The Pathological Role Of S1P/S1PR2-mediated Pericyte Loss In The Blood Brain Barrier Dysfunction Of NPSLE

Objective:To investigate the role of abnormal activation of S1P/S1PR2 pathway in the pathogenesis of NPSLE induced by pericyte loss in MRL/lpr mice and its pathological mechanism.Methods:The Neuroethology experiment was carried out at the age of 10 weeks in MRL/lpr female mice and its results were taken as the baseline.The Neuroethology experiment was carried out again two weeks later.Those whose Neuroethology to baseline ratio changes were more than 20%in MRL/lpr female mice were defined as NPSLE mice,and the mice with abnormal neurobehavior and those without Neuroethology abnormalities were screened out.Establish a non NPSLE control group,NPSLE-S1P antagonist group,NPSLE-S1PR2 blockade group,and a model group treated with NPSLE physiological saline at the same week of age(n=6).By administering S1P antagonist(2mg/kg FTY720,gavage administration),S1PR2 blocker(8mg/kg JTE-013,intraperitoneal injection),and the same dose of physiological saline three times a week,after 3 weeks of administration:① The expressions of S1P,IL-6 and IFN-α in serum were detected by ELISA.② The changes of blood-brain barrier permeability were evaluated by Evans blue.③HE staining was used to observe the inflammation of brain tissue,Nisi staining was used to observe the damage of brain neurons,and immunofluorescence staining was used to observe the expression of peripheral cells(NG2)and endothelial cells(CD31)in microvessels.④ The expression levels of S1P,S1PR2,PDGFR-β and ZO-1 were detected by Western blot,and the mRNA levels of S1PR2 and PDGFR-β were detected by RT-PCR to evaluate the pathological mechanism of peri-cell loss regulated by S1P/S1PR2 signaling pathway in neuropsychiatric lupus.Results:Neurobehavioral experiments were carried out on mice in each group after administration,and it was found that in the model group,the latency and resting time in water increased,while in the S1P antagonist group and S1PR2 blocking group,the latency and resting time in water decreased compared with the model group.In the open field experiment,the autonomous exploration ability of mice in the model group decreased significantly,which was reflected in the decrease of the exploration distance in the central region.The exploration distance of the central region of mice in S1P antagonist group and S1PR2 blocking group was higher than that in model group.The results showed that in HE staining,the ventricle inflammatory exudation increased significantly in the model group compared with the control group,and the inflammatory exudation decreased in the S1P antagonist group and S1PR2 blocking group compared with the model group.In the Niger staining,compared with the control group,the Niger staining was shallow and irregular in the model group,and the neuron damage was obvious.The concentrations of S1P,IL-6 and IFN-α in serum of mice were determined by ELISA.The results showed that the levels of S1P,IL-6 and IFN-α were significantly increased in the model group compared with the control group,while the levels of S1P antagonist group and S1PR2 blocking group were significantly decreased compared with the model group,and the difference was statistically significant.Western blot results showed that compared with the control group,the expressions of S1P and S1PR2 in the brain tissue of the model group were significantly increased,and the tight junction protein ZO-1 and peripheral cell surface molecule PDGFR-β in the model group were significantly decreased,with statistical significance.The increase of S1P and S1PR2 expression in S1P antagonist group and S1PR2 blocking group was alleviated compared with the model group,and the decrease of ZO-1 and PDGFR-βwas alleviated compared with the model group.The results of immunofluorescence staining of mouse brain tissue showed that the number of pericellular cells in the model group was significantly lower than that in the control group,and the loss of pericellular cells in the S1P antagonist group and the S1PR2 blocking group was improved.Conclusion:① Inhibition of S1P/S1PR2 can improve the neurobehavior of MRL/lpr mice with depression and anxiety symptoms.② Inhibition of S1P/S1PR2 can improve blood-brain barrier permeability③IL-6 and IFN-α may be directly or indirectly involved in the pathogenesis and progression of NPSLE.④S1P/S1PR2 may down regulate tight junction protein ZO-1,promote pericyte to lose pathological status,promote NPSLE blood brain barrier leakage,and cause NPSLE disease progression.