[Gastrodin alleviates microglia-mediated inflammatory responses in neonatal mice with hypoxic-ischemic brain damage by regulating CCR5/AKT signaling]

Abstract

Objective: To investigate the mechanism behind the protective effects of gastrodin against microglia-mediated inflammatory responses following hypoxic-ischemic brain damage (HIBD) in neonatal mice.

Methods: Thirty-six 10-day-old C57BL/6J mice were randomized into sham-operated group, HIBD (induced by ligation of the left common carotid artery followed by hypoxia for 40 min) group, and HIBD with gastrodin treatment groups (n=12). In gastrodin treatment group, 100 mg/kg gastrodin was injected intraperitoneally 1 h before and at 2 and 12 h after hypoxia. After the treatments, the expressions of CCR5, AKT, p-AKT, and TNF-α and the co-expression of IBA1 and CCR5 in the corpus callosum of the mice were detected with Western blotting and immunofluorescence double staining. In a BV2 microglial cell model of oxygen-glucose deprivation (OGD), the effects of pretreatment with gastrodin and Maraviroc (an CCR5 antagonist) on protein expressions of CCR5, AKT, p-AKT, TNF-α and IL-1β were evaluated using Western blotting and immunofluorescence double staining.

Results: The neonatal mice with HIBD showed significantly increased expressions of CCR5 and TNF-α with lowered p-AKT expression in the brain tissues, and GAS treatment obviously reversed these changes. HIBD also significantly increased the co-expression of IBA1 and CCR5 in the corpus callosum of the mice, which was obviously lowered by gastrodin treatment. In BV2 cells, OGD significantly increased the expressions of CCR5, TNF-α, and IL-1β and decreased the expression of p-AKT, and these changes were inhibited by treatment with gastrodin, Maraviroc or their combination; the inhibitory effect of the combined treatment did not differ significantly from that of gastrodin or Maraviroc alone.

Conclusion: Gastrodin can produce neuroprotective effects in neonatal mice with HIBD by inhibiting inflammatory cytokine production and activate AKT phosphorylation via inhibiting CCR5.

Keywords: CCR5/AKT; gastrodin; hypoxic-ischemic brain damage; microglia; oxygen-glucose deprivation.

图1

Western blotting检测新生小鼠胼胝体区AKT、p-AKT、CCR5和TNF-α的蛋白表达情况 Fig.1 Western blotting for detecting the protein expressions of AKT, p-AKT, CCR5 and TNF-α in the corpus callosum of the mice at 1 day (A-D) and 3 days (E-H) after HIBD and gastrodin (GAS) treatment (Mean±SD, n=3). HIBD: Hypoxic-ischemic brain damage. *P<0.05, **P<0.01, ***P<0.001 vs Sham group; ^# P<0.05, ^## P<0.01 vs HIBD group.

图2

免疫荧光双标染色法检测新生小鼠胼胝体中小胶质细胞标记物IBA1和CCR5的共表达情况 Fig.2 Co-expression of CCR5 and IBA1 proteins (white arrows) in the corpus callosum of the mice detected by immunofluorescence staining (Scale bar=50 μm). A: 1 day after HIBD and GAS treatment. B: 3 days after HIBD and GAS treatment.

图3

Western blotting检测BV2小胶质细胞 p-AKT、CCR5、TNF-α和IL-1β的蛋白表达情况 Fig.3 Expression levels of CCR5, p-AKT, TNF-α and IL-1β proteins in BV2 microglia after oxygen-glucose deprivation (OGD) and GAS treatment (Mean±SD, n=3). *P<0.05, **P<0.01, ***P<0.001 vs Control group; ^# P<0.05, ^## P<0.01 vs OGD group.

图4

免疫荧光双标染色法检测BV2小胶质细胞中CCR5和p-AKT表达情况 Fig.4 Expression levels of CCR5 (A)and p-AKT (B) proteins in BV2 microglia after OGD and GAS treatment detected by immunofluorescence staining (Scale bar=20 μm).

图5

OGD和Maraviroc处理BV2小胶质细胞后AKT、p-AKT、TNF-α和IL-1β的蛋白表达情况 Fig.5 Expression levels of AKT, p-AKT, TNF-α and IL-1β proteins in BV2 microglia after OGD and Maraviroc treatment detected by Western blotting (Mean±SD, n=3). M: Maraviroc. ^# P<0.05 vs OGD.