二黄胶囊对实验性自身免疫性脑脊髓炎大鼠中枢神经系统细胞因子和淋巴细胞亚群免疫组化表达的影响

Influence of Erhuang Capsule on the Immunohistochemical Expression of IFN-γ,TGF-β,MMP-9 and Lymphocyte Subsets in Central Nervous System of EAE Rat

摘要:

目的: 观察大鼠实验性自身免疫性脑脊髓炎(Experimental Autoimmune Encephalomyelitis, EAE)模型脑、脊髓组织转化生长因子-β(transforming growth factor-β,TGF-β)、干扰素-γ(interferon-γ,IFN-γ)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)免疫组化和T淋巴细胞亚群的表达,比较二黄方两种剂型(汤剂、胶囊剂)作用差异。 方法: 用髓鞘碱性蛋白(Myelin Basic Protein,MBP)免疫Lewis大鼠诱导EAE模型,随机分为正常组、模型组、泼尼松组、二黄汤组及二黄胶囊组,分别于免疫后15 d(急性期发病)和免疫后27 d(缓解期)随机选取各组大鼠,取脑和脊髓组织切片进行IFN-γ,TGF-β,MMP-9,CD4⁺,CD8⁺,CD3⁺,CD19⁺免疫组化染色。 结果: 与模型组比,二黄汤(免疫后27 d)和二黄胶囊(免疫后15 d和27 d)显著提高脑组织TGF-β表达;二黄汤和胶囊均能显著降低免疫后15 d,27 d脑和脊髓组织IFN-γ,MMP-9表达。二黄汤、二黄胶囊组免疫后15 d,27 d脑和脊髓组织IFN-γ、免疫后15 d脑和脊髓组织MMP-9表达降低作用强于泼尼松组。与模型组比,二黄汤组免疫后15 d、27 d 脑组织CD4⁺表达显著降低、CD8⁺表达显著升高,CD4⁺/CD8⁺显著降低,免疫后15 d CD19⁺表达降低;二黄胶囊组除免疫后15 d脑组织CD8⁺与模型组无显著差异外,其他均同二黄汤组。与模型组比,二黄汤组免疫后15 d、27 d脊髓组织CD4⁺表达显著降低、CD8⁺表达显著升高,CD4⁺/CD8⁺显著降低,二黄胶囊组免疫后15 d,脊髓组织CD4⁺表达显著降低、CD8⁺表达显著升高,CD4⁺/CD8⁺显著降低,27 d CD4⁺表达显著降低、CD4⁺/CD8⁺显著降低。二黄汤和二黄胶囊组间比较无显著差异。 结论: 二黄汤、二黄胶囊可上调EAE大鼠中枢神经系统TGF-β表达,作用同泼尼松。两者均可显著降低脑和脊髓组织IFN-γ、MMP-9表达,作用优于泼尼松。二黄汤、胶囊均可降低CD4⁺表达、升高CD8⁺表达,改善CD4⁺/CD8⁺比值。二黄方对上述指标的影响,可能是其减轻EAE神经损伤,改善患者临床症状的部分机理所在。

Abstract:

Objective: To observe the immunohistochemical expression of interferon-γ(IFN-γ),transforming growth factor-β(TGF-β),matrix metalloproteinase-9(MMP-9) and lymphocyte subsets in brain and spinal cord tissue of experimental autoimmune encephalomyelitis(EAE) rats,and explore the mechanism of Erhuang Capsule,comparing the differences in action for the two dosage forms(capsule and decoction). Method: Lewis rats were immunized with the myelin basic protein (MBP). Rats were divided randomly into nomal-group,EAE-group,prednisone-group,Erhuang Decoction group and Erhuang Capusule group after post immunization (PI). On the day 15^th and the day 27^th of PI,rats were killed and the immunohistochemical staining of IFN-γ、TGF-β、MMP-9、CD4⁺、CD8⁺、CD3⁺ and CD19⁺was performed on the sections of brain and spinal cord. Result: Compared with the EAE-group,Erhuang Decoction (on day 27^th PI)and Erhuang Capusule(on day 15 and 27 PI)both can raise the expression of TGF-β in brain tissue. Erhuang Decoction, Erhuang Capusule and prednisone can decrease the expression of IFN-γ and MMP-9 in brain tissue and spinal cord tissue on day 15^th and 27^th PI;compared with the prednisone,Erhuang Decoction and Erhuang Capusule can decrease the expression of IFN-γ in brain tissue and spinal cord tissue on day 15^th and 27^th PI,they can also decrease the expression of MMP-9 in brain tissue and spinal cord tissue on day 15^th PI. Compared with the EAE-group,the expresstion of CD4⁺in brain tissue of Erhuang Decoction group was significantly lower than that of EAE-group on day 15^th and 27^th PI,while the expresstion of CD8⁺raised and CD4⁺/CD8⁺decreased(The expresstion of CD19⁺ decreased on day 15^th PI). On day 15th PI,there is no difference in the expression of CD8⁺between the Erhuang Capusule group and EAE-group,others were the same as Erhuang Decoction group. Compared with the EAE-group,the expresstion of CD4⁺in spinal cord tissue of Erhuang Capusule group was significantly lower than that of EAE-group on day 15^th,while the expresstion of CD8⁺raised and CD4⁺/CD8⁺decreased,on day 27^th PI,the expresstion of CD4⁺and CD4⁺/CD8⁺ was decreased. There was no statistical difference between Erhuang Decoction and Erhuang Capusule. Conclusion: Erhuang Decoction and Erhuang Capusule both have the effect on upregulating the immunohistochemical expression of TGF-β in central nervous system of EAE rats,playing the same role as prednisone. They can also downregulate the expression of IFN-γ and MMP-9,are better than prednisone. Erhuang Decoction and Erhuang Capusule also can downregulate the expression of CD4⁺and upregulate the expression of CD8⁺,to improve the ratio of CD4⁺/CD8⁺. The impact on indexs described above may be the functional mechanism of Erhuang Decoction and Erhuang Capusule. There was no significant difference between Erhuang Decoction and Erhuang Capusule.