出版簡訊|內源性硫化氫通過NOS/NO通路在斑馬魚腦血管發育中的作用

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以下文章來源於SVN俱樂部 ，作者蔣偉青，王永剛

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H2S promotes developmental brain angiogenesis via the NOS/NO pathway in zebrafish

Weiqing Jiang, Chen Liu, Mingzhu Deng, Fei Wang, Xiao Ren, Yilin Fan, Jiulin Du, Yonggang Wang

doi: 10.1136/svn-2020-000584

Stroke & Vascular Neurology（SVN）新近上線文章「H2S promotes developmental brain angiogenesis via the NOS/NO pathway in zebrafish」，來自上海交通大學醫學院附屬仁濟醫院王永剛教授團隊。

硫化氫已被廣泛認為是繼一氧化氮和一氧化碳後的第三個氣體遞質家族成員。細胞實驗和動物缺血模型已證明，硫化氫可通過促進內皮細胞的生長、遷移和管狀結構形成等方面達到促血管生成的作用；在體實驗也表明生理性硫化氫具有促進肌肉組織血管生成的作用。然而，內源性硫化氫在腦血管新生中的作用尚不明確。本研究利用斑馬魚為模式生物，旨在明確內源性硫化氫對腦血管發育的作用。

研究發現，利用反義嗎啉環寡聚核苷酸（morpholino oligomer, MO）技術和CRISPR/Cas9技術分別下調和敲除斑馬魚硫化氫產生酶 —— 胱硫醚β合成酶（Cystathionine-β-synthase, cbs）和胱硫醚γ裂解酶（Cystathionine-γ-lyase, cth），均能抑制斑馬魚胚胎腦血管發育。

Figure 1. Knockdown and knockout of cbs or cth impair brain vascular development of larval zebrafish. (A) In situ hybridisation of whole zebrafish larvae at 3 dpf showing the ubiquitous expression of cbs and cth in the brain (lateral view). (B and C) Effects of morpholino oligonucleotide-mediated cbs or cth knockdown on brain vascular development. (B) Representative midbrain vessel structures reconstructed from confocal images of cbs or cth morphants at 3–5 dpf of the same larva. (C) Summary of data. The experiments were repeated three times, and six embryos were examined for each group at each time. (D and E) Effects of cbs or cth knockout (F0) on brain vascular development. (D) Representative midbrain vessel structures reconstructed from confocal images of the same F0 mutant larva at 3–5 dpf. (E) Summary of data. Six embryos were examined for each group. (F and G) Effects of cbs or cth homomutants on brain vascular development. (F) Representative midbrain vessel structures reconstructed from confocal images of the same cbs or cth homomutant larva at 3 and 5 dpf. (G) Summary of data. Six embryos were examined for each group. Scale bar, 300 µm (A), 25 µm (B, D, F). Error bars, SEM. *P<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student’s t-test).

硫化氫供體GYY4137可改善下調cbs和cth所導致的腦血管發育障礙。

Figure 2. Rescue effect of GYY4137 on the defects of brain vascular development in cbs and cth morphants. (A) Representative projected confocal images showing that GYY4137 treatment ameliorated the impaired brain vascular development in cbs and cth morphants. Confocal images were taken at 3–5 dpf of the same larva. (B) Summary of the rescue effect of GYY4137. Six embryos were examined for each group. Scale bar, 50 µm (A). Error bars, SEM. *P<0.05, **p<0.01, ***p<0.001 (one-way analysis of variance). MO, morpholino oligonucleotide.

利用軟體分析斑馬魚中腦血管網絡特徵，結果顯示，硫化氫在腦血管發育過程中主要作用在促血管生成方面，而並非腦血管網絡拓撲結構的形成。

Figure 3. Structure analysis of the midbrain vasculature in cbs and cth morphants. (A) Image of a 3-dpf larva showing the midbrain position delineated with dashed lines (upper left), and representative midbrain vasculature centerlines of 3-dpf larvae of Ctrl morpholino oligonucleotide (MO; upper right), cbs MO (lower left), and cth MO (lower right). C, caudal; L, lateral. (B–E) Summary of changes in the total vessel length (B), vessel segment number (C), weighted average segment Strahler order (D) and internal vessel loop number (E) of the midbrain vasculature in groups of Ctrl MO, cbs MO and cth MO. Six embryos were examined for each group. Error bars, SEM. ***P<0.001 (unpaired two-tailed Student’s t-test).

下調cbs和cth後，斑馬魚胚胎編碼一氧化氮合酶（nitric oxide synthase, NOS）的基因nos2a mRNA表達和總NO產生明顯減少，且通過共注射斑馬魚nos2a mRNA可以改善下調cbs和cth所導致的腦血管發育障礙。

Figure 4. The nitric oxide synthase (NOS)/nitric oxide (NO) pathway is involved in cbs and cth knockdown-induced defects of brain vascular development. (A and B) Effects of cbs and cth knockdown on the NOS/NO signalling pathway. Summary of nos1, nos2a and nos2b RNA expression (A) and total NO production (B) in 3-days post fertilisation (dpf) embryos. (C and D) Rescue effect of nos2a mRNA on the brain vascular developmental defects in cbs and cth morphants. Summary of increased total NO production (C) and midbrain vessel density (D) in 3-dpf embryos coinjected with cbs or cth MO and nos2a mRNA in comparison with those injected with cbs or cth MO. Six embryos were examined for each group. (E and F) No effect of cbs and cth knockdown on the protein expression of p-ERK1/2, ERK1/2 and VEGF. Representative blots (E) and summary (F) of Western blotting data. Error bars, SEM. *P<0.05, **p<0.01 (unpaired two-tailed Student’s t-test).

以上研究結果表明，內源性硫化氫通過NOS/NO通路促進斑馬魚腦血管生成。

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原標題：《出版簡訊｜內源性硫化氫通過NOS/NO通路在斑馬魚腦血管發育中的作用》

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