抑制S1P/S1PR2介导的周细胞脱失可缓解NPSLE小鼠血脑屏障功能障碍

Inhibition of S1P/S1PR2-mediated pericyte loss alleviates blood-brain barrier dysfunction in NPSLE mice

目的:探讨在MRL/lpr小鼠模型中鞘氨醇-1-磷酸(S1P)/S1P受体2(S1PR2)信号通路的异常激活引起周细胞丢失并进一步影响神经精神性系统性红斑狼疮(NPSLE)小鼠血脑屏障功能障碍的病理机制。方法:采用旷场实验、新物体识别实验和强迫游泳实验评估6周龄开始的雌性MRL/lpr小鼠行为学改变,将神经行为学与基线比改变大于20%者定义为NPSLE小鼠。将NPSLE小鼠随机分为NPSLE-S1P拮抗组、NPSLE-S1PR2阻断组和NPSLE生理盐水处理组,每组6只。另将行为学无异常改变的6只小鼠作为对照组。NPSLE-S1P拮抗组给予S1P拮抗剂FTY720(2 mg/kg,灌胃),NPSLE-S1PR2阻断组给予S1PR2阻断剂JTE-013(8 mg/kg,腹腔注射),NPSLE生理盐水处理组和对照组给予等体积的生理盐水。每周给药3次,持续3周。通过旷场实验、新物体识别实验和强迫游泳实验评估小鼠的行为学改变。采用酶联免疫吸附实验(ELISA)检测血清中S1P、白细胞介素6(IL-6)和干扰素α(IFN-α)水平;伊文思蓝法评估血脑屏障通透性改变;苏木精-伊红(HE)染色观察脑组织炎症情况;尼氏染色观察脑神经元受损情况;免疫荧光染色观察微血管中周细胞标志物神经胶质抗原2(NG2)和内皮细胞标志物CD31的表达;Western blot检测脑组织中S1P、S1PR2、血小板源性生长因子受体β(PDGFR-β)和闭锁小带蛋白1(ZO-1)的蛋白表达水平;RT-qPCR检测S1PR2和PDGFR-β的mRNA表达水平。结果:与模型组比较,S1P拮抗组和S1PR2阻断组小鼠水中潜伏和静止时间显著缩短(P<0.05),中央区探索距离显著增加(P<0.05),脑室炎性渗出和神经元受损减少,周细胞(NG2,绿色)和内皮细胞(CD31,红色)脱失情况减少,血清S1P、IL-6和IFN-α水平显著下降(P<0.05),S1PR2的mRNA表达水平显著下降(P<0.05),PDGFR-β的mRNA表达水平显著升高(P<0.05),S1P和S1PR2蛋白表达水平显著下降(P<0.05),PDGFR-β和ZO-1蛋白表达水平显著升高(P<0.05)。结论:抑制S1P/S1PR2途径介导的周细胞脱失可缓解NPSLE小鼠的神经行为症状,降低血脑屏障通透性,并减轻炎症反应。

AIM:To investigate the pathological mechanism by which abnormal activation of sphingosine-1-phosphate(S1P)/S1P receptor 2(S1PR2)signaling pathway induces pericyte loss and subsequently affects blood-brain barrier dysfunction in neuropsychiatric systemic lupus erythematosus(NPSLE)mice using MRL/lpr mouse model.METHODS:Behavioral changes in female MRL/lpr mice from 6 weeks of age were assessed using open-field test(OFT),novel object recognition test(NORT)and forced swimming test(FST).The mice with neurobehavioral changes exceeding 20%compared with baseline were defined as NPSLE mice,and were randomly divided into 3 groups,each with 6 mice:NPSLE-S1P antagonist group,NPSLE-S1PR2 blocker group,and NPSLE saline treatment group.Additional 6 mice with no abnormal behavioral changes served as control group.The mice in NPSLE-S1P antagonist group were treated with S1P antagonist FTY720(2 mg/kg,orally),those in NPSLE-S1PR2 blocker group received treatment with S1PR2 blocker JTE-013(8 mg/kg,intraperitoneally),while those in NPSLE saline treatment group and control group received an equivalent volume of saline.Treatments were conducted 3 times per week for 3 weeks.Behavioral changes were reassessed using OFT,NORT and FST.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum levels of S1P,interleukin-6(IL-6)and interferon-α(IFN-α).Blood-brain barrier permeability changes were evaluated by Evans blue staining.Hematoxylin-eosin(HE)staining was employed to observe brain tissue inflammation,Nissl staining was used to examine neuronal damage,and immunofluorescence staining was performed to assess the expression of pericyte marker neural/glial antigen 2(NG2)and endothelial cell marker CD31 in microvessels.Western blot analysis was conducted to detect the expression levels of S1P,S1PR2,platelet-derived growth factor receptor-β(PDGFR-β)and zonula occludens-1(ZO-1)in brain tissues,while RT-qPCR was utilized to measure S1PR2 and PDGFR-βmRNA levels.RESULTS:Compared with model group,the mice in S1P antagonist and S1PR2 blocker groups exhibited reduced latency and immobility time in the water(P<0.05)and increased central zone exploration distance(P<0.05).There was reduced inflammatory exudation and neuronal damage in the ventricles,along with attenuated retention of pericytes(NG2,green)and endothelial cells(CD31,red).Serum levels of S1P,IL-6 and IFN-αwere significantly decreased(P<0.05).The S1PR2 mRNA expression was significantly reduced,whereas PDGFR-βmRNA expression was significantly increased(P<0.05).Protein expression levels of S1P and S1PR2 were significantly decreased,while PDGFR-βand ZO-1 protein levels were significantly increased(P<0.05).CONCLUSION:Inhibition of the S1P/S1PR2 pathway attenuates neurobehavioral symptoms,reduces blood-brain barrier permeability,and suppresses inflammation in NPSLE mice.