左归丸和右归丸对自身免疫性脑脊髓炎大鼠中枢神经系统淋巴细胞亚群免疫组化表达的影响

Effects of Zuoguiwan and Youguiwan on Immunohistochemical Expression of Lymphocyte Subpopulation in Central Nervous System of EAE Rats

目的通过观察左归丸与右归丸对实验性自身免疫性脑脊髓炎(EAE)模型大鼠脑、脊髓组织淋巴细胞亚群免疫组化表达的影响,探讨滋阴补肾法与温阳补肾法治疗EAE作用机制。方法应用髓鞘碱性蛋白与完全福氏佐剂,按体积比1:1制成抗原并于Lewis大鼠双后足垫下注射,建立EAE模型。120只Lewis大鼠随机分为正常组、模型组、激素组、左归丸组及右归丸组,每组24只,正常组和模型组给予生理盐水灌胃,3mL/(只·d);激素组免疫后给予生理盐水灌胃,3mL/(只·d),发病后改为醋酸泼尼松混悬液灌胃,5mg/(kg·d);左归丸组免疫后给予左归丸混悬液,2g/(kg·d);右归丸组免疫后给予右归丸混悬液,3g/(kg·d)。直至处死。分别于急性期(免疫后15d)和缓解期(免疫后27d)随机选取各组大鼠,取脑和脊髓组织切片进行CD4+、CD8+、CD3+、CD19+免疫组化染色。结果造模后第15日及第27日,模型组脑和脊髓组织中CD4+免疫组化表达均较正常组升高(P<0.01),CD8+表达降低(P<0.05),CD4+/CD8+比值升高(P<0.01);同模型组比较,左归丸组、右归丸组、激素组CD4+免疫组化表达均降低(P<0.01),且在造模后第27日,左归丸组和右归丸组脊髓组织CD4+表达较激素组显著降低(P<0.01);造模后第15日,右归丸组和激素组CD8+表达较模型组升高(P<0.01),造模后第27日,左归丸组和激素组CD8+表达较模型组升高(P<0.01);不同时点激素组、左归丸组及右归丸组CD4+/CD8+比值均显著低于模型组(P<0.05);造模后第15日及第27日,模型组中枢神经系统CD19+免疫组化表达均较正常组升高(P<0.01),急性期左归丸组、右归丸组、激素组脊髓组织CD19+表达较模型组降低(P<0.01),缓解期则见左归丸组和右归丸组CD19+表达减少(P<0.01)。结论 EAE大鼠CD4+/CD8+比值升高,这在EAE发病中发挥重要作用。滋阴补肾法和温阳补肾法可下调CD4+表达,下调CD4+/CD8+比值来治疗EAE。

Objective To observe the irnmunohistochemical expression of lymphocyte subpopulationin in brain and spinal cord tissue, and explore the mechanism of Zuogui wan and Yougui wan on rats with experimental autoimmune encephalomyelitis （EAE）. Methods Lewis rats were immunized with the myelin basic protein （MBP）. 120 rats were grouping randomly into normai group, EAE group, prednisone group, Zuoguiwan group and Youguiwan group after post immunization （PI）. Rats in normal group and EAE group were administered normal soline, each 3 mL/d. Rats in prednisone group were administered suspension of prednisone after developed clinical signs, each 5 mg/kg. Rats in Zuoguiwan group were administered suspension of Zuoguiwan, each 2 g/kg, and rats in Youguiwan group were administered suspension of Youguiwan, each 3 g/kg. On 15^th and 27^th day after PI, rats were killed and the immunohistochemical staining was performed on the sections of brain and spinal cord. Results On 15^th and 27^th day after PI, the expression of CD4^＋ in brain and spinal cord tissue of EAE group were significantly higher than that in normal group （P〈0.01）, while the expression of CD8^＋ was significantly lower than normal group （P〈0.05）, CD4^＋/CD8^＋ was significantly lower too （P〈0.01）. Compared with EAE group, the expression of CD4^＋ in prednisone group, Zuoguiwan group and Youguiwan group were significantly lower （P〈0.01）. On 27^th day after PI, the expression of CD4^＋ in spinal cord tissue of Zuoguiwan group and Youguiwan group were significantly lower than that on 15^th day after PI （P〈0.01）. On 15^th day after PI, the expression of CD8^＋ in prednisone group and Youguiwan group were significantly higher than that in EAE group （P〈0.01）, while on 27^th day after PI, the expression of CD8^＋ in prednisone group and Zuoguiwan group were higher than that in EAE group （P 〈0.01）. On both points, CD4^＋/CD8^＋ in prednisone group, Zuoguiwan group and Youguiwan group were significantly lower than EAE group （P 〈 0.05）. On 15^th and 27^th day after PI, the expression of CD19^＋ in brain and spinal cord tissue of EAE group were significantly higherthan that in normal group （P〈0.01）. In acute stage, the expression of CD19^＋ in spinal cord tissue of prednisone group, Zuoguiwan group and Youguiwan group were significantly lower than that in EAE group （P〈0.01）. In chronic phase, the expression of CD19^＋ in spinal cord tissue of Zuoguiwan group and Youguiwan group were significantly lower than EAE group （P〈0.01）. Conclusion Zuoguiwan and Youguiwan both have the effect of down-regulating the immunohistochemicai expression of CD4^＋ in central nervous system of EAE rats, the down-regulation of CD4^＋/CD8^＋ may be the mechanism for the method of nourishing yin and invigorating yang.