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Dl-3-n-butylphthalide promotes angiogenesis in ischemic stroke mice through upregulating autocrine and paracrine sonic hedgehog

Acta Pharmacologica Sinica volume 44, pages 2404–2417 (2023)Cite this article

Abstract

Dl-3-n-butylphthalide (NBP) is a small-molecule drug used in the treatment of ischemic stroke in China, which is proven to ameliorate the symptoms of ischemic stroke and improve the prognosis of patients. Previous studies have shown that NBP accelerates recovery after stroke by promoting angiogenesis. In this study, we investigated the mechanisms underlying the angiogenesis-promoting effects of NBP in ischemic stroke models in vitro and in vivo. OGD/R model was established in human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs), while the tMCAO model was established in mice. The cells were pretreated with NBP (10, 50, 100 µM); the mice were administered NBP (4, 8 mg/kg, i.v.) twice after tMCAO. We showed that NBP treatment significantly stimulated angiogenesis by inducing massive production of angiogenic growth factors VEGFA and CD31 in both in vitro and in vivo models of ischemic stroke. NBP also increased the tubule formation rate and migration capability of HUVECs in vitro. By conducting the weighted gene co-expression network analysis, we found that these effects were achieved by upregulating the expression of a hedgehog signaling pathway. We demonstrated that NBP treatment not only changed the levels of regulators of the hedgehog signaling pathway but also activated the transcription factor Gli1. The pro-angiogenesis effect of NBP was abolished when the hedgehog signaling pathway was inhibited by GDC-0449 in HUVECs, by Sonic Hedgehog(Shh) knockdown in HUVECs, or by intracerebroventricular injection of AAV-shRNA(shh)-CMV in tMCAO mice. Furthermore, we found that HUVECs produced a pro-angiogenic response not only to autocrine Shh, but also to paracrine Shh secreted by astrocytes. Together, we demonstrate that NBP promotes angiogenesis via upregulating the hedgehog signaling pathway. Our results provide an experimental basis for the clinical use of NBP.

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Fig. 1: NBP increases the expression of angiogenic growth factors in HUVECs after OGD/R (n = 3, mean ± SEM).
Fig. 2: NBP significantly improves the neurological function and reduces the cerebral infarction volume ratio of C57BL/6 J mice after tMCAO (n = 10, mean ± SEM).
Fig. 3: NBP enhances the activity of the Hedgehog signaling pathway (n = 3, mean ± SEM).
Fig. 4: Downregulating Sonic Hedgehog expression inhibits the effect of NBP (n = 3, mean ± SEM).
Fig. 5: NBP stimulates paracrine expression of Shh by astrocytes to promote angiogenesis (n = 3, mean ± SEM).
Fig. 6: Knockdown of Shh inhibits the angiogenesis-promoting effect of NBP in mice (n = 10, mean ± SEM).

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Acknowledgements

This work was supported by grants from the key program of the Educational Department of Liao Ning Province (Nos. LJKZZ20220100).

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  1. Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, 110016, China

    Mei-jie Dai, Xing-xing Gui, Shu-miao Jia, Shu-ting Lv, Hao Dou & Wei Cui

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Contributions

WC, MJD, and STL designed the research. MJD and XXG performed most of the experiments and data analyses. SMJ and STL performed some experiments. HD was responsible for bioinformatics analyses, and MJD wrote and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Wei Cui.

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Dai, Mj., Gui, Xx., Jia, Sm. et al. Dl-3-n-butylphthalide promotes angiogenesis in ischemic stroke mice through upregulating autocrine and paracrine sonic hedgehog. Acta Pharmacol Sin 44, 2404–2417 (2023). https://doi.org/10.1038/s41401-023-01137-z

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