Neuroinflammation is associated with the progression of multiple neurological diseases. Many studies show that SIRT2 involves in multiple inflammatory processes. While, the mechanisms remain unclear. The purpose of this study was to explore the effect of SIRT2 inhibitor AGK2 on inflammatory responses and MAPK signaling pathways in LPS activated microglia in vitro and in vivo. The effect of AGK2 on cell viability of BV2 microglial cells was detected by CCK-8 assay. The expression of inflammatory cytokine iNOS was analyzed by western blotting and immunofluorescence. The mRNA expressions of iNOS, TNF-α, and IL-1β were detected by real-time polymerase chain reaction (RT-PCR). The SIRT2, phospho-P38, P38, phospho-JNK, JNK, phospho-ERK, ERK, α-tubulin, and acetyl-α-tubulin were analyzed by western blotting respectively. The interaction between SIRT2 and MKP-1 was measured by Co-immunoprecipitation (Co-IP) assay. Double immunofluorescent staining was performed to detect the expressions of CD11b and iNOS or SIRT2 in brain tissues. We found that AGK2 could suppress LPS-induced inflammatory cytokines (iNOS, TNF-α, and IL-1β) expression levels in BV2 microglial cells. Moreover, it could effectively reduce the expression of SIRT2 and increase the acetylation of α-tubulin in LPS activated BV2 microglial cells and LPS induced mice neuroinflammation. In addition, our results showed that AGK2 could reduce the increase of phosphorylation p38, JNK, and ERK after LPS challenge. Co-IP results showed that there was no direct interaction between MKP-1 and SIRT2. However, AGK2 by inhibition of SIRT2 could increase the expression of MKP-1. Furthermore, AGK2 could inhibit the activation of BV2 microglia and expression of iNOS and SIRT2 in LPS treated mice brain tissue. Taken together, our results suggested that AGK2 might alleviate lipopolysaccharide induced neuroinflammation through regulation of mitogen-activated protein kinase phosphatase-1.

Graphical abstract

中文翻译：

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AGK2通过调节丝裂原活化的蛋白激酶磷酸酶-1减轻脂多糖诱导的神经炎症。

神经炎症与多种神经系统疾病的进展有关。许多研究表明，SIRT2参与多种炎症过程。虽然，机​​制仍不清楚。这项研究的目的是探讨SIRT2抑制剂AGK2在体外和体内对LPS激活的小胶质细胞的炎症反应和MAPK信号通路的影响。通过CCK-8分析检测AGK2对BV2小胶质细胞的细胞活力的影响。通过蛋白质印迹和免疫荧光分析炎症细胞因子iNOS的表达。实时聚合酶链反应（RT-PCR）检测iNOS，TNF-α和IL-1β的mRNA表达。通过蛋白质印迹分别分析了SIRT2，磷酸化-P38，P38，磷酸化-JNK，JNK，磷酸化-ERK，ERK，α-微管蛋白和乙酰基-α-微管蛋白。SIRT2和MKP-1之间的相互作用通过共免疫沉淀（Co-IP）分析进行了测量。进行了双重免疫荧光染色以检测脑组织中CD11b和iNOS或SIRT2的表达。我们发现AGK2可以抑制LPS诱导的BV2小胶质细胞中炎性细胞因子（iNOS，TNF-α和IL-1β）的表达水平。此外，它可以有效降低SIRT2的表达并增加LPS激活的BV2小胶质细胞和LPS诱导的小鼠神经炎症中α-微管蛋白的乙酰化。此外，我们的结果表明，AGK2可以减少LPS攻击后p38，JNK和ERK磷酸化的增加。Co-IP结果表明，MKP-1和SIRT2之间没有直接相互作用。但是，通过抑制SIRT2，AGK2可以增加MKP-1的表达。此外，AGK2可以抑制LPS处理的小鼠脑组织中BV2小胶质细胞的活化以及iNOS和SIRT2的表达。两者合计，我们的结果表明，AGK2可能通过调节有丝分裂原激活的蛋白激酶磷酸酶-1减轻脂多糖诱导的神经炎症。

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