背景:-酒精诱导的促炎激活可能会影响细胞和突触病理学,从而导致与酒精使用障碍相关的行为表型。在本研究中,N-[(4-三氟甲基)-苄基]4-甲氧基丁酰胺(GET73)的可能的抗炎特性,一种有前途的治疗酒精使用障碍的药物,在大鼠皮质小胶质细胞的原代培养物中进行了评估。
方法:-用100ng/ml脂多糖处理大脑皮层小胶质细胞的原代培养物(LPS;8小时,37°C)或75mM乙醇(EtOH;4天,37°C)单独或在GET73(1-30µM)存在的情况下。在潜伏期结束时,使用xMAP技术和Luminex平台对细胞因子/趋化因子进行多参数定量.此外,用EtOH和GET73单独或组合处理后培养的小胶质细胞活力,已经通过比色测定(即MTT测定)测量。
结果:-GET73(10和30µM)部分或完全阻止了LPS诱导的IL-6,IL-1β的增加,RANTES/CCL5蛋白和MCP-1/CCL2水平。相反,GET73未能减弱LPS诱导的TNF-α水平升高。此外,GET73治疗(10-30µM)显着减弱或阻止EtOH诱导的TNF-α增加,IL-6、IL-1β和MCP-1/CCL2水平。最后,在所有测试浓度(1-30µM)下,GET73治疗未改变EtOH诱导的小胶质细胞活力降低.
结论:-目前的结果提供了GET73对EtOH诱导的神经炎症的保护特性的第一个体外证据。这些数据增加了有关化合物作用的复杂和多因素特征的更多信息,进一步支持将GET73开发为治疗酒精使用障碍个体的治疗工具的重要性。
BACKGROUND: - Alcohol-induced pro-inflammatory activation might influence cellular and synaptic pathology, thus contributing to the behavioral phenotypes associated with alcohol use disorders. In the present study, the possible anti-inflammatory properties of N-[(4-trifluoromethyl)-benzyl]4-methoxybutyramide (GET73), a promising therapeutic agent for alcohol use disorder treatment, were evaluated in primary cultures of rat cortical microglia.
METHODS: - Primary cultures of cerebral cortex microglial cells were treated with 100 ng/ml lipopolysaccharide (LPS; 8 h, 37 °C) or 75 mM ethanol (EtOH; 4 days, 37 °C) alone or in the presence of GET73 (1-30 µM). At the end of the incubation period, multiparametric quantification of cytokines/chemokines was performed by using the xMAP technology and Luminex platform. Furthermore, cultured microglial cell viability following the treatment with EtOH and GET73, alone or in combination, has been measured by a colorimetric assay (i.e. MTT assay).
RESULTS: - GET73 (10 and 30 µM) partially or fully prevented the LPS-induced increase of IL-6, IL-1β, RANTES/CCL5 protein and MCP-1/CCL2 levels. On the contrary, GET73 failed to attenuate the TNF-α level increase induced by LPS. Furthermore, GET73 treatment (10-30 µM) significantly attenuated or prevented the EtOH-induced increase of TNF-α, IL-6, IL-1β and MCP-1/CCL2 levels. Finally, at all the concentrations tested (1-30 µM), the GET73 treatment did not alter the EtOH-induced reduction of microglial cell viability.
CONCLUSIONS: - The current results provide the first in vitro evidence of GET73 protective properties against EtOH-induced neuroinflammation. These data add more information on the complex and multifactorial profile of action of the compound, further supporting the significance of developing GET73 as a therapeutic tool for the treatment of individuals with alcohol use disorders.