天麻素通过调节CCR5/AKT信号传导缓解新生小鼠缺血缺氧后小胶质细胞介导的炎症反应

doi:10.12122/j.issn.1673-4254.2024.10.02

摘要/Abstract

摘要：

目的研究天麻素（GAS）通过CCR5/AKT信号对新生小鼠缺血缺氧（HIBD）后小胶质细胞介导炎症反应的影响。方法选用36只10 d龄的C57BL/6J小鼠，随机分为假手术组（Sham）、缺血缺氧模型组（HIBD）、缺血缺氧+天麻素治疗组（HIBD+GAS），12只/组。模型组和治疗组均行左颈总动脉分离并结扎，1 h后置于缺氧环境中40 min后放回母笼，治疗组在术前1 h、缺氧后2 h及缺氧后12 h腹腔注射剂量为100 mg/kg的GAS。体外培养BV2小胶质细胞验证天麻素对CCR5/AKT及炎症因子TNF-α、IL-1β的影响，将其分为：对照组（Control）、氧糖剥夺组（OGD）、OGD+GAS处理组（OGD+GAS）、GAS处理组（GAS）；为进一步验证CCR5拮抗剂Maraviroc（M）的作用以及其与GAS联合干预的作用，将细胞分为：Control组、（OGD）组、M组、OGD+M组、OGD+M+GAS组。Control组用高糖培养基正常培养，含GAS组用 GAS 0.34 μmol/L处理1 h，含M组用Maraviroc 10 μmol/L处理1 h，最后将含OGD组均更换为无糖培养基并置于缺氧小室2 h以构建OGD模型。通过Western blotting检测CCR5、AKT、p-AKT、TNF-α、IL-1β的蛋白表达，免疫荧光双标染色检测新生小鼠胼胝体CCR5以及BV2小胶质细胞中CCR5和p-AKT的表达变化。结果与Sham组相比，HIBD组中CCR5、TNF-α表达显著增加，p-AKT表达显著减低（P<0.05，0.01或0.001），GAS治疗后逆转了上述结果（P<0.05或0.01）。与Sham组相比，HIBD组中新生小鼠胼胝体区小胶质细胞标记物IBA1及CCR5的荧光强度明显升高，其共表达增加，而GAS干预后IBA1及CCR5的荧光强度显著降低，共表达减少。与Control组相比，OGD组CCR5、TNF-α、IL-1β表达显著增加，p-AKT表达显著减少（P<0.05，0.01或0.001）；GAS或Maraviroc治疗后，逆转了上述结果（P<0.05或0.01）。OGD+M组与OGD+M+GAS组比较，差异无统计学意义。结论 GAS可能通过靶向CCR5激活AKT的磷酸化表达水平，抑制炎症因子的表达，发挥神经保护作用。

关键词: 天麻素, 缺血缺氧性脑损伤, 氧糖剥夺, 小胶质细胞, CCR5/AKT

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.

Key words: gastrodin, hypoxic-ischemic brain damage, oxygen-glucose deprivation, microglia, CCR5/AKT

石金沙, 张皓南, 张幸霖, 石浩龙, 左涵珺, 郭涛, 王朝, 余航, 李娟娟. 天麻素通过调节CCR5/AKT信号传导缓解新生小鼠缺血缺氧后小胶质细胞介导的炎症反应[J]. 南方医科大学学报, 2024, 44(10): 1850-1857.

Jinsha SHI, Haonan ZHANG, Xinglin ZHANG, Haolong SHI, Hanjun ZUO, Tao GUO, Zhao WANG, Hang YU, Juanjuan LI. Gastrodin alleviates microglia-mediated inflammatory responses in neonatal mice with hypoxic-ischemic brain damage by regulating CCR5/AKT signaling[J]. Journal of Southern Medical University, 2024, 44(10): 1850-1857.

图/表 5

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.

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