S1PR3 inactivation aggravates cerebrovascular endothelial cell permeability mediated by cPLA2 and stat3 phosphorylation under oxidative stress

S1PR3 inactivation aggravates cerebrovascular endothelial cell permeability mediated by cPLA2 and stat3 phosphorylation under oxidative stress | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 6 August 2025 V1 Latest version Share on S1PR3 inactivation aggravates cerebrovascular endothelial cell permeability mediated by cPLA2 and stat3 phosphorylation under oxidative stress Authors : Junyu Mu , Liye Ge , Li Dai , and Jing Zhang 0000-0002-4700-6907 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175449454.49584287/v1 Published Basic & Clinical Pharmacology & Toxicology Version of record Peer review timeline 208 views 139 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Sphingosine-1-phosphate receptor-3 (S1PR3) has been implicated in the maintanence of cerebrovascular integrity during cerebral ischemia. This study aimed to elucidate the impact of S1PR3 inactivation on oxidative stress-induced cerebrovascular endothelial cell permeability and to explore the potential mechanisms. In bEnd3 cells, blockade of S1PR3 exacerbated H 2 O 2 -induced endothelial hyperpermeability and ZO-1 redistribution. Previous study indicated that the activation of p38 and ERK pathway was essential for H 2 O 2 -mediated cPLA 2 phosphorylation in bEnd3 cells. This study revealed that the activation of JNK is crucial for H 2 O 2 -induced stat3 phosphorylation. In bEnd3 monolayer, either blockade or genetic knockdown of S1PR3 further enhanced the phosphorylation level of p38, ERK, cPLA 2 , JNK and stat3 in response to H 2 O 2 exposure. Furthermore, lentivirus-mediated knockdown of S1PR3 intensified H 2 O 2 -induced ZO-1 redistribution and paracellular hyperpermeability. These results underscored the role of S1PR3 as a critical modulator of endothelial monolayer permeability via both cPLA 2 and stat3 phosphorylation in the context of oxidative stress. S1PR3 inactivation aggravates cerebrovascular endothelial cell permeability mediated by cPLA 2 and stat3 phosphorylation under oxidative stress Junyu Mu a,1 , Liye Ge b,1 , Li Dai c , Jing Zhang a,* \received DD MMMM YYYY \acceptedDD MMMM YYYY a. Department of Clinical Pharmacology, School of Pharmacy, Nanjing Medical University, Nanjing, China. \received DD MMMM YYYY \acceptedDD MMMM YYYY b. Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences, Shanghai, China \received DD MMMM YYYY \acceptedDD MMMM YYYY c. Jiangsu Medical College, Yancheng, Jiangsu, China * Corresponding author. E-mail Address: [email protected] 1. Junyu Mu and Liye Ge contributed equally to this work. Abstract Sphingosine-1-phosphate receptor-3 (S1PR3) has been implicated in the maintanence of cerebrovascular integrity during cerebral ischemia. This study aimed to elucidate the impact of S1PR3 inactivation on oxidative stress-induced cerebrovascular endothelial cell permeability and to explore the potential mechanisms. In bEnd3 cells, blockade of S1PR3 exacerbated H 2 O 2 -induced endothelial hyperpermeability and ZO-1 redistribution. Previous study indicated that the activation of p38 and ERK pathway was essential for H 2 O 2 -mediated cPLA 2 phosphorylation in bEnd3 cells. This study revealed that the activation of JNK is crucial for H 2 O 2 -induced stat3 phosphorylation. In bEnd3 monolayer, either blockade or genetic knockdown of S1PR3 further enhanced the phosphorylation level of p38, ERK, cPLA 2 , JNK and stat3 in response to H 2 O 2 exposure. Furthermore, lentivirus-mediated knockdown of S1PR3 intensified H 2 O 2 -induced ZO-1 redistribution and paracellular hyperpermeability. These results underscored the role of S1PR3 as a critical modulator of endothelial monolayer permeability via both cPLA 2 and stat3 phosphorylation in the context of oxidative stress. Keywords: S1PR3, Endothelial permeability, Oxidative stress, Phosphorylation, Tight junction 1. Introduction The blood-brain barrier (BBB) serves as a selective barrier structure that prevents harmful substances from entering the brain and maintaining its homeostasis[1, 2]. Brain microvascular endothelial cells (BMECs), which constitude the primary component of the BBB, play a crucial role in modulating vascular permeability and sustaining central nervous system (CNS) function[3]. Increased cerebrovascular permeability significantly contributes to neuronal injury during stroke, as it facilitates the entrance of toxic plasma components into the brain parenchyma, thereby further compromising microvascular integrity[4]. In the context of cerebral ischemia-reperfusion injury, BMECs impairment not only disrupts vascular barrier function[5] and elevates BBB permeability[6], but also induces the generation of reactive oxidative species (ROS)[7]. H 2 O 2 , a cell-permeant and stable form of ROS, is a critical regulator of endothelial cell homeostasis, influencing various molecular processes, including the maintanence of endothelial homeostasis function[8]. Consequently, as previously reported[6, 9], bEnd3 cells were exposed to H 2 O 2 to imitate the effects of oxidative stress on cerebrovascular endothelial cells in vitro. Sphingosine-1-phosphate (S1P) is a bioactive lipid that plays a crucial role in modulating vascular endothelial permeability and maintaining endothelial barrier integrity[10]. It exerts its effects through five specific G-protein-coupled receptors (S1PR1-5)[11]. Among these receptors, mainly three of them (S1PR1, S1PR2, S1PR3) are particularly important in endothelial cells for regulating permeability and cellular activation[12, 13]. The activation of S1PR1 and S1PR3 are associated with protective barrier effects, while S1PR2 tends to destabilize this barrier protection function[14]. S1PR1 is widely expressed throughout the body, whereas S1PR2 and S1PR3 are predominantly found in the CNS[15]. During ischemic stroke, S1PR1 can rapidly mobilize and adhere to cerebrovascular endothelial cells in the ischemic area, thereby contributing to vascular injury and thrombosis[16]. In contrast, during atherosclerosis, S1P has been shown to enhance the expression of endothelial nitric oxide synthase (eNOS) by activating S1PR1 and S1PR3, thereby providing a protective effect on vascular endothelium[12]. Previous study has highlighted the opposite effects of S1PR2 to those of S1PR3 on the monolayer permeability of bEnd3 cells in response to H 2 O 2 [9]. Specifically, S1PR3 has been shown to promote adherens junction assembly and facilitate cell migration in endothelial cells, underscoring its critical role in maintaining endothelial integrity and function[4, 17]. MAPKs are extensively involved in various physiological processes, including the regulation of oxidative stress and ROS[18]. Previous study has demonstrated that S1PR2 modulated oxidative stress-induced paracellular permeability of BECs via p38 and ERK-activated cPLA 2 [6]. During cerebral ischemia, the pathogenic roles of S1PR3 were associated with MAPK pathways, particularly ERK and p38[19]. In the context of diabetic nephropathy, baicalin has been shown to mitigate oxidative stress and inflammation via the MAPK signaling pathway, including p38, ERK and JNK[20]. Additionally, the administration of Sodium tanshinone IIA silate enhances melanin synthesis by activating both MAPK and PKA pathways, thereby protecting melanocytes from oxidative stress induced by H 2 O 2 [21]. However, there is limited research on the relationship between S1PR3 and JNK in the regulation of oxidative stress. In this study, we investigated the role of S1PR3 in regulating cerebrovascular endothelial permeability in response to H 2 O 2 . Our findings revealed that oxidative stress activated the phosphorylation of MAPKs as well as both cPLA 2 and stat3. Moreover, pharmacological blockade or genetic knockdown of S1PR3 further exacerbated hyperpermeabiltiy and tight junction disruption in bEnd3 monolayer in response to H 2 O 2 primarily via the pathways involving both cPLA 2 and stat3 phosphorylation. These results suggested that S1PR3 may represent a novel target for therapeutic strategies aimed at mitigating oxidative stress-induced cerebrovascular diseases. 2. Materials and Methods The study was conducted in accordance with the Basic &Clinical Pharmacology & Toxicology Policy for Experimental and Clinical Studies[22]. 2.1 Cell culture and reagents Mouse BMECs bEnd3 were obtained from ATCC (American Type Culture Collection). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and maintained in a humidified atmosphere of 5% CO 2 at 37°C. For pharmacological interventions, SP600125 was sourced from Selleckchem Company (Houston, USA), while TY52156 was synthesized by MedChem Express (Nanjing). Antibodies to p-p38, p38, p-ERK, ERK, p-JNK, JNK, p-cPLA 2 , p-stat3 and stat3 were purchased from Cell Signaling Technology (Boston). Tight junction protein ZO-1 and GAPDH antibodies were from Proteintech (Chicago). Antibody against cPLA 2 was obtained from Santa Cruz Biotechnology (Texas, USA). These reagents and antibodies were employed to assess signaling pathways and junction integrity in response to H 2 O 2 . 2.2 Lentiviral transfection Lentiviral vectors containing S1PR3 short hairpin shRNA (shS1PR3) and negative control shRNA (shNC), both tagged with green fluorescent protein (GFP) were utilized to effectively knock down of S1PR3 in bEnd3 cells. The transfection procedure was conducted following the established protocols described in the previous work[9]. This approach allowed for the selective reduction of S1PR3 expression, facilitating the assessment of its role in cerebrovascular endothelial permeability under oxidative stress. 2.3 Transwell permeability assay Transendothelial permeability was assessed using a Transwell system as previously described[6, 9]. Briefly, bEnd3 cells (1×10 5 ) were seeded on 24-well hanging inserts (0.4 μm pore size, Millipore, Millicell) in 24-well dishes and cultured for 12 h. Following this, the cells were serum-starved for 4 h and treated with either vehicle (DMSO) or TY52156 for 2 h prior to stimulate with H 2 O 2 (2 mM) for 1 h. Immediately after H 2 O 2 treatment, 5 μL of FITC-dextran (molecular weight 40000, Sigma-Aldrich, final concentration 1 mg/mL) was added to the inserts. The fluorescence intensity of FITC-dextran in the samples collected from the lower chamber after H 2 O 2 treatment was measured in duplicate for each condition at excitation/emission wavelengths of 492/520 nm. This assay enabled the quantification of transendothelial permeability changes in response to oxidative stress. 2.4 Immunofluorescence staining The immunofluorescence staining was conducted following a standard immunofluorescence protocol. Briefly, bEnd3 cells were seeded in round culture dish and subsequently fixed, washed, permeabilized, and blocked by Goat Serum. The cells were then incubated overnight at 4°C with primary antibody anti-ZO-1 (21773-1-AP) (Proteintech) at a dilution of 1:50. After washing, the cells were treated with an Alexa Fluor 594(SA00006-4)-conjugated secondary antibody at a dilution of 1:200, (Proteintech). Nuclear staining was performed using DAPI to visualize the nuclei. The stained cells were then examined using a confocal laser-scanning microscope to capture images, allowing for the assessment of ZO-1 localization and expression in the context of each experimental conditions. 2.5 Western blot analysis Cells were lysed using RIPA buffer, supplemented with protease inhibitors, phosphatase inhibitors, and phenylmethanesulfonyl fluoride (PMSF). The protein concentration was determined using the BCA method. According to the standard protocols, equal amounts of protein were subjected to Western blot analysis. The following primary antibodies were used at a specified dilutions: p-p38(1:1000), p38(1:1000), p-ERK(1:1000), ERK(1:1000), p-JNK(1:1000), JNK(1:1000), p-cPLA 2 (1:1000), cPLA 2 (1:500), p-stat3(1:1000), stat3(1:1000) and GAPDH(1:5000) as a loading control. Immunoreactive proteins were visualized using enhanced chemiluminescence; and the resulting protein bands were analyzed by the chemiluminescent gel imaging system. This approach allowed for the quantification and comparison of protein expression levels across different experimental conditions. 2.6 Statistical analysis All data were presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 8, employing one-way ANOVA followed by Newman-Keuls tests to evaluate differences between multiple groups. All the experiments were conducted at least three times, yielding consistent similar results. P<0.05 was considered to be statistically significant. 3. Results 3.1 S1PR3 antagonist further enhanced H 2 O 2 -induced monolayer hyperpermeability in bEnd3 cells To assess the impact of the S1PR3 antagonist TY52156 on H 2 O 2 -induced brain endothelial permeability, the penetration level of high molecular weight FITC-dextran across bEnd3 cells monolayer was measured by the transwell assay. H 2 O 2 stimulation significantly increased the paracellular permeability. Notably, pretreatment with TY52156 further enhanced H 2 O 2 -induced endothelial hyperpermeability. However, TY52156 alone did not alter endothelial permeability (Figure 1A). To further explore the influence of TY52156 on H 2 O 2 -induced hyperpermeability, immunofluorescence staining was applied to examine the localization of tight junction protein ZO-1. As shown in Figure 1B, a continuous distribution of ZO-1 at cell junctions was observed in the absence of H 2 O 2 . However, H 2 O 2 stimulation disrupted this pattern, leading to a more discontinuous distribution of ZO-1. Furthermore, treatment with TY52156 exacerbated this effect, resulting in a complete loss of ZO-1 localization at cell junctions in the presence of H 2 O 2. Conversely, pretreatment with TY52156 alone did not affect the localization of ZO-1. These findings suggested that the S1PR3 antagonist further enhanced H 2 O 2 -induced hypermeability by ulteriorly disrupting tight junction integrity in bEnd3 cells. 3.2 Activation of JNK was required for H 2 O 2 -induced stat3 phosphorylation in bEnd3 cells Previous study has indicated that the activation of p38 and ERK was essential for H 2 O 2 -induced cPLA 2 phosphorylation in bEnd3 cells[6]. Given that JNK is another critical member of MAPK family, we investigated its involvement in regulating endothelial permeability under oxidative stress and explored the downstream signaling. As demonstrated in Figure 2A and B, H 2 O 2 treatment significantly elevated the phosphorylation of JNK. Pretreatment with JNK inhibitor, SP600125, effectively reduced the level of p-JNK in a dose-dependent manner, with a notable decrease observed at a concentration of 10 μM. Consequently, this concentration was selected for subsequent experiments. Furthermore, it has been reported that treatment of OGD/R condition increased the phosphorylation of stat3 in rat brain endothelial cells[2]. In the model of MCAO/R in rats, Xueshuantong injection has been shown to preserve microvascular integrity by modulating the stat3 signaling pathway via JNK[23]. To elucidate the potential relationship between JNK and stat3 in our model, bEnd3 cells were pretreated with SP600125. As shown in Figure 2C-E, H 2 O 2 stimulation increased the phosphorylation of JNK and stat3. However, preatment with SP600125 simultaneously inhibited the H 2 O 2 -induced increases in the phosphorylation levels of both JNK and stat3. These findings suggested that JNK activation is crucial for H 2 O 2 -induced stat3 phosphorylation in bEnd3 cells, indicating a potential pathway through which oxidative stress affects endothelial permeability. 3.3 S1PR3 regulated H 2 O 2 -induced cPLA 2 and stat3 phosphorylation in bEnd3 cells To determine the role of S1PR3 in the regulation of oxidative stress-induced cPLA 2 and stat3 phosphorylation, bEnd3 cells were pretreated with TY52156 prior to H 2 O 2 exposure. As depicted in Figure 3, H 2 O 2 treatment significant increased the phosphorylation of p38, ERK, cPLA 2 , JNK and stat3. Notably, pretreatment with TY52156 further enhanced the phosphorylation levels of all five proteins, suggesting that S1PR3 may exert a modulatory effect on these signaling pathways. To further validate the involvement of S1PR3, we utilized a shRNA approach by infecting bEnd3 cells with shS1PR3 lentivirus, effectively knocking down S1PR3 expression as described in the previous work[9]. As shown in Figure 4, H 2 O 2 stimulation led to even greater phosphorylation levels of p38, ERK, cPLA 2 , JNK and stat3 in shS1PR3-infected cells compared to those infected with the control shRNA (shNC). 3.4 S1PR3 knockdown aggravated tight junction disruption and monolayer hyperpermeability induced by H 2 O 2 in bEnd3 cells To investigate the effect of S1PR3 depletion on endothelial tight junction integrity, we next examined the localization of ZO-1 in the absence or presence of H 2 O 2 . As shown in Figure 5A, in the absence of H 2 O 2 , both shNC-infected and shS1PR3-infected cells displayed a continuous distribution of ZO-1 at cell junctions, indicating the tight junctions were intact. However, H 2 O 2 stimulation led to a partially discontinuous pattern of ZO-1 in shNC-infected cells, which was further exacerbated by S1PR3 knockdown, suggesting that S1PR3 depletion aggravated the tight junction disruption. We also assessed the role of S1PR3 on H 2 O 2 -induced monolayer hyperpermeability. H 2 O 2 stimulation increased the paracellular permeability in shNC-infected cells, and this effect was further raised by shS1PR3 treatment, as illustrated in Figure 5B. These results indicated that S1PR3 played a protective role in maintaining endothelial tight junction integrity and preventing monolayer hyperpermeability in response to oxidative stress. The knockdown of S1PR3 not only disrupted tight junction localization but also exacerbated the permeability of the endothelial monolayer, highlighting its potential importance in cerebrovascular barrier function. 4. Discussion The BBB is critical for maintaining the microvasculature homeostasis of the CNS and is characterized by a unique microvasculature composed of specialized CNS endothelial cells that possess distinct molecular properties essential for its function and integrity[24-26]. Damage of the BBB is closely associated with oxidative stress, a condition arising from the excessive generation of ROS, which can trigger neuroinflammation and contribute to neurological deterioration[27, 28]. H 2 O 2 , a cell-stable and permeant form of ROS, is commonly employed to induce endothelial injury and simulate the oxidative stress in vitro[29]. In this study, we focused on the role of S1PR3 in regulating H 2 O 2 -induced signaling pathways in bEnd3 cells. Our findings revealed that the activation of JNK played a critical role in mediating H 2 O 2 -induced stat3 phosphorylation. Furthermore, blockade/knockdown of S1PR3 further exacerbated H 2 O 2 -induced paracellular hyperpermeability and ZO-1 redistribution, a key tight junction proein. This was accompanied by increased phosphorylation levels of MAPK pathway including p38, ERK, cPLA 2 , JNK and stat3. These results underscored the importance of S1PR3 in maintaining BBB intigrity under oxidative stress. By modulating key signaling pathways, S1PR3 appeared to play a protective role against the detrimental effects of oxidative stress on endothelial function and tight junction integrity, thereby contributing to the overall stability of the BBB. As one of the most robust physical barriers in the body, the BBB is primarily composed of tight junction (TJ) proteins in brain microvascular endothelial cells[30]. The stability of these TJs is essential to maintaining the functional integrity of the BBB in a healthy brain[31]. During ischemic stroke, a significant consequence of brain endothelial cell activation, triggered by proinflammatory stimuli, is the increased BBB permeability due to the disruption of TJ integrity[32-34]. Among the key proteins involved in TJs, ZO-1 is a critical cytoplasmatic TJ protein, that is essential to the proper assembly of interendothelial junction complexes, thereby sustaining BBB integrity[35]. Previous study has revealed that cPLA 2 altered the subcellular localization of ZO-1 in response to H 2 O 2 [6]. Additionally, IL-6 has been shown to induce alterations in ZO-1 localization and endothelial barrier function, which are contingent upon stat3 phosphorylation[36]. This suggested that stat3 may play a regulatory role in endothalial integrity and permeability by influencing ZO-1 localization. Our data indicated that blockade or knockdown of S1PR3 exacerbated the loss of TJ integrity in the presence of H 2 O 2 , which implied that targeting S1PR3 could be a promising therapeutic strategy to mitigate endothelial barrier impairment under oxidative stress. By preserving the localization and functionality of ZO-1, S1PR3 may contribute to the maintenance of BBB integrity, particularly in pathological scenarios such as ischemic stroke. S1PRs are pivotal in modulating vascular endothelial permeability. The interplay between S1PR1 and S1PR2 within the endothelium is crucial for regulating vascular permeability[37]. In vascular endothelial cells, elevated expression of S1PR2 has been shown to exacerbate vascular injury during inflammation responses[38]. Moreover, S1PR2 is instrumental in the mechanisms that lead to increasing cerebrovascular permeability[6, 39]. However, the regulatory role of S1PR3 in cerebrovascular endothelium remains poorly understood. Consistant with prior findings[9], the current study corroborated that blockade or knockdown of S1PR3 further enhanced H 2 O 2 -induced endothelial monolayer permeability in bEnd3 cells. This suggested that S1PR3 may play an important role in maintaining endothelial barrier function under oxidative stress. It is well established that MAPKs play a pivotal role in regulating barrier function in endothelial cells. Notably, the activation of the p38 MAPK pathway has been implicated in the non-structural protein-induced impairment of endothelial barrier integrity[40]. Prior research has demonstrated that the alterations in phosphorylated p38, ERK and cPLA 2 levels in response to H 2 O 2 due to the suppression of both S1PR2 and S1PR3 could be reversed by CRH[9]. Among MAPK family, JNK is another critical member associated with various physiological functions. In the context of post-ischemic brain injury, the inhibition of S1PR2 has been shown to reduce the activation of M1-associated ERK and JNK activation[18]. Furthermore, the activation of the JNK signaling pathway contributed has been linked to the preservation of endothelial barrier function[41]. In PC12 cells, exposure to H 2 O 2 resulted in a modest activation of p38 and JNK, while significantly inhibiting ERK and AKT, indicating that the specific responses of MAPK pathways are ultimately determined by the cellular context and the nature of the stimuli[42]. Additionally, in mouse aortic smooth muscle cell (MOVAS), STRIP2 overexpression led to an increase in phosphorylated p38 and Akt levels, while simultaneously contributing to decreasing p-ERK and p-JNK expression[43]. Our present study further elucidated that JNK was also involved in regulating the monolayer permeability of bEnd3 cells in response to H 2 O 2 , exhibiting a similar trend to that observed for p38 and ERK. This finding underscore the significant role of the MAPK signaling pathway in modulating cerebrovascular endothelial cells permeability under oxidative stress. cPLA 2 activation has been shown to disrupt the BBB during cerebral ischemic injury, with its effects linked to p38 and ERK[44]. Furthermore, in the context of Alzheimer’s disease, a calcium channel blocker Azelnidipine was reported to suppress oxidative stress and inflammatory responses in bEnd3 cells via ERK-cPLA 2 pathway[45]. It was also documented that S1PR2 regulated oxidative stress-induced endothelial barrier impairment by attenuating the phosphorylation of cPLA 2 that is dependent on p38 and ERK signaling in vitro[6]. Consistant with previous study[9], this study further confirmed that blockade/knockdown of S1PR3 further exacerbated H 2 O 2 -induced endothelial hyperpermeability and ZO-1 redistribution via cPLA 2 phosphorylation. This highlights the critical role of S1PR3 in maintaining endothelial barrier integrity under oxidative stress. During ischemic stroke, the activation of stat3 was closely linked to the destruction of the BBB, making it a critical target for neuroprotective therapies[46]. It was reported that the activation of stat3 contributed to BBB dysfunction and the use of a stat3 inhibitor was shown to reduce endothelial monolayer hyperpermeability induced by OGD/R[2]. Additionally, a traditional Chinese medicine, xueshuantong, was demonstrated efficacy in improving cerebral microvascular structure and function in MCAO/R rats through JNK mediated stat3 signaling pathway[23]. This present study showed that pretreatment with SP600125, a selective JNK inhibitor, attenuated the phosphorylation of both JNK and stat3, suggesting stat3 is a direct downstream target of JNK signaling in bEnd3 cells. In light of these findings, our results revealed that the JNK-stat3 signaling pathway plays a significant role in regulating endothelial monolayer permeability in bEnd3 cells under oxidative stress. In conclusion, as illustrated in Fig. 6, our findings indicated that pharmacological blockade or genetic knockdown of S1PR3 further aggravated H 2 O 2 -induced endothelial hyperpermeability and TJ disruption in bEnd3 cell monolayer. This effect occurs through the activation of both the p38/ERK-cPLA 2 and JNK-stat3 signaling pathways. These insights may suggest potential strategies for addressing oxidative stress-induced cerebrovascular diseases, such as stroke. Acknowledgments This work was supported by National Science Foundation of China (No. KY109ZX20230072&82200426). Conflict of interests The authors declare that there is no conflict of interests Author contributions Junyu Mu and Liye Ge performed the experiments; Li Dai contributed essential reagents; Junyu Mu and Jing Zhang planned the experiments, analyzed the data, and wrote the paper. References 1. Ekhator C, Qureshi MQ, Zuberi AW, Hussain M, Sangroula N, Yerra S, et al. 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After 4 h of serum starvation, cells were treated with vehicle or TY52156 for 2 h before H 2 O 2 stimulation for 1 h. Scale bar, 20 μm. All experiments were performed more than three times independently and the data were expressed at the means ± S.E.M. (****P Figure 2 Activation of JNK was required for H 2 O 2 -induced stat3 phosphorylation. (A) The cells were treated with SP600125 at the indicated concentrations for 2 h before exposed to H 2 O 2 for 30 min. A representative immunoblot is shown. (B) Quantification of JNK phosphorylation in (A). (C) The cells were pretreated with SP600125 for 2 h before H 2 O 2 stimulation for 30 min. A representative immunoblot is shown. (D-E) Quantification of JNK and stat3 phosphorylation in (C). All experiments were performed more than three times independently and the data were expressed at the means ± S.E.M. (**P <0.01; ***P <0.001; ****P <0.0001. *versus the left group below the horizontal line.) Figure 3 S1PR3 antagonist TY52156 promoted H 2 O 2 -induced cPLA 2 and stat3 phosphorylation. The cells were pretreated with TY52156 for 2 h before exposed to H 2 O 2 for 30 min. (A) A representative immunoblot is shown. (B-F) Quantification of p38, ERK, cPLA 2 , JNK and stat3 phosphorylation in (A). All experiments were performed more than three times independently and the data were expressed at the means ± S.E.M. (**P <0.01; ***P horizontal line.) Figure 4 S1PR3 knockdown promoted H 2 O 2 -induced cPLA 2 and stat3 phosphorylation. The cells were exposed to H 2 O 2 for 30 min. (A) A representative immunoblot is shown. (B-F) Quantification of p38, ERK, cPLA 2 , JNK and stat3 phosphorylation in (A). All experiments were performed more than three times independently and the data were expressed at the means ± S.E.M. (**P <0.01; ****P Figure 5 S1PR3 knockdown aggravated tight junction disruption and monolayer hyperpermeability induced by H 2 O 2 . (A) Confocal images of ZO-1-immunolabeled (red) and DAPI-stained (blue) bEnd3 cells. (B) Endothelial permeability was measured by the FITC-dextran assay. After 96 h infection, the lentivirus-infected bEnd3 cells were serum-starved for 4 h and then exposed to H 2 O 2 for 1 h. Scale bar, 20 μm. Fluorescence intensity was normalized to vehicle-treated shNC-infected cells. All experiments were performed more than three times independently and the data were expressed at the means ± S.E.M. (****P <0.0001. *versus the left group below the horizontal line.) Figure 6 Schematic model of the effect of S1PR3 inactivation on cerebrovascular endothelial permeability under oxidative stress. Supplementary Material File (original image for checking.docx) Download 344.58 KB Information & Authors Information Version history V1 Version 1 06 August 2025 Peer review timeline Published Basic & Clinical Pharmacology & Toxicology Version of Record 2 Feb 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords endothelial permeability oxidative stress phosphorylation s1pr3 tight junction Authors Affiliations Junyu Mu Nanjing Medical University View all articles by this author Liye Ge Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences View all articles by this author Li Dai Jiangsu Medical College View all articles by this author Jing Zhang 0000-0002-4700-6907 [email protected] Nanjing Medical University View all articles by this author Metrics & Citations Metrics Article Usage 208 views 139 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Junyu Mu, Liye Ge, Li Dai, et al. 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