天麻素经PI3K/AKT通路改善新生大鼠缺氧缺血性脑损伤后小胶质细胞介导的炎症反应

doi:10.12122/j.issn.1673-4254.2024.09.11

摘要/Abstract

摘要：

目的探讨天麻素通过PI3K/AKT信号通路对新生大鼠缺氧缺血性脑损伤（HIBD）后活化小胶质细胞介导的炎症反应的作用机制。方法将39只3日龄新生SD大鼠随机分为假手术组（sham，n=9）、缺氧缺血模型组（HIBD，n=15）、天麻素处理组（HIBD+G，n=15）。采用Western blotting检测HIBD后TNF-α、IL-1β、IL-10和TGF-β1蛋白的表达；网络药理学筛选天麻素治疗HIBD的潜在作用靶点；Western blotting检测HIBD和氧糖剥夺（OGD）诱导活化的小胶质细胞中PI3K/AKT信号通路的表达；CCK8检测PI3K/AKT通路特异性抑制剂LY294002对BV-2小胶质细胞的细胞毒性作用；RT-qPCR检测LY294002干预后天麻素对TNF-α和TGF-β1的mRNA水平的影响。结果 Western blotting显示，与HIBD组相比，天麻素降低缺血侧胼胝体区TNF-α和IL-1β的蛋白表达（P<0.05），促进IL-10和TGF-β1的蛋白表达（P<0.05）；网络药理学显示，PI3K/AKT信号通路显著富集，且天麻素与PI3K之间具有较好的结合能力；Western blotting显示，与HIBD组、OGD组相比，天麻素促进PI3K和AKT的磷酸化水平（P<0.05）；CCK8结果显示LY294002在0~120 μmol/L浓度范围内对BV-2小胶质细胞没有细胞毒性作用；RT-qPCR结果显示，与对照组相比，OGD组TNF-α的mRNA水平升高，TGF-β1的mRNA水平降低（P<0.05）；天麻素干预后降低TNF-α的mRNA水平，升高TGF-β1的mRNA水平（P<0.05）；LY294002处理后TNF-α的mRNA水平进一步升高，TGF-β1的mRNA水平进一步降低（P<0.05）；而LY294002与天麻素联合用药后TNF-α和TGF-β1的mRNA水平无明显变化。结论天麻素能抑制HIBD后活化小胶质细胞介导的炎症反应，其作用机制与PI3K/AKT信号通路有关。

关键词: 缺氧缺血性脑损伤, 天麻素, 炎症反应, 氧糖剥夺, 小胶质细胞, PI3K/AKT

Abstract:

Objective To investigate the mechanism of gastrodin for inhibiting microglia-mediated inflammation after hypoxic-ischemic brain damage (HIBD) in neonatal rats. Methods Thirty-nine 3-day-old SD rats were randomly divided into sham group, HIBD group and gastrodin treatment group. Western blotting was used to detect the expressions of TNF-α, IL-1β, IL-10 and TGF-β1 in the corpus callosum of the rats. The potential targets of gastrodin for treatment of HIBD were screened by network pharmacology analysis. The expressions of PI3K/AKT signaling pathway proteins following HIBD-induced microglial activation in the rats and in cultured microglial BV-2 cells with oxygen-glucose deprivation (OGD) were detected with Western blotting. The effects of LY294002 (a specific inhibitor of the PI3K/AKT pathway) and gastrodin on TNF-α and TGF-β1 mRNA levels in BV-2 cells with OGD was detected with RT-qPCR. Results In the neonatal rats with HIBD, gastrodin treatment significantly decreased TNF-α and IL-1β expressions and enhanced IL-10 and TGF-β1 expressions in the ischemic corpus callosum. Network pharmacology analysis showed significant enrichment of the PI3K/AKT signaling pathway and a strong binding between gastrodin and PI3K. Gastrodin significantly promoted PI3K and AKT phosphorylation in neonatal rats with HIBD and in BV-2 cells exposed to OGD. In BV-2 cells with OGD, gastrodin obviously suppressed OGD-induced increase of TNF‑α and reduction of TGF‑β1 mRNA expressions, and this effect was strongly attenuated by LY294002 treatment. Conclusion Gastrodin can inhibit microglia-mediated inflammation in neonatal rats with HIBD by regulating the PI3K/AKT signaling pathway.

Key words: hypoxic-ischemic brain damage, gastrodin, inflammatory response, oxygen and sugar deprivation, microglia, PI3K/AKT

左涵珺, 段兆达, 王朝, 郭涛, 石金沙, 石浩龙, 李娟娟. 天麻素经PI3K/AKT通路改善新生大鼠缺氧缺血性脑损伤后小胶质细胞介导的炎症反应[J]. 南方医科大学学报, 2024, 44(9): 1712-1719.

Hanjun ZUO, Zhaoda DUAN, Zhao WANG, Tao GUO, Jinsha SHI, Haolong SHI, Juanjuan LI. Gastrodin improves microglia-mediated inflammatory response after hypoxic-ischemic brain damage in neonatal rats via PI3K/AKT pathway[J]. Journal of Southern Medical University, 2024, 44(9): 1712-1719.

图/表 9

表1 引物序列

Tab.1 Primer sequence for RT-qPCR

Primer	Sequence (5'-3')
TGF-β1 F	GCTGAACCAAGGAGACGGAA
TGF-β1 R	TCTTCTCTGTGGAGCGTTGAT
TNF-α F	ACCCTCACACTCACAAACCA
TNF-α R	GGCAGAGAGGAGGTTGACTTT
β-actin F	GTGGGAATGGGTCAGAAGGA
β-actin R	TACATGGCTGGGGTGTTGAA

图1 蛋白印迹法检测HIBD后天麻素对炎性因子TNF-α、IL-1β、抗炎因子IL-10和转化生长因子TGF-β1蛋白表达的影响

Fig.1 Effect of gastrodin on expressions of TNF-α, IL-1β, IL-10 and TGF-β1 proteins in the in brain tissue of neonatal rats at 1 day, 3 days, and 7 days after HIBD detected by Western blotting. A, C, E: Protein blots of TNF-α, IL-1β, IL-10, and TGF-β1. B, D, F: Quantification of TNF-α, IL-1β, IL-10, and TGF-β1 expression levels. *P<0.05, **P<0.01, ***P<0.001 (n=3).

图2 天麻素治疗HIBD潜在核心靶点的PPI网络图和GO富集分析

Fig.2 PPI network diagram and GO enrichment analysis of potential core target of gastrodin treatment of HIBD. A: PPI network diagram. B: GO enrichment analysis.

图3 天麻素治疗HIBD潜在核心靶点的KEGG富集分析

Fig.3 KEGG enrichment analysis of potential core targets for gastrodin treatment of HIBD. A, B: KEGG pathway enrichment analysis.

图4 天麻素与PI3K信号通路蛋白分子对接

Fig.4 Gastrodin and PI3K signaling pathway visualization protein molecular docking.

图5 蛋白印迹法检测HIBD后天麻素对活化小胶质细胞中PI3K/AKT信号通路表达的影响

Fig.5 Effect of gastrodin on expressions of PI3K/AKT signaling pathway in activated microglia of neonatal rats at 1 day, 3 days, and 7 days after HIBD detected by Western blotting. A, C, E: Western blots of the related proteins in PI3K/AKT signaling pathway. B, D, F: Quantification of P-PI3K, PI3K, P-AKT, and AKT protein levels. *P<0.05, **P<0.01, ***P<0.001 (n=3).

图6 蛋白印迹法检测OGD后天麻素对活化小胶质细胞中PI3K/AKT信号通路表达的影响

Fig.6 Effect of gastrodin on expression of PI3K/AKT signaling pathway in activated microglia BV-2 cells after OGD detected by western blotting. A: Western blots of the related proteins in PI3K/AKT signaling pathway in BV-2 cells. B: Quantification of P-PI3K, PI3K, P-AKT, and AKT protein levels. *P<0.05, **P<0.01 (n=3).

图7 CCK8 法检测LY294002 对BV-2细胞细胞毒性作用的影响

Fig.7 The cytotoxic effect of LY294002 on BV-2 cells was detected by CCK8 kit.

图8 RT-qPCR检测OGD后天麻素通过PI3K/AKT信号通路对活化小胶质细胞中炎症因子TNF-α和转化生长因子TGF-β1 mRNA水平表达变化的影响

Fig.8 Effect of gastrodin and LY294002 on TNF-α (A) and TGF-β1 mRNA (B) expressions in BV-2 cells with OGD detected by RT-qPCR. *P<0.05, **P<0.01, ***P<0.001 (n=3).

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