虫草素抑制脂多糖诱导的小胶质细胞活化及神经保护作用

Cordycepin Inhibits Microglial Activation Induced by Lipopolysaccharide and Provides Neuroprotection

目的:研究虫草素抑制脂多糖（Clipopolysaccharides,LPS）诱导小胶质细胞激活及其神经保护作用。方法:培养原代小鼠小胶质细胞,以LPS激活小胶质细胞使NO大量释放,同时给予不同浓度的虫草素（0.110μmol/L）处理,四甲基偶氮唑蓝（3-（4,5-dimethyl-2-thiazolyl）-2,5-Biphenyl-2-H-tetrazolium bromide,MTT）法检测细胞存活率,Griess法测定小胶质细胞NO释放,逆转录聚合酶链式反应法检测细胞内诱导型一氧化氮合酶（inducible nitric oxide synthase,i NOS）的mRNA表达。以硝普钠作为NO供体,以1,1-二苯基苦基苯肼（1,1-Biphenyl-2-picrylhydrazyl,DPPH）自由基作为自由基的供体,分别考察虫草素对NO和DPP日自由基的直接清除作用。分别以H2O2和以LPs激活的小胶质细胞条件培养液损伤小鼠PC12神经元细胞,MTT法考察虫草素对PC12细胞损伤的保护作用。此外,以羟胺法测定超氧化物歧化酶（superoxide dismutase,SOD）活性。结果:虫草素能够显著抑制LPS激活的原代小鼠小胶质细胞NO产生及iN05 mRNA表达,同时不影响细胞的存活率;虫草素具有显著的NO清除和DPPH自由基清除作用;虫草素本身对PC12小鼠神经元细胞的生长没有显著影响,但能够显著抑制H2O2或活化小胶质细胞的条件培养液引起的PC12细胞损伤。此外,虫草素可显著提高H2O2损伤的PC12细胞中SOD活性。结论:虫草素可通过抑制iNOS转录和直接清除NO而抑制LPS激活小胶质细胞的NO产生;虫草素还可通过抑制小胶质细胞激活而对PC12神经元细胞产生保护作用,也可通过提高SOD活性而保护H2O2损伤的PC12细胞。因此,虫草素可能对与小胶质细胞激活相关的神经退行性疾病具有潜在的防治作用。

Objective:To evaluate the inhibition of cordycepin on microglial activation induced by lipopolysaccharide(LPS)and the protection of neuronal cells by cordycepin.Methods:The effect of cordycepin(0.1-10 μmol/L) on cell viability was evaluated by MTT assay.Nitrite levels in culture supernatants were examined by Griess assay.The mRNA expression of inducible nitric oxide synthase(iNOS) was evaluated by RT-PCR.The NO-scavenging and DPPH free radical-scavenging activities of cordycepin were measured using SNP as a NO donor and DPPH as a free radical donor,respectively.The effect of cordycepin on the decreased viability of PC12 neurons induced by H2O2 or conditioned medium obtained from LPSactivated microglial cells was evaluated by MTT assay.In addition,the superoxide dismutase(SOD) activity was examined by hydroxylamine method.Results:Cordycepin could inhibit the production of NO and the mRNA expression of iNOS in LPS-activated primary mouse microglial cells obviously.However,treatment with cordycepin(0.1-10 mol/L) had no significant cytotoxicity.Cordycepin showed significant scavenging activity towards nitric oxide radical(NO) and DPPH free radicals.Cordycepin alone did not influence the viability of PC12 neurons;however,it improved the decreased viability of PC12 neurons induced by H2O2 or conditioned medium from LPS-activated microglia.In addition,the SOD activity in H2O2-treated PC12 cells could be upgraded.Conclusion:It is suggested that cordycepin can inhibit LPS-induced NO production by blocking the transcriptional levels of iNOS in microglial cells.Cordycepin can protect PC12 neurons from toxic influence by activated microglia,and protect H2O2-impaired PC12 cells by improving SOD activity.Cordycepin may have therapeutic potential for the treatment of neurodegenerative diseases accompanied by microglial activation.