Sani Farnaz, Soufi Zomorrod Mina, Azarpira Negar, Soleimani Masoud
Hematology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Transplant Research Center, Shiraz University of Medical Sciences, Khalili Street P.O. Box 7193711351, Shiraz, Iran.
Stem Cells Int. 2023 Aug 4;2023:8836452. doi: 10.1155/2023/8836452. eCollection 2023.
Although several studies have been conducted on modeling human liver disease, it is still challenging to mimic nonalcoholic fatty liver disease in vitro. Here, we aimed to develop a fibrotic liver microtissue composed of hepatocytes, hepatic stellate, and endothelial cells. In addition, the therapeutic effects of umbilical cord mesenchymal stem cell-derived exosomes (UC-MSC-EXO) and anti-miR17-5p as new antifibrotic drugs were investigated.
To create an effective preclinical fibrosis model, multicellular liver microtissues (MLMs) consisting of HepG2, LX2, and HUVECs were cultured and supplemented with a mixture of palmitic acid and oleic acid for 96 hr. Then, MLMs were exposed to UC-MSC-EXO and anti-miR17-5p in different groups. The results of cell viability, reactive oxygen species (ROS) production, liver enzyme levels, inflammation, and histopathology were analyzed to assess the treatment efficacy. Furthermore, the expression of collagen I (COL I) and -smooth muscle actin (-SMA) as critical matrix components, transforming growth factor beta (TGF-), and miR-17-5p were measured.
Free fatty acid supplementation causes fibrosis in MLMs. Our results demonstrated that UC-MSC-EXO and anti-miR17-5p attenuated TGF-1, interleukin-1, and interleukin-6 in all experimental groups. According to the suppression of the TGF-1 pathway, LX2 activation was inhibited, reducing extracellular matrix proteins, including COL I and -SMA. Also, miR-17-5p expression was elevated in fibrosis conditions. Furthermore, we showed that our treatments decreased alanine aminotransferase and aspartate aminotransferase, and increased albumin levels in the culture supernatant. We also found that both MSC-EXO and MSC-EXO + anti-miR17-5p treatments could reduce ROS production.
Our findings indicated that anti-miR17-5p and MSC-EXO might be promising therapeutic options for treating liver fibrosis. Furthermore, EXO + anti-miR had the best effects on boosting the fibrotic markers. Therefore, we propose this novel MLM model to understand fibrosis mechanisms better and develop new drugs.
尽管已经开展了多项关于人类肝脏疾病建模的研究，但在体外模拟非酒精性脂肪性肝病仍具有挑战性。在此，我们旨在构建一种由肝细胞、肝星状细胞和内皮细胞组成的纤维化肝脏微组织。此外，还研究了脐带间充质干细胞衍生的外泌体（UC-MSC-EXO）和抗miR17-5p作为新型抗纤维化药物的治疗效果。
为创建一个有效的临床前纤维化模型，培养由HepG2、LX2和人脐静脉内皮细胞（HUVECs）组成的多细胞肝脏微组织（MLMs），并添加棕榈酸和油酸混合物培养96小时。然后，将不同组的MLMs分别暴露于UC-MSC-EXO和抗miR17-5p中。分析细胞活力、活性氧（ROS）产生、肝酶水平、炎症和组织病理学结果以评估治疗效果。此外，检测了作为关键基质成分的I型胶原（COL I）和α-平滑肌肌动蛋白（α-SMA）、转化生长因子β（TGF-β）以及miR-17-5p的表达。
补充游离脂肪酸会导致MLMs发生纤维化。我们的结果表明，UC-MSC-EXO和抗miR17-5p在所有实验组中均减弱了TGF-β1、白细胞介素-1和白细胞介素-6。根据对TGF-β1信号通路的抑制作用，LX2的激活受到抑制，从而减少了包括COL I和α-SMA在内的细胞外基质蛋白。此外，在纤维化状态下miR-17-5p表达升高。此外，我们还表明，我们的治疗降低了培养上清液中的丙氨酸氨基转移酶和天冬氨酸氨基转移酶水平，并提高了白蛋白水平。我们还发现，MSC-EXO和MSC-EXO +抗miR17-5p两种治疗均可减少ROS产生。
我们的研究结果表明，抗miR17-5p和MSC-EXO可能是治疗肝纤维化的有前景的治疗选择。此外，EXO +抗miR对增强纤维化标志物的作用最佳。因此，我们提出这种新型的MLM模型，以更好地理解纤维化机制并开发新药。
