目的 探究华支睾吸虫外泌体中关键miRNA分子在肝纤维化中的作用，以期为华支睾吸虫致肝纤维化的机制提供新的线索。方法 从阳性家猫肝脏剖取华支睾吸虫成虫，体外培养，从排泄/分泌产物中提取外泌体，透射电子显微镜观察并对粒径分布进行鉴定。提取外泌体总RNA并测序，miRDeep2软件预测miRNA，与miRBase数据库中序列进行比对，测算比较每个miRNA的表达量并筛选。使用miRanda、miRDB、PITA软件分别对选定miRNA进行靶基因预测分析，topGO、clusterProfiler软件分别对靶基因的基因本体和信号通路进行富集分析。分别用浓度为5、10、20 μg/ml外泌体与肝星状细胞（HSC）共培养24、48 h，设置空白对照组；终浓度为50 nmol/L的选定miRNA（Csi-miR-125a）转染HSC 48 h，设置阴性对照组，均采用细胞计数试剂盒（CCK-8）检测各组HSC增殖情况。使用小鼠抗人β-actin、兔抗人α-平滑肌肌动蛋白（α-SMA）、兔抗人Ⅲ型胶原、兔抗人微管相关蛋白1A/1B-轻链3（LC3）、兔抗人p62、兔抗人细胞外信号调节激酶（ERK）、兔抗人p-ERK抗体（1∶1 000）作为一抗，辣根过氧化物酶（HRP）标记的山羊抗小鼠或抗兔IgG（1∶10 000）作为二抗，蛋白质免疫印迹（Western blotting）检测HSC的活化相关蛋白α-SMA、Ⅲ型胶原，自噬相关蛋白LC3、p62的表达水平及信号通路相关ERK蛋白磷酸化水平。结果 华支睾吸虫外泌体呈小泡状，具有磷脂双分子层结构，粒径分布主峰值为72 nm，分布范围主要为30~150 nm。外泌体中含有多种miRNA，选择表达量次高且与已知miRNA序列相似度高的Csi-miR-125a进行后续研究。Csi-miR-125a存在301个靶基因预测位点重合，主要富集在细胞质、胞浆等细胞组件，参与蛋白结合、离子结合等分子功能，涉及生物调节、系统发育、神经系统发育等生物过程；靶基因信号通路大多富集于Hedgehog信号通路、细胞外基质-受体互作的相关通路、NOD样受体信号通路等。CCK-8法检测结果表明，外泌体浓度为10 μg/ml、孵育24 h时A₄₅₀达最高，为1.707；Csi-miR-125a共孵育组A₄₅₀为1.122，高于阴性对照组（0.492）。Western blotting检测结果显示，阴性对照组及Csi-miR-125a组的α-SMA条带灰度值分别为0.588和0.904（t = 10.560，P < 0.01）；Ⅲ型胶原条带灰度值分别为0.783和0.997（t = 6.483，P < 0.05）。阴性对照组和Csi-miR-125a组LC3Ⅱ/LC3Ⅰ条带灰度比值分别为1.093和2.215（t = 5.206，P < 0.05），p62条带灰度值分别为0.537和1.369（t = 13.100，P < 0.01），p-ERK/ERK条带灰度比值分别为0.791和1.037（t = 5.067，P < 0.05）。结论 华支睾吸虫外泌体中miRNA种类丰富多样，且涉及多种细胞功能和多条信号通路。Csi-miR-125a可能通过阻断HSC自噬流和激活ERK信号通路促进HSC增殖活化从而在肝纤维化的发生中发挥一定的作用。

Objective To examine the role of hub miRNAs from Clonorchis sinensis exosomes in hepatic fibrosis, so as to provide new insights into deciphering the mechanisms underlying hepatic fibrosis induced by C. sinensis. Methods Adult worms of C. sinensis was collected from liver specimens of a domestic cat infected with C. sinensis, cultured in vitro, and excretory/secretory products were collected, centrifuged and filtered. Exosomes were extracted and observed under a transmission electron microscope, and their particle size distribution was analyzed. Total RNA was extracted from exosomes, and sequenced, and miRNA was predicted using the miRDeep2 software. The miRNA sequences were aligned with those in the miRBase database, and the expression of each miRNA was quantified and screened. The target genes of selected miRNAs were predicted using the miRanda, miRDB, and PITA software, and the Gene Ontology (GO) and signaling pathways of target genes were subjected to enrichment analyses using the topGO and clusterProfiler software. Hepatic stellate cells (HSCs) were incubated with exosomes at concentrations of 5, 10, and 20 μg/ml for 24 and 48 h, and a blank control group was assigned. HSCs were transfected with a selected miRNA (Csi-miR-125a) at a final concentration of 50 nmol/L for 48 h, and a negative control group was assigned. The proliferation of HSCs was measured using the cell counting kit-8 (CCK-8) assay, and the expression of HSC activation-related protein α-smooth muscle actin (α-SMA) and type Ⅲ collagen, autophagy-related proteins microtubule associated protein 1A/1B light chain 3 (LC3) and p62, and the phosphorylation of the signaling pathway-related extracellular signal regulated kinase (ERK) protein was determined using Western blotting with mouse anti-human β-actin, rabbit anti-human α-SMA, rabbit anti-human type Ⅲ collagen, rabbit an-human LC3, rabbit anti-human p62, rabbit anti-human ERK, and rabbit anti-human p-ERK primary antibodies (1∶1 000 dilution), and the secondary horseradish peroxidase (HRP) conjugated goat anti-mouse or anti-rabbit IgG antibody (1∶1 000 dilution). Results The C. sinensis exosomes appeared a small vesicular shape with a phospholipid bilayer structure, and the main peak particle size was 72 nm (range, 30 to 150 nm). The C. sinensis exosomes contained multiple miRNAs, and Csi-miR-125a, which had the second highest expression and a high similarity to known miRNA sequences, was selected for subsequent experiment. Csi-miR-125a had overlapping of 301 predicted sites of target genes, and these target genes were significantly enriched in cellular components such as cytoplasm, involved in molecular functions of protein binding and ion binding, and biological processes of biological regulation, systematic development, and nervous system development, and significantly enriched in Hedgehog signaling pathways, extracellular matrix- receptor interaction related pathways, and NOD like receptor signaling pathways. The CCK-8 assay measured the highest A₄₅₀ value (1.707) at an exosome concentration of 10 μg/ml for 24 h incubation, and the A₄₅₀ value was higher in the Csi-miR-125a co-culture group (1.122) than in the negative control group (0.492). Western blotting determined that the grayscale values of the α-SMA bands were 0.588 and 0.904 in the negative control group and the Csi-miR-125a group (t = 10.560, P < 0.01), and the grayscale values of type Ⅲ collagen bands were 0.783 and 0.997 in the negative control group and the Csi-miR-125a group (t = 6.483, P < 0.05).The grayscale ratios of LC3 Ⅱ/LC3 Ⅰ bands were 1.093 and 2.215 in the negative control group and the Csi-miR-125a group (t = 5.206, P < 0.05), and the grayscale values of p62 bands were 0.537 and 1.369 in the negative control group and the Csi-miR-125a group (t = 13.100, P < 0.01), while the grayscale ratios of pERK/ERK bands were 0.791 and 1.037 in the negative control group and the Csi-miR-125a group, respectively (t = 5.067, P < 0.05). Conclusion There are diverse miRNAs in C. sinensis exosomes, which are involved in multiple cellular functions and signaling pathways. Csi-miR-125a may contribute to hepatic fibrosis through blocking the autophagic flux of HSCs and activating the ERK signaling pathway to promote HSCs proliferation and activation.