Abstract:

Objective To discuss the effect of Xuebijing on brain tissue damage and immune imbalance of helper T lymphocyte 17 （Th17）/regulatory T lymphocyte （Treg） in cerebrospinal fluid （CSF） of the N-methyl-D-aspartate （NMDA） receptor encephalitis model mice， and to clarify its therapeutic effect. Methods Sixty healthy male C57BL/6J mice were randomly divided into control group， model group， low dose of Xuebijing group， and high dose of Xuebijing group， and there were 15 mice in each group. Except for control group， the mice in the other three groups were injected with the antigen combined with immunostimulation to establish the NMDA receptor encephalitis models. The mice in low and high doses of Xuebijing groups were injected intraperitoneally with 5 and 10 mL·kg^-1 of Xuebijing injection， respectively. HE staining was used to observe the pathomorphology of brain tissue of the mice in various groups； TUNEL assay was used to detect the apoptotic rates of the neurons in hippocampus CA1 region of brain tissue of the mice in various groups；enzyme-linked immunosorbent assay （ELISA） method was used to detect the levels of interleukin （IL）-6， IL-10， IL-17， and transforming growth factor β （TGF-β） in serum of the mice in various groups； flow cytometry was used to detect the percentages of Th17 and Treg cells in CSF of the mice in various groups； Western blotting method was used to detect the expression levels of retinoic acid-related orphan receptor γt （RORγt）， forkhead box protein 3 （Foxp3）， IL-10， and IL-17 proteins in brain tissue of the mice in various groups；immunohistochemistry method was used to detect the rates of IL-17 and Foxp3 positive cells in brain tissue of the mice in various groups. Results The HE staining results showed that the hippocampus CA1 region of brain tissue of the mice in control group had a clear structure without obvious lesions； compared with control group， the mice in model group showed partial pyramidal cell shrinkage， elongation of apical dendrites， loss of a few neurons， and sparse tissue in the hippocampus CA1 region of brain tissue； compared with model group， the mice in low and high doses of Xuebijing groups showed that the damage of the cells in the hippocampus CA1 region of brain tissue was decreased， and the morphological recovery， more orderly arrangement， and more significant improvement could be seen in hippocampus CA1 region of the mice in high dose of Xuebijing group. The TUNEL assay results showed that compared with control group， the apoptotic rate of the neurons in hippocampus CA1 region of brain tissue of the mice in model group was significantly increased （P<0.05）； compared with model group， the apoptotic rate of the neurons in hippocampus CA1 region of brain tissue of the mice in low and high doses of Xuebijing groups were significantly decreased （P<0.05）； compared with low dose of Xuebijing group， the apoptotic rate of the neurons in hippocampus CA1 region of brain tissue of the mice in high dose of Xuebijing group was significantly decreased （P<0.05）. The ELISA results showed that compared with control group， the levels of IL-6 and IL-17 in serum of the mice in model group were significantly increased （P<0.05）， while the levels of IL-10 and TGF-β were significantly decreased （P<0.05）； compared with model group， the levels of IL-6 and IL-17 in serum of the mice in low and high doses of Xuebijing groups were significantly decreased （P<0.05）， while the levels of IL-10 and TGF-β were significantly increased （P<0.05）； compared with low dose of Xuebijing group， the levels of IL-6 and IL-17 in serum of the mice in high dose of Xuebijing group were significantly decreased （P<0.05）， while the levels of IL-10 and TGF-β were significantly increased （P<0.05）. The flow cytometry results showed that compared with control group， the percentage of CD4+IL-17A+ Th17 cells in CSF of the mice in model group was significantly increased （P<0.05）， while the percentage of CD25+Foxp3+ Treg cells was significantly decreased （P<0.05）； compared with model group， the percentages of CD4+IL-17A+ Th17 cells in CSF of the mice in low and high doses of Xuebijing groups were significantly decreased （P<0.05）， while the percentage of CD25+Foxp3+ Treg cells was significantly increased （P<0.05）； compared with low dose of Xuebijing group， the percentage of CD4+IL-17A+ Th17 cells in CSF of the mice in high dose of Xuebijing group was significantly decreased （P<0.05）， while the percentage of CD25+Foxp3+ Treg cells was significantly increased （P<0.05）. The Western blotting results showed that compared with control group， the expression levels of RORγt and IL-17 proteins in brain tissue of the mice in model group were significantly increased （P<0.05）， while the expression levels of Foxp3 and IL-10 proteins were significantly decreased （P<0.05）； compared with model group， the expression levels of RORγt and IL-17 proteins in brain tissue of the mice in low and high doses of Xuebijing groups were significantly decreased （P<0.05）， while the expression levels of Foxp3 and IL-10 proteins were significantly increased （P<0.05）； compared with low dose of Xuebijing group， the expression levels of RORγt and IL-17 proteins in brain tissue of the mice in high dose of Xuebijing group were significantly decreased （P<0.05）， while the expression levels of Foxp3 and IL-10 proteins were significantly increased （P<0.05）. The immunohistochemistry results showed that compared with control group， the rate of IL-17 positive cells in brain tissue of the mice in model group was significantly increased （P<0.05）， while the rate of Foxp3 positive cells was significantly decreased （P<0.05）； compared with model group， the rates of IL-17 positive cells in brain tissue of the mice in low and high doses of Xuebijing groups were significantly decreased （P<0.05）， while the rates of Foxp3 positive cells were significantly increased （P<0.05）； compared with low dose of Xuebijing group， the rate of IL-17 positive cells in brain tissue of the mice in high dose of Xuebijing group was significantly decreased （P<0.05）， while the rate of Foxp3 positive cells was significantly increased （P<0.05）. Conclusion Xuebijing can effectively ameliorate the brain tissue injury， regulate the cytokine levels， and intervene in immune imbalance of Th17/Treg in the mice with anti-NMDA receptor encephalitis.

Key words: Anti-N-methyl-D-aspartate receptor encephalitis, Xuebijing, Neuronal apoptosis, Helper T lymphocyte 17, Regulatory T lymphocyte, Immune imbalance