雷公藤多甙对实验性自身免疫性脑脊髓炎大鼠保护作用的探讨
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摘要
目的:
建立实验性自身免疫性脑脊髓炎(EAE)动物模型,观察大鼠脊髓中caspase-3和IFN-γ的表达,探讨雷公藤多甙对EAE大鼠保护作用,为多发性硬化的治疗寻找有效途径。
方法:
将60只雌性Wistar大鼠随机分成四组:正常组(CON组)、EAE模型组(EAE组)、TWP1组(EAE+TWP10mg/kg.d)、TWP2组(EAE+TWP30mg/kg.d)。用豚鼠脊髓匀浆(GPSCH)制备EAE模型。CON组不予任何处理,在免疫后第21天全部处死。EAE组、TWP1组和TWP2组大鼠在抗原免疫后根据处死时间不同,各自在组内又分为第7天、第14天、第21天处死的三个亚组,从免疫后第1天开始,分别每天给予药物干预一次,直到处死。每天观察各组大鼠发病情况并进行神经功能评分。取大鼠脊髓组织切片进行苏木素-伊红(hematoxylin-eosin,HE)染色,在光镜下进行炎性病灶计数。免疫组化方法检测各组大鼠脊髓组织中caspase-3和IFN-γ的表达情况。
结果:
1.动物发病率观察:CON组无大鼠发病; EAE组发病率为86.67%;TWP1组和TWP2组动物发病率明显低于EAE组(P <0.05);TWP2组发病率低于TWP1组发病率(P <0.05)。
2.行为学观察:在免疫后第7天、第14天、第21天分别进行神经功能评分,并进行比较,在第14天、第21天TWP1、TWP2组评分均小于EAE组(P <0.05),第14天TWP2组神经功能评分小于TWP1组(P <0.05)。各组三个时间点比较,7天组、21天组与14天组相比较有统计学差异(P <0.05);7天与21天组比较无统计学差异(P >0.05)。
3.组织病理学观察:CON组大鼠脊髓内无炎性细胞浸润;在疾病各期EAE组、TWP1组和TWP2组,大鼠脊髓中均存在炎性细胞浸润。第7天、第14天、第21天,TWP1组和TWP2组炎症细胞浸润形成的血管袖套数目少于同期的EAE组(P <0.05); TWP2组少于同期TWP1组,差异有统计学意义(P <0.05)。各组中三个时间点比较,7天组、21天组与14天组相比较有统计学差异(P <0.05);7天与21天组比较无统计学差异(P >0.05)。
4.脊髓caspase-3和IFN-γ表达:CON组脊髓无caspase-3和IFN-γ阳性表达; EAE组、TWP1组、TWP2组均存在caspase-3、IFN-γ的阳性表达。阳性细胞数比较:TWP1组、TWP2组caspase-3和IFN-γ表达低于同期EAE组(P <0.05);TWP2组与同期的TWP1组比较,第14天TWP2组中阳性细胞数目低于TWP1组,差异有统计学意义(P <0.05)。各组中三个时间点比较,7天组、21天组与14天组相比较有统计学差异(P <0.05);7天与21天组比较无统计学差异(P >0.05)。
结论:
雷公藤多甙干预对EAE大鼠有保护作用,可减轻EAE大鼠发病,并呈现剂量相关性,其机制可能与减少EAE大鼠脊髓内caspase-3和IFN-γ表达有关。
Objective To establish the animal model of experimental autoimmune encephalomyelitis(EAE) and observe the expression of caspase-3 and IFN-γin spinal cord of EAE rats, explore the possible protective function mechanism of Tripterygium wilfordii Ployglycosidium (TWP) to EAE rats, seek for the efficient path of multiple sclerosis's treatment .
Methods Sixty female Wistar rats were randomly divided into four groups: normal control group (CON), EAE mode group (EAE), TWP1 group, TWP2 group. The EAE animal model was established in rats by immunizing rats with guinea pig spinal cord homogenate (GPSCH) and complete freund’s adjuvant(CFA). The CON group rats were sacrificed in the 21st day. The animals of other groups were divided into three subgroups :the 7th day sacrificed ,the 14th sacrificed and the 21st sacrificed. The Rats were fed with TWP(10mg/kg.d) in TWP1 group, TWP (30mg/kg.d) in TWP2 group , starting from the 1st day after immunization to the day rats were Sacrificed. Nerve function scores of rats were examined daily. Histological examinations were performed on the sections of spinal cord with the aid of hematoxylin- eosin staining. The number of inflammation in the central nervous system (CNS) was counted under the optical microscope .And the expressions of caspase-3 and IFN-γin spinal cord were detected by using immunohistochemistry technique.
Results
1.Incidence of a disease: No ill rats in CON group .The incidence of EAE group was 86.67%. The incidence of TWP1 and TWP2 groups was lower than that of the EAE group (P <0.05), the incidence of TWP2 group was lower comparing with the TWP1 group (P<0.05).
2.Ethology appraisal:The neurological function score of TWP1 and TWP2 groups was lower than that of the EAE group at the 14th day and the 21th day (P <0.05), and that of the 14th day of TWP2 group was lower comparing with the TWP1 group(P <0.05). Comparing the three time points in each group, the 7th day of group, the 21st day of group compared with the 14th day of group was significantly different (P <0.05); the 7th day and the 21st day of group, no significant difference between groups (P > 0.05).
3.Histopathological findings: There was no inflammatory cell infiltration of the CON group, but the inflammatory cell was found in the spinal cord of the each stage group of EAE group and TWP1、TWP2 group. The nurnber of inflammatory cell of TWP1、TWP2 group was fewer than that of the EAE group at the 14th day and the 21th day (P <0.05),and that of the TWP2 group was lower than the TWP1 group (P <0.05). Comparing the three time points in each group, the 7th day of group, the 21st day of group compared with the 14th day of group was significantly different (P <0.05); the 7th day and the 21st day of group, no significant difference between groups (P > 0.05).
4.Caspase-3 and IFN-γexpression: There was no caspase-3 and IFN-γcells in spinal cord of the CON group; but the caspase-3 and IFN-γcell could be seen in the spinal cord of the EAE group and TWP1、TWP2 group,and the number of caspase-3 and IFN-γcell of TWP1、TWP2 group was fewer than that of the EAE group (P <0.05),that of the 14th day of TWP2 group was lower than the TWP1 group (P <0.05). Comparing the three time points in each group, the 7th day of group, the 21st day of group compared with the 14th day of group was significantly different (P <0.05); the 7th day and the 21st day of group, no significant difference between groups (P > 0.05).
Conclusion
TWP intervention has a protective effect on EAE in rats, reduce the incidence of EAE rats and showed dose-dependent, which may reduce the EAE rat spinal cord with caspase-3 and IFN-γexpression.
建立实验性自身免疫性脑脊髓炎(EAE)动物模型,观察大鼠脊髓中caspase-3和IFN-γ的表达,探讨雷公藤多甙对EAE大鼠保护作用,为多发性硬化的治疗寻找有效途径。
方法:
将60只雌性Wistar大鼠随机分成四组:正常组(CON组)、EAE模型组(EAE组)、TWP1组(EAE+TWP10mg/kg.d)、TWP2组(EAE+TWP30mg/kg.d)。用豚鼠脊髓匀浆(GPSCH)制备EAE模型。CON组不予任何处理,在免疫后第21天全部处死。EAE组、TWP1组和TWP2组大鼠在抗原免疫后根据处死时间不同,各自在组内又分为第7天、第14天、第21天处死的三个亚组,从免疫后第1天开始,分别每天给予药物干预一次,直到处死。每天观察各组大鼠发病情况并进行神经功能评分。取大鼠脊髓组织切片进行苏木素-伊红(hematoxylin-eosin,HE)染色,在光镜下进行炎性病灶计数。免疫组化方法检测各组大鼠脊髓组织中caspase-3和IFN-γ的表达情况。
结果:
1.动物发病率观察:CON组无大鼠发病; EAE组发病率为86.67%;TWP1组和TWP2组动物发病率明显低于EAE组(P <0.05);TWP2组发病率低于TWP1组发病率(P <0.05)。
2.行为学观察:在免疫后第7天、第14天、第21天分别进行神经功能评分,并进行比较,在第14天、第21天TWP1、TWP2组评分均小于EAE组(P <0.05),第14天TWP2组神经功能评分小于TWP1组(P <0.05)。各组三个时间点比较,7天组、21天组与14天组相比较有统计学差异(P <0.05);7天与21天组比较无统计学差异(P >0.05)。
3.组织病理学观察:CON组大鼠脊髓内无炎性细胞浸润;在疾病各期EAE组、TWP1组和TWP2组,大鼠脊髓中均存在炎性细胞浸润。第7天、第14天、第21天,TWP1组和TWP2组炎症细胞浸润形成的血管袖套数目少于同期的EAE组(P <0.05); TWP2组少于同期TWP1组,差异有统计学意义(P <0.05)。各组中三个时间点比较,7天组、21天组与14天组相比较有统计学差异(P <0.05);7天与21天组比较无统计学差异(P >0.05)。
4.脊髓caspase-3和IFN-γ表达:CON组脊髓无caspase-3和IFN-γ阳性表达; EAE组、TWP1组、TWP2组均存在caspase-3、IFN-γ的阳性表达。阳性细胞数比较:TWP1组、TWP2组caspase-3和IFN-γ表达低于同期EAE组(P <0.05);TWP2组与同期的TWP1组比较,第14天TWP2组中阳性细胞数目低于TWP1组,差异有统计学意义(P <0.05)。各组中三个时间点比较,7天组、21天组与14天组相比较有统计学差异(P <0.05);7天与21天组比较无统计学差异(P >0.05)。
结论:
雷公藤多甙干预对EAE大鼠有保护作用,可减轻EAE大鼠发病,并呈现剂量相关性,其机制可能与减少EAE大鼠脊髓内caspase-3和IFN-γ表达有关。
Objective To establish the animal model of experimental autoimmune encephalomyelitis(EAE) and observe the expression of caspase-3 and IFN-γin spinal cord of EAE rats, explore the possible protective function mechanism of Tripterygium wilfordii Ployglycosidium (TWP) to EAE rats, seek for the efficient path of multiple sclerosis's treatment .
Methods Sixty female Wistar rats were randomly divided into four groups: normal control group (CON), EAE mode group (EAE), TWP1 group, TWP2 group. The EAE animal model was established in rats by immunizing rats with guinea pig spinal cord homogenate (GPSCH) and complete freund’s adjuvant(CFA). The CON group rats were sacrificed in the 21st day. The animals of other groups were divided into three subgroups :the 7th day sacrificed ,the 14th sacrificed and the 21st sacrificed. The Rats were fed with TWP(10mg/kg.d) in TWP1 group, TWP (30mg/kg.d) in TWP2 group , starting from the 1st day after immunization to the day rats were Sacrificed. Nerve function scores of rats were examined daily. Histological examinations were performed on the sections of spinal cord with the aid of hematoxylin- eosin staining. The number of inflammation in the central nervous system (CNS) was counted under the optical microscope .And the expressions of caspase-3 and IFN-γin spinal cord were detected by using immunohistochemistry technique.
Results
1.Incidence of a disease: No ill rats in CON group .The incidence of EAE group was 86.67%. The incidence of TWP1 and TWP2 groups was lower than that of the EAE group (P <0.05), the incidence of TWP2 group was lower comparing with the TWP1 group (P<0.05).
2.Ethology appraisal:The neurological function score of TWP1 and TWP2 groups was lower than that of the EAE group at the 14th day and the 21th day (P <0.05), and that of the 14th day of TWP2 group was lower comparing with the TWP1 group(P <0.05). Comparing the three time points in each group, the 7th day of group, the 21st day of group compared with the 14th day of group was significantly different (P <0.05); the 7th day and the 21st day of group, no significant difference between groups (P > 0.05).
3.Histopathological findings: There was no inflammatory cell infiltration of the CON group, but the inflammatory cell was found in the spinal cord of the each stage group of EAE group and TWP1、TWP2 group. The nurnber of inflammatory cell of TWP1、TWP2 group was fewer than that of the EAE group at the 14th day and the 21th day (P <0.05),and that of the TWP2 group was lower than the TWP1 group (P <0.05). Comparing the three time points in each group, the 7th day of group, the 21st day of group compared with the 14th day of group was significantly different (P <0.05); the 7th day and the 21st day of group, no significant difference between groups (P > 0.05).
4.Caspase-3 and IFN-γexpression: There was no caspase-3 and IFN-γcells in spinal cord of the CON group; but the caspase-3 and IFN-γcell could be seen in the spinal cord of the EAE group and TWP1、TWP2 group,and the number of caspase-3 and IFN-γcell of TWP1、TWP2 group was fewer than that of the EAE group (P <0.05),that of the 14th day of TWP2 group was lower than the TWP1 group (P <0.05). Comparing the three time points in each group, the 7th day of group, the 21st day of group compared with the 14th day of group was significantly different (P <0.05); the 7th day and the 21st day of group, no significant difference between groups (P > 0.05).
Conclusion
TWP intervention has a protective effect on EAE in rats, reduce the incidence of EAE rats and showed dose-dependent, which may reduce the EAE rat spinal cord with caspase-3 and IFN-γexpression.
引文
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[25] Kim S, Moon C, Wie MB, et al. Enhanced exp ression of constitutive and inducible forms of nitric oxide synthase in autoimmune encephalomyelitis [ J ]. J Vet Sci, 2000, 1 (1) : 11 - 17.
[26] Bechtold DA, Smith KJ. Sodium 2 mediate axonal degeneration in inflammatory demyelinating disease [J]. J Neurol Sci, 2005, 233 (1-2) : 27 - 35.
[27] ZhaoW, XieW, LeW, et al. Activatedmicroglia initiatemotor neuron injury by a nitric oxide and glutamate2mediated mechanism [J]. J Neuropathol Exp Neurol, 2004, 63 (9) : 964 - 977.
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[31] Cid C,Alvarez, Cermeno JC, Regidor I, et a1.Caspase inhibitors protect against neuronal apoptosis induced by cerebrospinal fluid from multiple sclerosis patients[J].J Neuroimmunol,2003,136:119-124
[32] Greter M, Heppner F L ,Lemos M P et al . Dendritic cells permit immune invasion of the CNS in an animal model of multiple sclerosis [J] . Nat Med , 2005 ;11 (3) :328-334.
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