百日咳毒素减轻实验性自身免疫性脑脊髓炎小鼠模型炎症反应的机制研究

Mechanism of action of pertussis toxin in alleviating inflammatory response in the mouse model of experimental autoimmune encephalomyelitis

目的探讨百日咳毒素(PTx)对实验性自身免疫性脑脊髓炎(EAE)小鼠模型的作用及机制。方法将C57BL/6小鼠随机分为正常对照组、EAE组和PTx治疗组,每组12只。EAE组和PTx治疗组用MOG 35-55诱导EAE模型。PTx组于建模后第7天给予腹腔注射1000 ng的PTx。随后观察两组临床症状,组织学染色评价炎症反应和脱髓鞘改变,并观察VEGF和血管新生现象,并用Western blot检测脊髓VEGF和Collagen IV的整体水平。在体外,用PTx刺激原代培养的神经元,评价PTx对在体外对神经元VEGF表达的影响。结果 PTx能减轻EAE模型的炎症反应和脱髓鞘改变,在大脑PTx将炎症评分从(3.4±0.55)分降至(1.2+0.45)分,P<0.01;脱髓鞘评分从(3.6+0.55)分降至(1.4+0.55)分,P<0.01。在脊髓PTx将炎症评分从(3.6+0.55)分降至(1.0+0.71)分,P<0.01;脱髓鞘评分从(4.2+0.84)分降至(1.4+0.55)分,P<0.01。Western blot检测显示,与正常对照组比较,EAE组VEGF下降49.0%(P<0.01),Collagen IV下降36.0%(P<0.01);PTx治疗后,与EAE组比较,VEGF上升59.5%(P<0.01),Collagen IV上升45.0%(P<0.01)。体外实验显示,PTx治疗24 h后VEGF在神经元上的表达增加,与正常对照相比,100 ng/ml组上调34.6%(P<0.01),400 ng/ml组上调76.9%(P<0.01)。结论 PTx可上调神经元内源VEGF的表达和血管新生现象,继而在EAE模型中起保护作用。

Objective To investigate the role and mechanism of action of pertussis toxin（ PTx） in the mouse model of experimental autoimmune encephalomyelitis（ EAE）. Methods C57 BL /6 mice were randomly divided into normal control group,EAE group,and PTx treatment group,with 12 mice in each group. In the EAE group and PTx treatment group,EAE model was established using MOG 35- 55,and the mice in the PTx treatment group were given 1000 ng intraperitoneally injected PTx at 7 days after modeling. The clinical symptoms were observed in these two groups. Histological staining was used to evaluate inflammatory response and changes in demyelination and observe vascular endothelial growth factor（ VEGF） and angiogenesis. Western blot was used to measure the levels of VEGF and collagen IV. PTx was used to stimulate primary cultured neurons in vitro to assess the in vitro effect of PTx on VEGF expression in neurons. Results PTx alleviated the inflammatory response and demyelination in EAE model,and in the brain,PTx treatment reduced the inflammation score from 3. 4 ± 0. 55 to1. 2 ± 0. 45（ P 0. 01） and the demyelination score from 3. 6 ± 0. 55 to 1. 4 ± 0. 55（ P 0. 01）. In the spinal cord,PTx treatment reduced the inflammation score from 3. 6 ± 0. 55 to 1. 0 ± 0. 71（ P 0. 01） and the demyelination score from 4. 2 ± 0. 84 to 1. 4 ± 0. 55（ P 0. 01）. The results of Western blot showed that compared with the normal control group,the EAE group showed a 49. 0% reduction in VEGF（ P 0. 01）and a 36. 0% reduction in Collagen IV（ P 0. 01）; compared with the EAE group,the PTx treatment group showed a 59. 5% increase in VEGF（ P 0. 01） and a 45. 0% increase in collagen IV（ P 0. 01）. The in vitro experiment showed that at 24 hours after PTx treatment,the expression of VEGF in neurons was upregulated; compared with the normal control group,the 100 ng / ml group showed a 34. 6% increase in the expression of VEGF,and the 400 ng / ml group showed a 76. 9% increase. Conclusions PTx can upregulate endogenous VEGF in neurons and angiogenesis,and thus exerts a protective effect in EAE model.