Table_3_Glycine-Histidine-Lysine (GHK) Alleviates Astrocytes Injury of Intracerebral Hemorrhage via the Akt/miR-146a-3p/AQP4 Pathway.DOCX

Intracerebral hemorrhage (ICH) is a major type of cerebrovascular disease with poor prognosis. Recent studies have shown that Glycyl-l-histidyl-l-lysine (GHK) is a kind of natural human tripeptide which could inhibit inflammation and against neurodegenerative diseases, but neither its role nor the mechanisms in ICH have yet been explicit. Currently, we investigated the possible strategies of GHK on ICH injury. Neurological deficit scores, brain water content, Nissl staining, and aquaporin 4 (AQP4) immunohistochemistry were detected in different groups of rats. The expression of microRNAs (miRNAs) was examined by real-time PCR. Inflammatory factors were detected using enzyme-linked immunosorbent assay (ELISA). Cell viability and cell proliferation were detected by Cell Counting Kit-8 (CCK-8). Matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitors of metalloproteinase-1 (TIMP1), AQP4 expression were detected/assessed using western blot. We observed that 5 and 10 μg/g of GHK improved neurological recovery by significantly reducing brain water content, improving neurological deficits, and promoting neuron survival. Besides, GHK alleviated inflammatory reaction and downregulated AQP4 expression. Furthermore, the effects of GHK on astrocyte were associated with the upregulation of miRNA-146a-3p, which partially regulated the expression of AQP4. Our results demonstrated that the phosphatidylinositol 3-kinase (PI3K)/AKT pathway participated in the GHK-induced upregulation of miR-146a-3p and miR-146a-3p/AQP4 interaction plays a role in the injury following ICH. These findings suggested that GHK could provide a novel therapeutic strategy for ICH.

脑出血（Intracerebral hemorrhage, ICH）是一类预后较差的重要脑血管疾病。现有研究表明，甘氨酰-L-组氨酰-L-赖氨酸（Glycyl-l-histidyl-l-lysine, GHK）是一种天然人源三肽，可抑制炎症反应并对抗神经退行性疾病，但其在脑出血中的作用与分子机制尚未明确。本研究旨在探讨GHK对脑出血损伤的潜在干预策略。本研究对不同组别大鼠开展了神经功能缺损评分、脑含水量检测、尼氏染色（Nissl staining）以及水通道蛋白4（aquaporin 4, AQP4）免疫组化检测；采用实时荧光定量PCR（real-time PCR）检测微小RNA（microRNAs, miRNAs）的表达水平；采用酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）检测炎症因子水平；采用细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）检测细胞活力与细胞增殖能力；采用蛋白质印迹法（western blot）检测基质金属蛋白酶2（Matrix metalloproteinase 2, MMP2）、MMP9、金属蛋白酶组织抑制剂-1（tissue inhibitors of metalloproteinase-1, TIMP1）以及AQP4的蛋白表达水平。结果显示，5 μg/g与10 μg/g剂量的GHK可通过显著降低脑含水量、改善神经功能缺损以及促进神经元存活，提升神经功能恢复效果。此外，GHK可缓解炎症反应并下调AQP4的蛋白表达。进一步研究发现，GHK对星形胶质细胞的调控作用与miRNA-146a-3p的上调有关，而miRNA-146a-3p可部分调控AQP4的表达。本研究结果证实，磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase, PI3K）/AKT通路参与了GHK诱导的miR-146a-3p上调过程，且miR-146a-3p与AQP4的相互作用在脑出血后继发性损伤中发挥调控作用。上述研究结果表明，GHK可为脑出血的治疗提供全新的潜在干预策略。