栀子甙在炎症诱导的多巴胺能神经元损伤中的作用及机制

Effect of gardenoside on the damage of dopaminergic neurons induced by inflammation and its mechanism

目的研究栀子甙在脂多糖（LPS）介导的星形胶质细胞（AC）过度激活时对多巴胺能神经元保护作用的影响及可能机制。方法建立高纯度多巴胺能神经元培养体系、多巴胺能神经元和AC混合培养体系并分别经栀子甙预处理后再予以LPS作用24h，同时设立对照组，观察多巴胺能神经元的生存率，TH mRNA的表达及培养基中TNF-α、NO、IL-6、基质金属蛋白酶-9（MMP-9）和胶质源性神经生长因子（GDNF）含量的变化。结果AC促进多巴胺能神经元的存活。栀子甙不能增加高纯度多巴胺能神经元培养体系中多巴胺能神经元的存活率，却可剂量依赖地增加混合培养体系中多巴胺能神经元存活率，同对照组相比，40mg／L组增加细胞存活率从203．0％±17．4％升高到256．7％±15、2％（F=17．22，P=0．001）。LPS作用24h后混合培养体系中TNF-α、NO、GDNF含量并无显著变化，IL-6、MMP-9含量显著上升，栀子甙可明显下调这种反应，同对照组相比，40mg／L组IL-6含量下降到原来的67．2％±6．4％（F=12．89，P=0．001），MMP-9下降到原来的77．3％±9．8％（F=8．27，P=0．001）。结论LPS通过过度激活AC而增加炎性因子的分泌，从而削弱其对多巴胺能神经元的保护作用，栀子甙通过抑制AC分泌炎性介质，不增加GDNF的分泌，上调AC对多巴胺能神经元的保护作用。

Objective To explore the protective effect and mechanism of gardenoside on the damage of dopaminergic neurons induced by lipopolysaccharide （LPS）. Methods Both neuron-enriched cultures and neuron-astrocyte cultures were pretreated with vehicle or gardenosides （ 10, 20 and 40 mg/L） for 30 min at 37 ℃. The culture media were subsequently renewed in order to remove gardenosides. LPS was then added into all culture media at a final concentration of 10 mg/L. Twenty-four hours later, the culture media was collected to measure TNF-α, NO, IL-6, GDNF and MMP-9; the cells were collected to count the number of cells labeled with an antibody against tyrosine hydroxylase （TH） and to assess the expression of TH mRNA using reverse transcription-polymerase chain reaction. Results Gardenoside didn＇ t promote the survival of dopaminergic neurons in neuron-enriched culture, but significantly increased the survival of dopaminergic neurons in neuron-astrocyte culture, compared with the vehicle group, the survival of dopaminergic neurons increased from 203.0% ± 17.4% to 256. 7% ± 15.2% （ F = 17.22, P = 0. 001 ） in 40 mg/L gardenoside group. The amount of TNF-α, NO and GDNF released from the neuron-astrocyte cultures after 24 h of addition of LPS was not changed significantly, while the expression of IL-6 and MMP-9 was increased significantly. In this study, the gardenoside concentration-dependently attenuated the LPS- induced increase of the expression of IL-6 and MMP-9, compared with the vehicle group, the expression of IL-6 and MMP-9 decreased to 67.2%±6. 4% （ F = 12. 89 ,P = 0.001 ）, 77.3% ± 9. 8% （ F = 8. 27,P = 0. 001 ） respectively in 40 mg/L gardenoside group. Conclusions Astrocytes play a neuroprotective role on dopaminergic neurons, which is decreased by LPS via inducing the secretion of pro-inflammatory factors . Gardenoside may protect dopaminergic neurons from LPS-induced injury in an astrocyte-dependent manner and it inhibits the production of proinflammatory factors instead of promoting the secretion of GDNF. From the point of view that a very low toxicity of gardenosides has been well documented, this report may reveal a new way of developing therapeutic interventions for inflammation-related diseases such as Parkinson＇ s disease.