Table9_Ferulic Acid Combined With Bone Marrow Mesenchymal Stem Cells Attenuates the Activation of Hepatic Stellate Cells and Alleviates Liver Fibrosis.XLSX

Bone marrow mesenchymal stem cells (BMSCs) can effectively alleviate liver fibrosis, but the efficacy of cell therapy alone is insufficient. In recent years, a combination of traditional Chinese medicine (TCM) and cell therapy has been increasingly used to treat diseases in clinical trials. Ferulic acid (FA) is highly effective in treating liver fibrosis, and a combination of cells and drugs is being tested in clinical trials. Therefore, we combined BMSCs and Ferulic acid to treat CCl4-induced fibrosis and determine whether this combination was more effective than single treatment. We used BMSCs and FA to treat CCl4-induced fibrosis in rat models, observed their therapeutic effects, and investigated the specific mechanism of this combination therapy in liver fibrosis. We created a BMSC/hepatic stellate cell (HSC) coculture system and used FA to treat activated HSCs to verify the specific mechanism. Then, we used cytochalasin D and angiotensin II to investigate whether BMSCs and FA inactivate HSCs through cytoskeletal rearrangement. MiR-19b-3p was enriched in BMSCs and targeted TGF-β receptor II (TGF-βR2). We separately transfected miR-19b-3p into HSCs and BMSCs and detected hepatic stellate cell activation. We found that the expression of the profibrotic markers α-SMA and COL1-A1 was significantly decreased in the combination group of rats. α-SMA and COL1-A1 levels were also significantly decreased in the HSCs with the combination treatment. Cytoskeletal rearrangement of HSCs was inhibited in the combination group, and RhoA/ROCK pathway gene expression was decreased. Following angiotensin II treatment, COL1-A1 and α-SMA expression increased, while with cytochalasin D treatment, profibrotic gene expression decreased in HSCs. The expression of COL1-A1, α-SMA and RhoA/ROCK pathway genes was decreased in the activated HSCs treated with a miR-19b-3p mimic, indicating that miR-19b-3p inactivated HSCs by suppressing RhoA/ROCK signalling. In contrast, profibrotic gene expression was significantly decreased in the BMSCs treated with the miR-19b-3p mimic and FA or a miR-19b-3p inhibitor and FA compared with the BMSCs treated with the miR-19b-3p mimic alone. In conclusion, the combination therapy had better effects than FA or BMSCs alone. BMSC and FA treatment attenuated HSC activation and liver fibrosis by inhibiting cytoskeletal rearrangement and delivering miR-19b-3p to activated HSCs, inactivating RhoA/ROCK signalling. FA-based combination therapy showed better inhibitory effects on HSC activation.

骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）可有效缓解肝纤维化，但单一细胞治疗的疗效仍存在不足。近年来，中医药（traditional Chinese medicine, TCM）联合细胞治疗的方案在疾病临床试验中的应用愈发广泛。阿魏酸（Ferulic acid, FA）在治疗肝纤维化方面疗效显著，且细胞-药物联合疗法已进入临床试验阶段。为此，本研究将BMSCs与阿魏酸联合用于治疗四氯化碳诱导的肝纤维化模型，旨在探究该联合疗法是否优于单一治疗方案。我们采用BMSCs与FA处理四氯化碳诱导的大鼠肝纤维化模型，观察其治疗效果，并深入研究该联合疗法抗肝纤维化的具体分子机制。我们构建了BMSC/肝星状细胞（hepatic stellate cell, HSC）共培养体系，使用FA处理活化的肝星状细胞以验证其具体作用机制。随后，我们利用细胞松弛素D与血管紧张素II，探究BMSCs与FA是否通过调控细胞骨架重排实现肝星状细胞的失活。微小RNA-19b-3p（MiR-19b-3p）在BMSCs中富集，且可靶向结合转化生长因子β受体II（transforming growth factor-β receptor II, TGF-βR2）。我们分别将MiR-19b-3p转染至肝星状细胞与BMSCs中，检测肝星状细胞的活化状态。研究发现，联合治疗组大鼠的促纤维化标志物α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）与Ⅰ型胶原α1链（collagen type I alpha 1 chain, COL1-A1）的表达水平显著降低。联合治疗组的肝星状细胞中，α-SMA与COL1-A1的表达水平同样显著下调。联合治疗组的肝星状细胞细胞骨架重排受到抑制，且RhoA/ROCK信号通路相关基因的表达水平降低。经血管紧张素II处理后，肝星状细胞中COL1-A1与α-SMA的表达水平升高；而经细胞松弛素D处理后，肝星状细胞的促纤维化基因表达水平则出现下降。在经MiR-19b-3p模拟物处理的活化肝星状细胞中，COL1-A1、α-SMA及RhoA/ROCK通路相关基因的表达水平均降低，表明MiR-19b-3p可通过抑制RhoA/ROCK信号通路实现肝星状细胞的失活。与之相反，相较于仅经MiR-19b-3p模拟物处理的BMSCs，经MiR-19b-3p模拟物联合FA处理，或经MiR-19b-3p抑制剂联合FA处理的BMSCs，其促纤维化基因的表达水平均显著降低。综上，该联合治疗方案的疗效优于单独使用FA或BMSCs的单一治疗方案。BMSCs与FA联合治疗可通过抑制细胞骨架重排，并将MiR-19b-3p递送至活化的肝星状细胞，进而抑制RhoA/ROCK信号通路，最终减轻肝星状细胞活化与肝纤维化。基于阿魏酸的联合疗法对肝星状细胞活化展现出更优异的抑制效果。