桂枝加葛根汤对神经炎症小鼠皮层小胶质细胞活化和神经元保护作用的研究

Study on effects of “Guizhi Plus Gegen Decoction ” on microglial activation and neuronal protection in cortex of neuroinflammatory mice

目的研究桂枝加葛根汤(GGD)对脂多糖(LPS)诱导的神经炎症小鼠皮层小胶质细胞活化及神经元损伤的保护作用及其机制。方法将63只雄性ICR小鼠随机分成5组:正常对照组(Control组,n=13)、模型组(Model组,n=13)、桂枝加葛根汤低剂量组(GGD-low组,n=10)、桂枝加葛根汤高剂量组(GGD-high组,n=14)、二甲基四环素阳性对照组(Positive control组,n=13)。腹腔注射LPS(0.33μg/g)建立阿尔茨海默病(Alzheimer’s disease,AD)神经炎症小鼠模型;GGD-low组(6 g/kg)和GGD-high组(12 g/kg)给予小鼠GGD灌胃治疗,共4周;阳性对照组给予二甲基四环素(0.05 g/kg)腹腔注射,共3天,药物治疗结束后小鼠处死前4 h注射LPS造模。分别采用免疫荧光法观察桂枝加葛根汤对神经炎症小鼠皮层小胶质细胞活化和神经元密度的影响;酶联免疫吸附实验(ELISA)检测皮层促炎细胞因子TNF-α和IL-1β含量。结果 LPS诱导后Model组小鼠神经炎症反应加剧,与Control组相比,皮层小胶质细胞数量增多、胞体肿大、突起回缩但分支增多,活化现象显著,而皮层NeuN阳性神经元密度明显减少,胞浆着色淡,细胞轮廓模糊;桂枝加葛根汤治疗后,与Model组相比,GGD-high组小胶质细胞活化现象明显抑制,数量少,分支也少,而皮层神经元密度增加,尤其GGD-high组密度增加更显著,胞浆染色加深明亮,说明桂枝加葛根汤对皮层的神经元损伤具有一定保护作用。LPS诱导后皮层TNF-α和IL-1β含量与Control组相比显著增高(P<0.05,P<0.01);桂枝加葛根汤治疗后与Model组小鼠比较,GGD-high组小鼠皮层TNF-α含量显著降低(P<0.05),GGD-low和GGD-high组皮层IL-1β含量均明显降低(P<0.05,P<0.01)。结论桂枝加葛根汤对LPS诱导的神经炎症小鼠皮层神经元损伤具有一定保护作用,其机制可能与抑制皮层小胶质细胞活化和调节促炎细胞因子TNF-α和IL-1β含量的作用有关。

Objective To study the effects of＂Guizhi Plus Gegen Decoction＂on microglial activation and neuronal protection in cortex of neuroinflammatory mice induced by LPS,and to explore its mechanism. Methods Sixty-three male ICR mice were randomly divided into five groups： normal control group（ n = 13）,model group（n = 13）,low-dose＂Guizhi Plus Gegen Decoction＂group（GGD-low group,n = 10）,high-dose＂Guizhi Plus Gegen Decoction＂group（GGD-high group,n = 14） and positive control group（ n = 13）. Mice were intraperitoneally injected with LPS（0. 33 mg /kg） to induce neuroinflammatiory model with Alzheimer＇s disease（AD）. Mice in the high and the low dose GGD groups were administered with 6 g /kg and 12 g /kg by gastrogavage for 4 weeks. Mice in the positive control group were intraperitoneally injected with minocycline（0. 05 g /kg） for 3 days. By the end of treatment,LPS were injected in mice 4 h before sacrificed. Immunofluoresent assay was conducted in contex of neuroinflammatory mice to observe the effects of GGD on activated microglia and neuronal density.Enzyme-linked immuno sorbent assay（ELISA） was tested the effects of GGD on proinflammatory cytokine in cortex,i. e,tumor necrosis factor（ TNF-α） and interleukin-1β（IL-1β）. Results After LPS induced,there were intensified neuroinflammatory reaction in the model group. Compared with the control group,the number of microglia was increased,the cell body swelling,protuberant retraction,but the branches were grow. There were significant microglial activation phenomena. While the density of NeuN positive neuron in cortex reduced evidently,and pale cytoplasm shaded,contour is fuzzy. After GGD treatment,microglial activation phenomena were suppressed significantly in cortex of GGD-high group mice,including the decreased number of microglia and branch. However,mice in the low and high dose GGD groups had obvious intensified density of neuron in cortex. Especially in GGD-high mice,the neuronal density in cortex was increased more significantly,cytoplasm staining more deepen and bright than GGD-low mice. The results showed GGD had certain protective effect on cortical neuron injury.Also,LPS raised the level of TNF-α and IL-1β in cortex at some degree（P〈0.05,P〈0.01）. After GGD treatment,the level of TNF-α was obviously lower in cortex of GGD-high mice than the model group mice（P〈0.05）. However,mice in the low and high dose GGD groups had obvious decreased level of IL-1β in cortex（P〈0.05,P〈0.01）. Conclusion GGD has certain protective effects on cortical neuronal injury induced by LPS. Its mechanism might be possibly correlated with its inhibiting effects of GGD on activated microglia and regulatory effects on level of proinflammatory cytokines i. e. TNF-α and IL-1β in cortex.