海风藤提取物对激活的小胶质细胞IL-1β和TNF-α表达的影响

Effects of Piper Kadsura Ohwi extracts on IL-1β and TNF-α expressions in activated microglia

目的研究海风藤提取物对不同聚集状态的β淀粉样蛋白1-42(Aβ1-42)诱导小胶质细胞中炎性因子白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)表达的影响。方法体外培养小鼠小胶质细胞株BV2,制备寡聚体和纤丝状Aβ1-42,应用四甲基偶氮唑蓝(MTT)比色法检测不同浓度的海风藤提取物对BV2细胞活力的影响;BV2细胞分为4个实验组,分别采用寡聚体Aβ1-42、寡聚体Aβ1-42+海风藤提取物、纤丝状Aβ1-42、纤丝状Aβ1-42+海风藤提取物干预48 h,并设空白对照组,应用实时荧光定量PCR测定各组IL-1β和TNF-α的表达水平。结果海风藤提取物在10～80 g/L范围内对细胞活性无影响;实时荧光定量结果显示,4个实验组的小胶质细胞表达IL-1β和TNF-α的水平均高于空白对照组。寡聚体Aβ1-42+海风藤提取物组IL-1β和TNF-α的表达水平低于寡聚体Aβ1-42组,纤丝状Aβ1-42+海风藤提取物组IL-1β和TNF-α的表达水平低于纤丝状Aβ1-42组。结论炎性指标IL-1β和TNF-α的表达增高证明寡聚体和纤丝状Aβ1-42均能激活小胶质细胞;IL-1β和TNF-α的表达降低,表明海风藤提取物可以抑制寡聚体和纤丝状Aβ1-42对小胶质细胞的激活。

Objective To explore the inhibitory effect of Piper Kadsura Ohwi extracts on the releases of proinflammatory cytokines, IL-1β and TNF-a, in microglia cells which were activated by Aβ142 with different aggregation state. Methods Mice microglia cell line BV2 was cultured in vitro. Consistent soluble oligomeric and fibrillar Aβ1.42 were prepared. The cytotoxicity of different extracts from Piper Kadsura Ohwi was assessed by methyl thiazolyl tetrazolium （MTT）. BV2 cells were divided into 4 experimental groups, intervened respectively by oligomeric Aβ1-42, oligomeric Aβ1-42 ＋ Piper Kadsura Ohwi extracts, fibrillar Aβ1-42 and fibrillar Aβ1-42 ＋ Piper Kadsura Ohwi extracts. Besides, a blank control group was set up without any intervention. Expressions of IL-β1 and TNF-a were assessed by Real-time PCR. Results There was no effect on the survival of BV2 cells with various concentrations （ 10-80 g/L） of Piper Kadsura Ohwi extracts. Real-time PCR showed that each of the 4 experimental groups had higher expressions of IL-1β and TNF-a, compared with blank control group. The oligomeric Aβ1-42 ＋ Piper kadsura Ohwi extract group had lower expressions of IL-1 β and TNF-a than the oligomeric Aβ1-42 group; likewise, the fibrillar Aβ1-42 ＋ Piper Kadsura Ohwi extract group had lower expressions of IL-1β and TNF-a than the fibrillar Aβ1-42 group. Conclusion Both fibrillar and oligomeric A13142 can activate the microglia cell BV2, and their activating effects can be inhibited by Piper Kadsura Ohwi extracts.