory cytokines by LPS stimulated primary microglia IL-1β and TNF-α mainly originated from activated microglia or monocytes / macrophages in ischemic brain [13]. We further investigated weather vanillin regulated the expression of cytokines in LPS stimulated primary microglia. Immuno uorescence staining showed that the purity of isolated primary microglia was beyond 95% (Fig. 3a). Besides vanillin treatment, we used dexamethasone (Dexo) as a positive control, which was reported to inhibit microglia / macrophage activation stimulated by LPS in vitro. As shown in Fig. 3b-i, mRNA and protein levels of pro-in ammatory cytokines IL-1β and TNF-α increased signi cantly (P < 0.05) but anti-in ammatory cytokines IL-4 and IL-10 did not be affected by LPS stimulation. Compared with LPS treatment group, vanillin decreased mRNA and protein levels of IL-1β and TNF-α although mRNA level of TNF-α and secretion level of IL-1β did not have statistically signi cant difference. As for dexamethasone group, it also decreased the mRNA and protein levels of IL-1β and TNF-α in primary microglia stimulated by LPS. Furthermore, dexamethasone signi cantly increased expression of anti-in ammatory IL-4 in primary microglia cells.

Vanillin inhibited expression of pro-in ammatory cytokines by LPS stimulated BV2 cells
In order to further prove that vanillin reduced expression of pro-in ammatory cytokines in vitro, we also veri ed it in BV2 microglia cell. As shown in Fig. 4a-h, by using LPS stimulated, the mRNA and protein levels of IL-1β and TNF-α signi cantly elevated in BV2 cells (P < 0 0.05). Vanillin signi cantly decreased mRNA levels of IL-1β and TNF-α in LPS stimulated BV2 cells. Unlike primary microglia, the mRNA levels of IL-4 and IL-10 also elevated, and vanillin reversed these changes. However, the protein levels of IL-4 and IL-10 did not be regulated in vanillin treatment, compared with LPS group. Similar to primary microglia, dexamethasone not only signi cantly decreased mRNA and protein levels of IL-1β and TNF-α, but also regulated the expression of IL-10.

Vanillin inhibited activation of microglia by inhibiting TLR4/NF-κB signal pathway
It was reported that TLR4/NF-κB signaling pathway plays an important role in microglia activation, which regulates the expression of in ammatory mediators [14], [15]. Hence, we investigated whether vanillin participated in the activation of microglia through TLR4/NF-κB signaling pathway. We used LPS to stimulate BV2 cells or primary microglia, and 30 minutes later, cell protein samples were collected for Western blot. As shown in Fig. 5a-d, LPS signi cantly increased the expression of TLR4 as well as phosphorylated NF-κB p56. Vanillin pretreatment signi cantly reduced the expression of TLR4 and decreased phosphorylated NF-κB p65. These results suggested that vanillin may regulate proin ammatory cytokine expression in microglia by inhibiting TLR4/NF-κB pathway.

Discussion
Vanillin is a avouring agent, which has been widely used in the food, pharmaceutical and cosmetics industries. At present, more and more studies have shown that vanillin could improve neurological impairment, such as potassium bromate-induced neurotoxicity, LPS-induced motor dysfunction, rotenone induced in rat model of Parkinson's disease, neuroin ammation induced by β-amyloid and so on[16], [17], [18], [19]. It was reported that vanillin could cross the blood-brain barrier, making it possible to play its neuroprotective and anti-in ammatory effects in CNS system. Furthermore, Xiaobing Lan et al. showed that vanillin reduces hypoxic ischemic brain damage and improved neurological function in neonatal rats [11]. Our study found that vanillin decreased infract volume and improved motor function in tMCAO mice.
Cytokine is a kind of small molecule protein with extensive biological activity. A series of proin ammatory cytokines and anti-in ammatory cytokines involved in ischemic stroke. Pro-in ammatory factors were considered to relating in immune cell activation, tissue injury and necrosis, while antiin ammatory factors inhibiting and ultimately reversing the in ammatory process [20]. IL-1β and TNF-α were the widely studied pro-in ammatory cytokines in in ammatory response. Lambertsen KL revealed that IL-1β and TNF-α increase in serum and CSF during acute ischemic stroke. It was also reported that both the mRNA expression of IL-1β and TNF-α increase in damaged brain tissues of MCAO mice [21]. In accordance with those studies [22], [23], our results indicated mRNA and protein levels of IL-1β and TNF-α increased in damaged brain of tMCAO mice. Furthermore, vanillin attenuated in ammation by decreasing expressions of IL-1β and TNF-α in brain of tMCAO mice. Previous studies suggested that vanillin plays an anti-in ammatory role by suppressing the expression of proin ammatory cytokines. Compared with acute lung injury model group, vanillin inhibits expression levels of pro-in ammatory mediators TNF-α and IL-1β in lung tissue [24]. Moreover, vanillin treatment decreases the protein levels of TNF-α and IL-1β in mastitis mice compared with model group [25]. Similar to those researches, we demonstrated that vanillin decreased the expression of TNF-α and IL-1β in tMCAO mice.
IL-4 and IL-10 are important anti-in ammatory cytokines which play a key role in promoting in ammation resolution and tissue repair. Xiong X found that the ischemic damage in brain is more severe in IL-4de cient mice, but it can be reversed via intraventricular injection of IL-4[26]. IL-10 is a kind of multidirectional anti-in ammatory cytokine which inhibits the expression of TNF-α and IL-6 in monocytes / macrophages and inhibits the release of MMPs and ICAM-1 [27]. It was reported that exogenous supplement of IL-10 signi cantly reduces infarct volume and IL-10 could decrease apoptosis and axonal injury after glucose deprivation in vitro[28], [29]. However, our results suggested that vanillin plays its therapeutic effect by selectively regulating the expression of pro-in ammatory cytokines without affecting anti-in ammatory cytokines.
IL-1β and TNF-α mainly originated from activated microglia and monocytes / macrophages after ischemic stroke [13]. Hence, we wondered whether vanillin regulated the expression of cytokines via microglia. LPS-stimulated microglia have been widely used to investigate various in ammatory diseases such as ischemic stroke, multiple sclerosis and Alzheimer's disease [30]. Using LPS-stimulated primary microglia and BV2 microglia cells, our results indicated vanillin signi cantly decreased the expression of IL-1β and TNF-α. This coincided with previous study, which suggested that vanillin reduces the mRNA and protein levels of IL-1β and TNF-α in LPS-activated THP-1 cells [31]. However, our study further disclosed that vanillin did not regulate expression of anti-in ammatory cytokines in vitro. In addition, activated microglia can be divided into pro-in ammatory M1 type and anti-in ammatory M2 type according to different state, function and secreted factors [32]. Whether the anti-in ammatory mechanism of vanillin involves the transformation between M1 and M2 needs to be further investigated.
TLR4/NF-κB signaling pathway participates in in ammatory processes of many CNS diseases. Recent studies indicated that TLR4, MyD88 and NF-κB may involve in pathophysiology after ischemic stroke [7]. Compared with other TLRs, TLR4 is more important in ischemic stroke [33]. TLR4 activates NF-κB when it combines with its ligand such as LPS. Next, activated NF-κB could translocate into nucleus and then regulate multiple in ammatory response, such as the expression of IL-1β and TNF-α [34]. It was reported LPS-stimulated microglia involves in the activation of TLR4/NF-κB signaling pathway, and inhibiting the activation of TLR4/NF-κB pathway reduces the expression of in ammatory mediators in microglia [15]. Guo W indicated that vanillin reduces in ammation in mastitis mice by inhibiting TLR4/NF-κB pathway [25]. Consistent with other studies, our ndings showed that vanillin decreases the expression of TLR4 and phosphorylated NF-κB p65, which indicated vanillin might decrease expression of IL-1β and TNF-α in microglia via inhibiting TLR4/ NF-κB signal pathway.
In conclusion, we found that vanillin decreased the activation of microglia by inhibiting TLR4 /NF-κB signal pathway, and then reduced the expression of pro-in ammatory cytokines IL-1β and TNF-α, and nally reduced infarct volume and improved motor function in tMCAO mice. Moreover, our study also found that vanillin played its anti-in ammatory effects without involving anti-in ammatory regulation, which hinted a promising drug for vanillin in the treatment of ischemic stroke. Author Contributions: L-Z designed research, intellectual input, supervision; P-W and CY-L performed research, analyzed data, wrote the paper; GL-L, YH-H, XX-L and XD-L performed research, corrected the paper; WL-C: corrected the paper, provided directions for experimental design. L-Z, P-W, CY-L and WL-C contribute equal to this manuscript. All authors read and approved the nal manuscript. Figure 1 Vanillin reduced infarct volume and promoted motor function in tMCAO mice (a) TTC staining after 90 minutes of occlusion and 24 hours of reperfusion in tMCAO mice. (b) were the corrected infarct volume and (c) were the Berderson neurological function score in different group of tMCAO mice. n (Sham) = 3, n (tMCAO) = 7, n (7mg/kg) = 6, n (28mg/kg) = 6, n (56mg/kg) = 6. * p <0.05, *** p < 0.001.

Figure 5
Vanillin inhibited activation of microglia by inhibiting TLR4 / NF-κB signal pathway Cells were pre-treated with different treatment for 30 min before the stimulation by 100 ng/mL LPS for 30 min individually. The protein levels of TLR4, NF-κB p65 and phosphorylated NF-κB p65 in primary microglia(a) and BV2 microglia (c) were measured using western blot. (b) and (d) were statistical graphs of A and C, respectively. Values are mean ± SD of three independent experiments. ns, no signi cance. * p <0.05, ** p <0.01, *** p < 0.001.

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