Mechanism of adipose tissue-derived mesenchymal stem cell-derived exosomes regulating autophagy of hepatic stellate cells

doi:10.12307/2025.524

Abstract

Abstract: BACKGROUND: Adipose tissue-derived mesenchymal stem cells release a large amount of exosomes to participate in various pathophysiological processes, but the impact and precise mechanism of exosomes derived from adipose tissue-derived mesenchymal stem cells on autophagy of hepatic stellate cells have not been fully elucidated.
OBJECTIVE: To explore the targeted regulatory effect and molecular mechanism of adipose tissue-derived mesenchymal stem cell-derived exosomes on autophagy of hepatic stellate cells through miR-15a-5p.
METHODS: Adipose tissue was collected from inguinal region of 8-week male C57BL/6 mice. Adipose tissue-derived mesenchymal stem cells were extracted by collagenase digestion. Adipose tissue-derived mesenchymal stem cell-derived exosomes were extracted by ultracentrifugation. Mouse liver tissue was obtained, and hepatic stellate cells were isolated and extracted using collagenase perfusion digestion and density gradient centrifugation. The experiment was divided into two groups. In control group, hepatic stellate cells were cultured alone for 48 hours. In the exosome group, hepatic stellate cells were co-cultured with adipose tissue-derived mesenchymal stem cell-derived exosomes for 48 hours. The effects of exosomes on hepatic stellate cell proliferation, activation, autophagy, and expression of fibrosis markers were detected by western blot assay, RT-qPCR, and immunofluorescence staining. RT-qPCR and western blot assay were used to detect the effect of exosomes on the mRNA and protein expression of miR-15a-5p and the downstream signaling pathway Bcl-2, Beclin-1, and Rubicon in hepatic stellate cells.
RESULTS AND CONCLUSION: (1) Compared with the control group, the ratio of autophagy markers LC3-II expression decreased, the number of autophagosome was also significantly decreased, and the intracellular lipid droplets were regenerated, simultaneously, cell volume diminished with the weakening of proliferation in hepatic stellate cells of the exosome group, indicated that the hepatic stellate cell activation was significantly inhibited. (2) Compared with the control group, the expressions of α-smooth actin and type I collagen were significantly decreased (P < 0.01), and the expression of miR-15a-5p was significantly increased in hepatic stellate cells of the exosome group (P < 0.01). At the same time, the expression of its downstream target gene Bcl-2 was significantly decreased (P < 0.01), while the expressions of autophagy genes Beclin-1 and Rubicon were significantly increased in hepatic stellate cells of the exosome group (P < 0.01). The results indicate that adipose tissue-derived mesenchymal stem cell-derived exosomes inhibits the expression of Bcl-2 in hepatic stellate cells by targeting miR-15a-5p and increases the expression of downstream autophagy genes Beclin-1 and Rubicon, thereby inhibiting the autophagy of hepatic stellate cells.

Key words: ">adipose tissue-derived mesenchymal stem cell, exosome, hepatic stellate cell, autophagy, miR-15a-5p, Bcl-2, Beclin-1, Rubicon

CLC Number:

Chen Zhenkun, Zhu Shiwei, Xiao Jingnan, Tang Weiping. Mechanism of adipose tissue-derived mesenchymal stem cell-derived exosomes regulating autophagy of hepatic stellate cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(25): 5296-5303.

Figures/Tables 2

2.1 脂肪间充质干细胞及ADSC-exo的鉴定结果从小鼠脂肪组织获取脂肪间充质干细胞，培养72 h后倒置显微镜下观察见细胞贴壁生长，呈纺锤形，传代培养后选取第3代细胞经成脂肪诱导分化7 d，油红O染色可见细胞内脂滴呈红色，经成软骨诱导分化14 d，Alcian Blue染色阳性(图1A)。细胞免疫荧光染色提示Vimentin(波形蛋白)和N-cadherin(神经型钙黏蛋白)表达阳性，而血管内皮细胞标志物CD31呈阴性(图1B)。流式细胞仪检测显示间充质干细胞标志物CD29和CD90阳性，而血管内皮细胞共同标志物CD31和CD45阴性(图1C)，符合脂肪间充质干细胞的表型特征。收集脂肪间充质干细胞的上清液，通过超速离心法分离提取上清液中的外泌体，透射电子显微镜观察外泌体呈现双层膜盘状囊泡的外形，形状为椭圆形或不规则圆形，均一性良好，大小基本分布在80-130 nm之间(图1D)。Western blot检测外泌体表面标志物CD63和CD81表达阳性(图1E)。 2.2 ADSC-exo抑制肝星状细胞活化从小鼠肝组织中获取的肝星状细胞呈扁圆形，内含较多光亮的脂滴，折光性强，在327 nm波长下可见蓝色荧光(图2A左)，培养5 d后肝星状细胞体积增大，呈纤维细胞样形态，胞浆中脂滴明显减少，α-平滑肌肌动蛋白活化，细胞呈完全活化状态(图2A右)；PKH26标记的ADSC-exo与肝星状细胞共孵育48 h后，PKH26红色荧光标记的ADSC-exo与DAPI标记的蓝色荧光在肝星状细胞中共定位，而作为对照组的无外泌体培养基培养的肝星状细胞则未见红色荧光(图2B)，表明ADSC-exo可以被肝星状细胞所摄取。 ADSC-exo与肝星状细胞共孵育48 h后，肝星状细胞中可见脂滴形成，细胞体积缩小，提示肝星状细胞由活化型向静息型转化(图2C)。RT-qPCR结果显示外泌体组miR-15a-5p的表达量相较于对照组显著升高，提示ADSC-exo可能是通过携带并释放miR-15a-5p至肝星状细胞中，从而使肝星状细胞中的miR-15a-5p水平升高(P < 0.01，图2D)。CCK-8检测提示ADSC-exo与肝星状细胞共孵育4 d后，细胞活力较对照组有显著降低，由此说明ADSC-exo可明显抑制肝星状细胞的增殖能力(P < 0.01，图2E)。RT-qPCR和Western blot检测结果显示ADSC-exo处理后α-平滑肌肌动蛋白和Ⅰ型胶原蛋白表达显著下调(图2F-H)，提示肝星状细胞活化明显受抑制。"

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