Gastrodin improves microglia-mediated inflammatory response after hypoxic-ischemic brain damage in neonatal rats via PI3K/AKT pathway

doi:10.12122/j.issn.1673-4254.2024.09.11

Abstract

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

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.

Figures/Tables 9

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

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).

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.

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

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

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).

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).

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

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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