Portal fibroblasts are mesenchyme-derived fibroblasts surrounding the bile ducts, and activated into portal myofibroblasts (pMF) during cholestatic liver injury. pMF express α-smooth muscle actin (α-SMA) and produce the fibrogenic extracellular matrix (ECM) collagen type I and fibronectin, playing important roles in portal fibrosis. A cholestatic bile duct-ligated (BDL) model is characterized by impaired hepatobiliary excretion of bile, leading to increased bile acid accumulation. Accumulation of bile acids is known to induce endoplasmic reticulum (ER) stress leading to liver damage and cell death. Additionally, a BDL fibrotic model is also associated with upregulation of CCN (CYR61, CTGF and NOV) matricellular proteins and reported to induce ER stress both in vitro and in vivo. To explore the effects of CCN proteins, we used adenovirus-mediated CCN1-4 (Ad-CCN1-4) gene transfers into cultured pMF. Overexpression of CCN proteins leads to protein accumulation in the ER lumen, causing ER stress and unfolded protein response (UPR). We further found ER stress and UPR to mitigate fibrogenesis in pMF by decreased cellular production of fibronectin, collagen type 1 and α-SMA. In this scenario, Tauroursodeoxycholic acid, a pharmaceutical chaperone and ER stress inhibitor, attenuated Ad-CCN1-4 induced pMF apoptosis and restored collagen and fibronectin levels. Since hepatic fibrogenesis is accompanied by ER stress and upregulation of CCN proteins in a BDL, we further evaluated ER stress responses after Ad-CCN1 gene transfer in such a model and found overexpressed CCN1 to enhance the ER stress-associated proteins BiP and CHOP with positive cleaved caspase 3 and 9 staining in periportal nonparenchymal cells. This indicates that these nonparenchymal cells, most likely pMF, have the tendency to undergo apoptosis during later stages of BDL. Ad-CCN1 transduction furthermore sensitized pMF for ER stress and apoptosis. We suggest that CCN proteins are key factors in the fibrotic microenvironment impacting pMF survival during fibrogenesis and pMF apoptosis during fibrosis resolution.

门脉成纤维细胞是在胆管周围的间充质来源的成纤维细胞，在胆汁淤积性肝损伤期间被激活为门脉成纤维细胞（pMF）。pMF表达α平滑肌肌动蛋白（α-SMA）并产生I型胶原纤维生成因子和纤连蛋白，在门脉纤维化中起重要作用。胆汁淤积性胆管结扎（BDL）模型的特征是胆汁肝胆排泄受损，导致胆汁酸蓄积增加。已知胆汁酸的积累会引起内质网（ER）应激，从而导致肝损伤和细胞死亡。此外，BDL纤维化模型还与CCN（CYR61，CTGF和NOV）基质细胞蛋白的上调相关，并且据报道在体外和体内均可诱导ER应激。为了探索CCN蛋白的作用，我们使用了腺病毒介导的CCN1-4（Ad-CCN1-4）基因转移到培养的pMF中。CCN蛋白的过度表达导致内质网腔中的蛋白质积聚，从而导致内质网应激和未反应的蛋白质反应（UPR）。我们进一步发现ER应激和UPR通过减少细胞中纤连蛋白，1型胶原和α-SMA的细胞生成来减轻pMF中的纤维生成。在这种情况下，牛磺去氧胆酸（一种药物伴侣和内质网应激抑制剂）减弱了Ad-CCN1-4诱导的pMF细胞凋亡并恢复了胶原蛋白和纤连蛋白的水平。由于肝纤维化伴有内质网应激和BDL中CCN蛋白的上调，我们在这种模型中进一步评估了Ad-CCN1基因转移后的ER应激反应，发现过表达的CCN1可以在门静脉周围的非实质细胞中以阳性裂解的caspase 3和9染色增强与ER应激相关的蛋白BiP和CHOP。这表明这些非实质细胞，最可能是pMF，有在BDL后期发生凋亡的趋势。Ad-CCN1转导进一步使pMF对内质网应激和细胞凋亡敏感。我们建议，CCN蛋白是纤维化微环境中影响纤维形成过程中pMF存活和纤维化解决过程中pMF凋亡的关键因素。Ad-CCN1转导进一步使pMF对内质网应激和细胞凋亡敏感。我们建议，CCN蛋白是纤维化微环境中影响纤维形成过程中pMF存活和纤维化解决过程中pMF凋亡的关键因素。Ad-CCN1转导进一步使pMF对内质网应激和细胞凋亡敏感。我们建议，CCN蛋白是纤维化微环境中影响纤维形成过程中pMF存活和纤维化解决过程中pMF凋亡的关键因素。