National Brain Research Centre, Manesar, Haryana-122050, India.
J Neuroinflammation. 2010 Oct 15;7:68. doi: 10.1186/1742-2094-7-68.
Activation of microglia, the resident macrophages of the central nervous system (CNS), is the hallmark of neuroinflammation in neurodegenerative diseases and other pathological conditions associated with CNS infection. The activation of microglia is often associated with bystander neuronal death. Nuclear factor-κB (NF-κB) is one of the important transcription factors known to be associated with microglial activation which upregulates the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and other pro-inflammatory cytokines. Recent studies have focused on the role of Krüppel-like factor 4 (Klf4), one of the zinc-finger transcription factors, in mediating inflammation. However, these studies were limited to peripheral system and its role in CNS is not understood. Our studies focused on the possible role of Klf4 in mediating CNS inflammation.
For in vitro studies, mouse microglial BV-2 cell lines were treated with 500 ng/ml Salmonella enterica lipopolysacchride (LPS). Brain tissues were isolated from BALB/c mice administered with 5 mg/kg body weight of LPS. Expressions of Klf4, Cox-2, iNOS and pNF-κB were evaluated using western blotting, quantitative real time PCR, and reverse transcriptase polymerase chain reactions (RT-PCRs). Klf4 knockdown was carried out using SiRNA specific for Klf4 mRNA and luciferase assays and electromobility shift assay (EMSA) were performed to study the interaction of Klf4 to iNOS promoter elements in vitro. Co-immunoprecipitation of Klf4 and pNF-κB was done in order to study a possible interaction between the two transcription factors.
LPS stimulation increased Klf4 expression in microglial cells in a time- and dose-dependent manner. Knockdown of Klf4 resulted in decreased levels of the pro-inflammatory cytokines TNF-α, MCP-1 and IL-6, along with a significant decrease in iNOS and Cox-2 expression. NO production also decreased as a result of Klf4 knockdown. We found that Klf4 can potentially interact with pNF-κB and is important for iNOS and Cox-2 promoter activity in vitro.
These studies demonstrate the role of Klf4 in microglia in mediating neuroinflammation in response to the bacterial endotoxin LPS.
小胶质细胞是中枢神经系统（CNS）的固有巨噬细胞，其激活是神经炎症的标志，存在于神经退行性疾病和其他与 CNS 感染相关的病理状况中。小胶质细胞的激活通常与旁观者神经元死亡有关。核因子-κB（NF-κB）是与小胶质细胞激活相关的重要转录因子之一，它上调诱导型一氧化氮合酶（iNOS）、环氧化酶-2（Cox-2）和其他促炎细胞因子的表达。最近的研究集中在锌指转录因子之一 Krüppel 样因子 4（Klf4）在介导炎症中的作用。然而，这些研究仅限于外周系统，其在 CNS 中的作用尚不清楚。我们的研究集中在 Klf4 在介导 CNS 炎症中的可能作用。
对于体外研究，用 500ng/ml 沙门氏菌 enterica 脂多糖（LPS）处理小鼠小胶质细胞 BV-2 细胞系。用 5mg/kg 体重 LPS 处理 BALB/c 小鼠分离脑组织。用 Western blot、实时定量 PCR 和逆转录聚合酶链反应（RT-PCR）评估 Klf4、Cox-2、iNOS 和 pNF-κB 的表达。使用针对 Klf4 mRNA 的 siRNA 进行 Klf4 敲低，并进行荧光素酶测定和电泳迁移率变动分析（EMSA），以研究 Klf4 与 iNOS 启动子元件在体外的相互作用。进行 Klf4 和 pNF-κB 的共免疫沉淀，以研究这两个转录因子之间可能的相互作用。
LPS 刺激以时间和剂量依赖的方式增加小胶质细胞中 Klf4 的表达。Klf4 敲低导致促炎细胞因子 TNF-α、MCP-1 和 IL-6 的水平降低，同时 iNOS 和 Cox-2 的表达也显著降低。NO 产生也因 Klf4 敲低而减少。我们发现，Klf4 可以与 pNF-κB 潜在相互作用，并在体外对 iNOS 和 Cox-2 启动子活性很重要。
这些研究表明，Klf4 在小胶质细胞中在对细菌内毒素 LPS 的反应中介导神经炎症。
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