摘要
目的:
本研究利用急性大鼠实验性自身免疫性脑脊髓炎(EAE)的动物模型和星形胶质细胞和脾脏淋巴细胞体外培养模型,观察青藤碱对模型的神经功能评分、病理改变及炎症因子表达的影响,并探讨其对EAE的预防治疗作用及可能机制,为临床应用治疗MS提供基础实验依据。
方法:
1.动物实验部分
(1).EAE模型制备:用髓鞘碱性蛋白(myelin basic protein,MBP)肽段68-82(MBP68-82)、含结核杆菌的完全弗氏佐剂(complete Freund's adguvant,CFA)和百日咳毒素(Pertussis Toxin,PTX)免疫Lewis大鼠,建立EAE模型。
(2).实验分组:本实验分为5组:模型对照组(Control组)、模型组(EAE组)、50mg/kg/d青藤碱治疗组(SIN50)、100mg/kg/d青藤碱治疗组(SIN100)、200mg/kg/d青藤碱治疗组(SIN200)。从免疫的第-1天至第3天连续5天腹腔注射不同剂量的SIN治疗EAE大鼠,观察21天并进行临床评价。
(3).青藤碱治疗作用评价及机制探讨:通过行为观察记录神经功能评分、体重改变;HE染色评价脊髓组织单个核细胞浸润;3H-TdR法检测脾细胞的增殖反应和ELISA法检测细胞因子分泌水平;用RT-PCR法和Western Blot法检测脊髓组织中T-bet、IFN-γ、iNOS mRNA和蛋白表达变化。
2.细胞实验部分
(1).建立星形胶质细胞体外培养诱生iNOS体系:用IFN-γ(10ng/mL)和TNF-α(10 ng/mL)共同诱导,用SIN (1mM)或iNOS选择性抑制剂L-canavanine (1mM)干预,6小时RT-PCR法检测iNOS mRNA水平,12小时Western Blot法检测其蛋白表达,以探讨青藤碱对iNOS是否有直接抑制作用。
(2).建立脾细胞体外培养诱生T-bet/IFN-γ体系:用抗CD3抗体(2μg/ml)和重组IL-12(10ng/ml)刺激体外培养的脾细胞,用SIN (1mM)干预,24小时RT-PCR法检测脾细胞T-bet/IFN-γmRNA水平,48小时Western Blot法检测T-bet蛋白表达,ELISA法检测培养上清中IFN-γ水平。
(3).建立脾细胞培养上清对星形胶质细胞诱生iNOS体系:以抗CD3(2μg/ml)和重组IL-12(10ng/ml)刺激体外培养的脾细胞,加或不加青藤碱处理。再以此培养上清作为刺激剂加入星形胶质细胞培养中,观察其对星形胶质细胞iNOS的影响。
结果:
1.动物实验部分
(1).EAE模型发病情况及临床评价:EAE组大鼠免疫后第7天开始发病,第15天达高峰,发病表现为:体重减轻、尾部无力、肢体瘫痪、甚至呈濒死状态,而正常对照组无一发病。青藤碱治疗组大鼠发病高峰期较EAE组延后1天,为免疫后第16天,发病大鼠临床表现与EAE组类同,但病情程度较EAE组轻。EAE组高峰期神经功能评分明显高于青藤碱治疗组(P<0.05 )。
(2).组织病理学改变:HE染色显示EAE组脊髓组织内有大量单个核细胞浸润,血管周围炎性浸润明显,多数血管周围的浸润呈“袖套样”改变。而SIN治疗组脊髓组织淋巴细胞浸润及血管周围炎性细胞浸润随剂量依赖性减轻。
(3).脾细胞抗原MBP刺激增殖反应及炎性细胞分子分泌水平:特异性抗原MBP68-82刺激EAE组脾细胞,增殖反应明显高于未刺激对照组;SIN各剂量组对EAE大鼠脾细胞增殖均有抑制作用(P<0.05)。与正常对照组相比,MBP68-82再次活化的EAE组脾细胞培养上清中IFN-γ和TNF-α分泌水平显著增高;而青藤碱能剂量依赖性地抑制特异性抗原活化的脾细胞分泌炎性细胞因子IFN-γ和TNF-α(P<0.05)。
(4).脊髓组织中T-bet、IFN-γ、iNOS mRNA和蛋白表达变化:MBP诱导的大鼠EAE模型脊髓组织的T-bet、IFN-γ、iNOS mRNA和蛋白水平都较正常对照组显著增高。与EAE组比较,50mg/kg SIN组不能降低脊髓组织中IFN-γ的mRNA和蛋白水平,但100mg/kg、200mg/kg SIN组可有效抑制脊髓组织中IFN-γ的mRNA和蛋白水平,分别抑制了21.32%和55.29%。同样,100mg/kg、200mg/kg SIN组均可有效抑制脊髓组织中T-bet的mRNA和蛋白水平(P<0.05)。50mg/kg、100mg/kg、200mg/kg SIN组均可有效抑制脊髓组织中iNOS的mRNA和蛋白水平(P<0.05)。
2.细胞实验部分
(1).青藤碱对大鼠体外培养星形胶质细胞诱生iNOS的影响:用IFN-γ和TNF-α共同刺激星形胶质细胞,可诱导出iNOS mRNA和蛋白的高表达,但青藤碱不能直接抑制由炎性细胞因子诱导的iNOS的产生。而iNOS选择性抑制剂干预细胞可显著抑制iNOS mRNA和蛋白表达水平。
(2).青藤碱对大鼠体外培养脾细胞诱生T-bet/ IFN-γ的影响:正常初始脾细胞不加任何刺激,T-bet和IFN-γ的表达量水平较低,当用抗CD3抗体和向Th1细胞分化的细胞因子IL-12共同刺激时,T-bet和IFN-γ的产生可显著增加。在培养体系中用SIN(1mM)预处理细胞,发现青藤碱可抑制T-bet和IFN-γ的mRNA和蛋白的表达。
(3).青藤碱对抗CD3抗体和IL-12处理的脾细胞培养上清诱导星形胶质细胞产生iNOS的影响:用抗CD3抗体和细胞因子IL-12刺激脾细胞培养的上清可诱导星形胶质细胞产生高水平的iNOS,当在脾细胞培养体系中加用青藤碱预处理,脾细胞培养上清诱导星形胶质细胞表达iNOSmRNA和蛋白的能力均被抑制。
结论:
1.以髓鞘碱性蛋白肽段MBP68-82、含结核杆菌的完全弗氏佐剂和百日咳毒素为抗原混合物共同免疫Lewis大鼠建立了急性EAE模型,该模型大鼠的发病时间、体重变化、发病时行为改变、临床表现、神经功能评分及病理学改变均与文献报道一致,能有效模拟MS患者的急性发作。
2.青藤碱改善EAE大鼠的病情、降低发病时临床功能评分、抑制炎性细胞向CNS系统异常浸润;青藤碱可抑制抗原MBP活化的脾细胞增殖反应及分泌炎性细胞因子IFN-γ和TNF-α的能力,从而发挥对EAE的免疫抑制作用。
3.青藤碱有效抑制T-bet/IFN-γ/iNOS轴,从而缓解脱髓鞘症状。青藤碱抑制iNOS的机制并不是直接抑制星形胶质细胞产生iNOS,而是通过抑制淋巴细胞产生的iNOS诱生剂IFN-γ,而这一作用与青藤碱抑制Th1特异性转录因子T-bet的表达有关。
Objective:
This study was designed to explore the effect of Sinomenine on the treatment of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. To understand the possible mechanism about the effect of Sinomenine on EAE with animal model in vivo including clinical evaluation, histopathology and expression of cytokine and in vitro using astrocytes and splenocytes culture, so as to approach the Sinomenine's application in clinic.
Methods
1. In vivo
(1) Preparation for EAE model
Females Lewis rats were immunized with MBP68-82 plus complete Freund's adjuvant (CFA) and Pertussis Toxin(PTX)to make up the model.
(2)Groups
Five groups: Control rats,EAE rats,SIN50mg/kg/d treated EAE rats,SIN 100mg/kg/d treated EAE rats,SIN 200mg/kg/d treated EAE rats. The Lewis rats were treated with different doses of SIN with intraperitoneal injection from day -1 to 3 for 5 consecutive days after immunization.
(3)Effects of SIN on EAE and the mechanisms of the effects
The incident condition, clinical score and weight lose were used to evaluated severity of EAE. Histological changes were detected by HE and Luxol fast blue/periodic acid-Schiff (LFB/PAS) stain in spinal cords of each group rats. Splenocytes were isolated and used to test proliferation response with 3H-TdR and cytokine levels of splenocytes supernatant and spinal cord with ELISA. Using RT-PCR to test the expression of T-bet, IFN-γand iNOS mRNA level in spinal cords, T-bet and iNOS protein level were measured with Western Blot.
2. In Vitro
(1) Astrocyte cell culture and iNOS induction
Astrocytes were incubated with IFN-γ(10ng/mL) and TNF-α(10ng/mL) to induce expression of iNOS with or without SIN (1mM) or selective iNOS inhibitor, L-canavanine (1mM). SIN (1mM) or L-canavanine (1mM) was added 30 minutes ahead. These cells were cultured for 6 hours for RT-PCR and 12 hours for Western Blot.
(2) Splenocyte culture and T-bet, IFN-γinduction
Na?ve splenocytes were stimulated with plate-coated anti-CD3 antibody (2μg/ml) and recombinant rat IL-12 (10ng/ml). To determine the effects of SIN, the cells were pretreated with SIN (1mM) before being added to the plates for 30 minutes. After 48 hours, the culture supernatants were collected and assayed for IFN-γproduction by ELISA. Cells were analyzed for IFN-γand T-bet mRNA by RT-PCR at 24 hours and T-bet protein by western blot at 48 hours.
(3) The supernatants of rat splenocytes stimulated with anti-CD3 antibody (2μg/ml) and rmIL-12 (10ng/ml) in the presence and absence of SIN (1mM) were collected and then used to induce iNOS production by primary astrocytes. After being cultured for either 6 hours or 12 hours, the astrocytes were collected for preparation of RNA and protein extracts. RT-PCR was performed with the cellular RNA extracts to determine the expression levels of iNOS mRNA. Western blot was performed with the cellular protein extracts to assay the level of iNOS protein.
Results:
1. In vivo:
(1)The condition of the incident and clinical evaluation in each group rats
The rats in EAE group begin to fall ill after 7 days, the fiftieth day to the peak. The clinic manifestations are: losing weight, limbs palsy, even on the brink of death. The rats in control group have no incidence of EAE. The peak time of SIN treated group delayed 1 day, on the sixth day after immunization. The condition is likely the EAE group, but the clinical situation is better than the EAE group and the clinical scores are significantly reduced compared with EAE group (p<0.05).
(2) Histopathology change in each group rats
HE stain showed inflammatory cell infiltration is obviously in EAE rats, the number of mononuclear cells in spinal cords surrounding vascellum in EAE rats significantly increased. The spinal cords from SIN-treated rats showed reduced infiltration of inflammatory cells in the lumbar segments.
(3)Proliferation responses by stimulation with MBP68-82 and secretion of IFN-γand TNF-αin the supernatants of splenocytes in each group rats
Compared with unstimulated splenocytes of EAE rats, proliferation responses by MBP68-82 were obviously increased. Compared with EAE rats, proliferation of splenocytes in SIN treated rats were significantly inhibited (P<0.05). Besides, SIN also inhibited secretion of IFN-γand TNF-αin splenocytes supernatant (P<0.05).
(4) T-bet、IFN-γ、iNOS mRNA and protein levels in each group rats
MBP-induced EAE resulted in high expression levels of T-bet, IFN-γand iNOS compared with control rats. Compared with EAE rats, 50 mg/kg, 100 mg/kg and 200 mg/kg SIN inhibited the iNOS mRNA and protein level in spinal cord, but only 100 mg/kg and 200 mg/kg SIN blocked the T-bet and IFN-γmRNA and protein levels in spinal cord.
2. In Vitro
(1) iNOS induction in primary astrocytes culture
The primary astrocytes stimulated with combined IFN-γand TNF-αhad a high expression of iNOS mRNA and protein levels compared unstimulated cells. However, the levels of iNOS in the primary astrocytes cultures were reduced by L-canavanine but not by SIN (1mM).
(2) T-bet/ IFN-γinduction in primary splenocytes culture
The splenocytes cultured with anti-CD3 antibody and rmIL-12 in the presence or absence of SIN (1mM). Na?ve splenocytes expressed T-bet and IFN-γat basal levels. However, stimulation with CD3 antibody under polarized conditions with rmIL-12 induced their expression significantly. Treatment with SIN (1mM) markedly inhibited the expression of both IFN-γand T-bet.
(3) The ability of the splenocytes supernatants treated with anti-CD3 antibody and rmIL-12 in the presence of SIN to induce iNOS production by primary astrocytes is reduced
The supernatants from splenocytes stimulated with anti-CD3 antibody and rmIL-12 in the presence of SIN (1mM) induced significantly lower production of iNOS by primary astrocytes than stimulated only with anti-CD3 antibody and rmIL-12, without SIN (1mM).
Conclusion
1. EAE induced with peptide MBP68-82, complete Freund's adguvant and Pertussis Toxin in Lewis rats, whose incidence condition, mean day of onset, disease progression and severity were coincide with with many other reports.
2. SIN could effectively ameliorate EAE by reducing clinical severity and inflammatory infiltration, proliferation of antigen specific T cells and inflammatory cytokines, which exserted an immunesuppression property.
3. SIN could inhibit T-bet/IFN-γ/iNOS axis in spinal cord. The anti-iNOS effect of SIN is not associated with the directly suppression iNOS induction of astrocytes but correlated with its inducer IFN-γblocking, which resulted from the inhibition of the transcription factor T-bet.
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