Abstract:

Objective To investigate the effect of Xuebijing injection against blood-brain barrier （BBB） ??damage in the mice with anti-N-methyl-D-aspartate receptor （NMDAR） encephalitis， and to elucidate its regulatory effect on the imbalance of helper T cells 17 （Th17）/regulatory T cells （Treg）. Methods The active immunization models of anti-NMDAR encephalitis in the mice were established using glutamate receptor N1 subunit （GluN1） 356-385 antigen peptide， and the serum anti-NMDAR immunoglobulin G （IgG） antibody levels were detected by enzyme-linked immunosorbent assay（ELISA）. The healthy mice without modeling were served as control group， and the mice with successful modeling were randomly divided into model group， low dose of Xuebijing injection （XBJ-L） group， and high dose of Xuebijing injection （XBJ-H） group， with 10 mice in each group. After modeling， the mice in XBJ-L and XBJ-H groups were intraperitoneally injected with 5 and 10 mL·kg^-1 Xuebijing injection， respectively. The Longa score was used to assess the neurological impairment of the mice in various groups； evans blue （EB） staining was used to determine the BBB permeability； immunofluorescence staining was used to detect the expressions of zonula occludens 1 （ZO-1） and Occludin in cerebral cortex of the mice in various groups； Western blotting method was used to determine the expression levels of ZO-1， Occludin， Claudin-5， and neuron-specific nuclear protein （NeuN） in cerebral cortex of the mice in various groups； ELISA method was used to determine the levels of Th17- and Treg-related cytokines including interleukin （IL）-17， IL-22， and IL-10 in serum of the mice； flow cytometry was used to determine the percentages of Th17 and Treg cells in peripheral blood of the mice in various groups， and the Th17/Treg ratio was calculated. Results The serum of the mice induced with the GluN1 356-385 antigen peptide was positive for NMDAR IgG antibodies， indicating that the models were successfully established. Compared with control group， the neurological impairment score of the mice in model group was significantly increased （P<0.05）， and the EB level in brain tissue was significantly increased （P<0.05）； the fluorescence staining intensities of ZO-1 and Occludin in the cerebral cortex were decreased， and the expression levels of ZO-1， Occludin， Claudin-5， and NeuN proteins in the cerebral cortex were significantly decreased （P<0.05）； the serum levels of IL-17 and IL-22 were significantly increased （P<0.05）， while the IL-10 level was significantly decreased （P<0.05）； the percentage of Th17 cells in peripheral blood was significantly increased （P<0.05）， while the percentage of Treg cells was significantly decreased （P<0.05）， and the Th17/Treg ratio was significantly increased （P<0.05）. Compared with model group， the neurological impairment scores of the mice in XBJ-L and XBJ-H groups were significantly decreased （P<0.05）， the EB levels in brain tissue were significantly decreased （P<0.05）， the fluorescence staining intensities of ZO-1 and Occludin in cerebral cortex were increased， and the expression levels of ZO-1， Occludin， Claudin-5， and NeuN proteins were significantly increased （P<0.05）； the levels of IL-17 and IL-22 in serum were significantly decreased （P<0.05）， and the level of IL-10 was significantly increased （P<0.05）； the percentages of Th17 cells in peripheral blood were significantly decreased （P<0.05）， the percentages of Treg cells were significantly increased （P<0.05）， and the Th17/Treg ratios were significantly decreased （P<0.05）. Compared with XBJ-L group， the neurological function injury score of the mice in XBJ-H group was significantly decreased （P<0.05）， the EB level in brain tissue was significantly decreased （P<0.05）； the fluorescence staining intensities of ZO-1 and Occludin in the cerebral cortex were increased， and the expression levels of ZO-1， Occludin， Claudin-5， and NeuN proteins were significantly increased （P<0.05）； the serum levels of IL-17 and IL-22 were significantly decreased （P<0.05）， and the level of IL-10 was significantly increased （P<0.05）； the percentage of Th17 cells in peripheral blood was significantly decreased （P<0.05）， the percentage of Treg cells was significantly increased （P<0.05）， and the Th17/Treg ratio was significantly decreased （P<0.05）. Conclusion Xuebijing injection can improve BBB injury， regulate Th17/Treg balance， and thereby alleviate the neurological functional damage in anti-NMDAR encephalitis.

Key words: Xuebijing injection, Anti-N-methyl-D-aspartic acid receptor encephalitis, Blood-brain barrier, Neurological function, Helper T cell 17, Regulatory T cell