Institute of Biochemistry, Biological Research Centre, Temesvári krt. 62, Szeged, H-6726, Hungary.
Doctoral School in Biology, University of Szeged, Szeged, Hungary.
J Neuroinflammation. 2021 Jan 10;18(1):22. doi: 10.1186/s12974-020-02070-2.
Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury.
In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry.
Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions.
Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.
热休克蛋白 B1（HSPB1）是进化上保守的小分子热休克蛋白家族中最知名和用途最广泛的成员之一。它通过调节炎症反应，被认为对各种神经疾病具有神经保护作用，但它在神经炎症中的确切作用尚不清楚。为了阐明 HSPB1 对炎症调节的确切机制，我们在急性脑损伤的体内和体外模型中研究了 HSPB1 对神经炎症过程的影响。
在这项研究中，我们使用了一种过表达人 HSPB1 蛋白的转基因小鼠品系。在体内实验中，用乙醇处理 7 天龄的转基因和野生型小鼠。使用 TUNEL 检测凋亡细胞。用 RT-PCR 和免疫组织化学检测大脑中细胞因子和神经胶质细胞标志物的 mRNA 和蛋白水平。我们还建立了原代神经元、星形胶质细胞和小胶质细胞培养物，并用细胞因子和乙醇进行处理。用 ELISA 从上清液中测量 TNFα 和 hHSPB1 水平，并用荧光免疫组织化学分析细胞内 hHSPB1 表达。
乙醇处理后，与野生型大脑相比，hHSPB1 过表达小鼠的大脑中促炎细胞因子（TNF、IL1B）、小胶质细胞（CD68、Arg1）和星形胶质细胞（GFAP）标志物的 mRNA 水平显著升高。与野生型相比，乙醇处理的转基因小鼠的某些脑区的小胶质细胞激活和 1 周后反应性星形胶质细胞增生更高。尽管促凋亡 TNF 的表达水平显著升高，但 hHSPB1 过表达的小鼠并未表现出更高水平的凋亡。我们的数据表明，细胞内 hHSPB1（在原代星形胶质细胞中表达水平最高）负责调节炎症。在我们的模型中，小胶质细胞是 TNFα 的主要来源。在炎症条件下，从小鼠分离出的 hHSPB1 过表达的小胶质细胞释放的 TNFα 明显高于野生型细胞。
我们的工作提供了新的体内证据，表明 hHSPB1 过表达通过增强促炎细胞因子的表达和增强神经胶质细胞的激活，而不是增加神经元凋亡，对急性神经炎症具有调节作用。这些结果表明，hHSPB1 在调节乙醇诱导的神经炎症反应中可能发挥复杂的作用。
