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BACKGROUND

The liver's regenerative capacity allows it to repair itself after injury. Extracellular vesicles and particles (EVPs) in the liver's interstitial space are crucial for signal transduction, metabolism, and immune regulation. Understanding the role and mechanism of liver-derived EVPs in regeneration is significant, particularly after partial hepatectomy, where the mechanisms remain unclear.

METHODS

A 70% hepatectomy model was established in mice, and EVPs were isolated and characterized using electron microscopy, nanocharacterization, and Western blot analysis. Combined metabolomic and transcriptomic analyses revealed β-sitosterol enrichment in EVPs and activation of the Hedgehog signaling pathway during regeneration. The role of β-sitosterol in EVPs on the Hedgehog pathway and its targets were identified using qRT-PCR, Western blot analysis. The regulation of carnitine synthesis by this pathway was determined using a dual luciferase assay. The effect of a β-sitosterol diet on liver regeneration was verified in mice.

RESULTS

After 70% hepatectomy, the liver successfully regenerated without liver failure or death. At 24 hours post-surgery, tissue staining showed transient regeneration-associated steatosis (TRAS), with increased Ki67 positivity at 48 hours. EVPs displayed a spherical lipid bilayer structure with particle sizes of 70-130 nm. CD9, CD63, and CD81 in liver-derived EVPs were confirmed. Transcriptomic and metabolomic analyses showed EVPs supplementation significantly promoted carnitine synthesis and fatty acid oxidation. Tissue staining confirmed accelerated TRAS resolution and enhanced liver regeneration with EVP supplementation. Mass spectrometry identified β-sitosterol in EVPs, which binds to Smo protein, activating the Hedgehog pathway. This led to the nuclear transport of Gli3, stimulating Setd5 transcription and inducing carnitine synthesis, thereby accelerating fatty acid oxidation. Mice with increased β-sitosterol intake showed faster TRAS resolution and liver regeneration compared to controls.

CONCLUSION

Liver-derived EVPs promote regeneration after partial hepatectomy. β-sitosterol from EVPs accelerates fatty acid oxidation and promotes liver regeneration by activating Hedgehog signaling pathway.

BACKGROUND

METHODS

RESULTS

CONCLUSION

Liver-derived EVPs promote regeneration after partial hepatectomy. β-sitosterol from EVPs accelerates fatty acid oxidation and promotes liver regeneration by activating Hedgehog signaling pathway.

背景

肝脏具有再生能力，可在受伤后自行修复。肝脏间质空间中的细胞外囊泡和颗粒（EVPs）对于信号转导、代谢和免疫调节至关重要。了解肝源性 EVPs 在再生中的作用和机制具有重要意义，特别是在部分肝切除术后，其机制尚不清楚。

方法

在小鼠中建立 70%肝切除模型，使用电子显微镜、纳米特征分析和 Western blot 分析分离和表征 EVPs。结合代谢组学和转录组学分析，发现 EVPs 中β-谷甾醇富集，并在再生过程中激活 Hedgehog 信号通路。使用 qRT-PCR、Western blot 分析鉴定 EVPs 中β-谷甾醇对 Hedgehog 通路及其靶标的作用。使用双荧光素酶测定法确定该途径对肉碱合成的调节作用。在小鼠中验证β-谷甾醇饮食对肝脏再生的影响。

结果

70%肝切除后，肝脏成功再生，没有肝功能衰竭或死亡。手术后 24 小时，组织染色显示短暂的再生相关脂肪变性（TRAS），48 小时时 Ki67 阳性增加。肝源性 EVPs 显示出具有 70-130nm 粒径的球形脂质双层结构。在肝源性 EVPs 中确认了 CD9、CD63 和 CD81。转录组学和代谢组学分析表明，EVPs 补充显著促进肉碱合成和脂肪酸氧化。组织染色证实，EVPs 补充可加速 TRAS 缓解并增强肝脏再生。质谱分析鉴定出 EVPs 中的β-谷甾醇与 Smo 蛋白结合，激活 Hedgehog 通路。这导致 Gli3 的核转运，刺激 Setd5 转录并诱导肉碱合成，从而加速脂肪酸氧化。与对照组相比，摄入更多β-谷甾醇的小鼠显示出更快的 TRAS 缓解和肝脏再生。