Liver transplantation is the only radical treatment for decompensated cirrhosis, but its use is limited owing to a shortage of donors; hence, there is an urgent need for new treatments. Previously, we developed a liver-regeneration therapy using autologous bone marrow-derived mesenchymal stem cells (BMSCs), which is under clinical investigation. Cell–cell interactions between BMSCs and macrophages (Mφs) participate in the improvement of liver function and alleviation of liver fibrosis, although the associated mechanisms have not been elucidated. Therefore, in this study, we investigated phenotypic changes in Mφs caused by interactions with BMSCs, as well as the underlying mechanisms. Co-culturing lipopolysaccharide (LPS)-stimulated murine bone marrow-derived Mφs (BMDMs) with BMSCs substantially upregulated matrix metalloproteinase 9 (Mmp9), Mmp12, and Mmp13 expression, and downregulated tumor necrosis factor alpha (Tnfα) expression. To identify humoral factors involved in phenotypic changes occurring in Mφs, microarray analysis was performed with microRNAs (miRNAs) derived from extracellular vesicles in the supernatant of co-cultured BMSCs and LPS-stimulated BMDMs. We found that miR-6769b-5p was highly expressed and that transfecting miR-6769b-5p mimic upregulated MMP9 in LPS-stimulated BMDMs and downregulated Tnfα and interleukin-1 beta (Il-1β). MiR-6769b-5p expression in BMDMs was decreased by LPS stimulation but was increased by co-culture with BMSCs. Microarray and pathway analyses of gene expression in LPS-stimulated, miR-6769b-5p-transfected BMDMs revealed changes in the eukaryotic initiation factor 2-signaling pathway and decreased the expression of activating transcription factor 4 (Atf4). LPS-stimulated BMDMs exhibited increased MMP9 expression and decreased the expression of Tnfα and Il-1β by ATF4 knockdown. These findings indicate that upregulating miR-6769b-5p in BMDMs induced a fibrolytic phenotype, where MMP9 was highly expressed and inflammatory cytokine expression was decreased by the suppression of ATF4 expression. These findings imply that regulating miR-6769b-5p or ATF4 expression in BMDMs may be helpful for treating chronic liver disease.

中文翻译：

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间充质干细胞通过调节巨噬细胞中的 mmu-miR-6769b-5p 表达诱导纤维溶解表型。

肝移植是失代偿期肝硬化唯一的根治性治疗方法，但由于供体短缺，其应用受到限制；因此，迫切需要新的治疗方法。此前，我们开发了一种使用自体骨髓间充质干细胞 (BMSCs) 的肝脏再生疗法，目前正在临床研究中。BMSCs 和巨噬细胞 (Mφs) 之间的细胞-细胞相互作用参与了肝功能的改善和肝纤维化的缓解，尽管相关机制尚未阐明。因此，在本研究中，我们研究了与 BMSC 相互作用引起的 Mφ 的表型变化，以及潜在的机制。与 BMSC 共培养脂多糖 (LPS) 刺激的小鼠骨髓来源的 Mφ (BMDM) 显着上调基质金属蛋白酶 9。Mmp9 )、Mmp12和Mmp13表达，以及下调肿瘤坏死因子 α ( Tnfα ) 表达。为了确定 Mφ 中发生的表型变化所涉及的体液因素，使用来自共培养的 BMSC 和 LPS 刺激的 BMDM 上清液中的细胞外囊泡的 microRNA (miRNA) 进行微阵列分析。我们发现 miR-6769b-5p 高表达，转染 miR-6769b-5p 模拟 LPS 刺激的 BMDM 中上调 MMP9 和下调Tnfα和白细胞介素 1β（Il-1β）。BMDMs 中的 MiR-6769b-5p 表达因 LPS 刺激而降低，但与 BMSCs 共培养时增加。LPS 刺激的、miR-6769b-5p 转染的 BMDM 中基因表达的微阵列和通路分析揭示了真核起始因子 2 信号通路的变化，并降低了激活转录因子 4 ( Atf4 )的表达。LPS 刺激的 BMDMs 表现出增加的 MMP9 表达并降低Tnfα和Il-1β 的表达被 ATF4 击倒。这些发现表明，在 BMDM 中上调 miR-6769b-5p 诱导了纤维溶解表型，其中 MMP9 高表达，炎症细胞因子表达通过抑制 ATF4 表达而降低。这些发现意味着调节 BMDM 中 miR-6769b-5p 或 ATF4 的表达可能有助于治疗慢性肝病。