Hepatocyte Rho-Associated Kinase Signaling is Required to Survive Liver Injury. Hepatocyte Rho-Associated Kinase Signaling is Required to Survive Liver Injury

Rho-associated kinases 1 and 2 (ROCK1 and ROCK2) are downstream effectors of RhoA and regulate various critical cell functions such as actomyosin contractility, apoptosis, proliferation, and cell migration. Some studies utilizing inhibitors suggested that ROCK inhibition could serve as a treatment for liver fibrosis. However, more investigation is needed to understand the role of ROCK signaling in hepatocytes with injury in vivo. Rock1fl/fl, Rock2fl/fl, and Rock1fl/fl;Rock2fl/fl mice were injected with adeno-associated virus serotype 8 (AAV8)-thyroid hormone-binding globulin (TBG)-Cre for targeted gene deletion in hepatocytes, or injected with AAV8-TBG-Null to generate littermate controls (WT). Mice were then given a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce cholestatic liver injury. Serum liver function tests, liver histology, and RNAseq analyses were performed. Mice deficient in both ROCK1 and ROCK2 (DKO) showed early mortality, decreased hepatic synthetic function, and increased hepatocellular injury compared to WT. Mice deficient in ROCK1 only (R1KO) or ROCK2 only (R2KO) demonstrated minimal differences compared to WT, indicating that the two ROCK isoforms had redundant roles in this disease model. Compared to WT, histologic analysis showed that DKO mice had significantly greater loss of liver parenchyma. RNAseq analysis indicated upregulation of apoptotic and inflammatory genes in DKO compared to WT liver. Immunostaining showed an increase in p21 and cleaved caspase 3 in DKO mice compared to WT, indicating that deletion of ROCK potentially leads to p21 overexpression and cell death. These results demonstrate that hepatocyte ROCK signaling is essential in promoting cell survival in the setting of liver cholestatic injury. Overall design: We examined the role of liver epithelial ROCK by deleting the isoforms Rock1 and Rock2 separately or simultaneously in male mice using an inducible Cre/loxP-mediated gene knockout. Weinduced fibrotic liver disease using the 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) injury model to investigate the role of ROCK in the hepatocyte response to injury. Differential gene expression profiling analysis of RNA-seq data was conducted for WT vs R1KO and R2KO, and for WT vs DKO given DDC diet. Single KO WT samples, R1WT and R2WT, were combined into one WT group for analysis. DKO mice were separated into 2 week or 4 week collection due to the mice being in moribound state; however, all mice were combined into two groups, WT and DKO, for analysis.

Rho相关激酶1与2（Rho-associated kinases 1 and 2, ROCK1和ROCK2）是RhoA的下游效应分子，可调控多项关键细胞功能，包括肌动蛋白-肌球蛋白收缩性、细胞凋亡、细胞增殖与细胞迁移。部分使用抑制剂的研究表明，ROCK抑制可作为肝纤维化的治疗手段，但目前仍需更多研究以阐明体内损伤肝细胞中ROCK信号通路的作用。
本研究将Rock1条件性敲除纯合小鼠（Rock1fl/fl）、Rock2条件性敲除纯合小鼠（Rock2fl/fl）以及Rock1与Rock2双条件性敲除纯合小鼠（Rock1fl/fl;Rock2fl/fl）分别注射8型腺相关病毒-甲状腺激素结合球蛋白-Cre重组酶（adeno-associated virus serotype 8, AAV8-thyroid hormone-binding globulin, TBG-Cre）以实现肝细胞的靶向基因敲除，或注射8型腺相关病毒-甲状腺激素结合球蛋白-空载体（AAV8-TBG-Null）以构建同窝野生型对照（WT）。随后向所有小鼠饲喂0.1%的3,5-二乙氧羰基-1,4-二氢可力丁（3,5-diethoxycarbonyl-1,4-dihydrocollidine, DDC）饲料以诱导胆汁淤积性肝损伤。后续开展了血清肝功能检测、肝脏组织学分析与RNA测序（RNAseq）分析。
与野生型对照相比，同时敲除ROCK1与ROCK2的双敲除小鼠（DKO）出现早期死亡，肝脏合成功能下降，肝细胞损伤程度升高。仅敲除ROCK1（R1KO）或仅敲除ROCK2（R2KO）的小鼠与野生型对照相比差异极小，表明两种ROCK亚型在该疾病模型中存在功能冗余。
组织学分析显示，相较于野生型小鼠，DKO小鼠的肝实质丢失情况显著更严重。RNA测序分析表明，DKO小鼠肝脏中的凋亡相关基因与炎症相关基因表达上调。免疫染色结果显示，DKO小鼠的p21蛋白与裂解型半胱氨酸天冬氨酸蛋白酶3（cleaved caspase 3）表达水平升高，提示敲除ROCK可能导致p21过表达与细胞死亡。
上述结果证实，肝细胞ROCK信号通路在胆汁淤积性肝损伤环境中对维持细胞存活至关重要。
整体实验设计：本研究通过可诱导的Cre/loxP介导的基因敲除系统，在雄性小鼠中分别或同时敲除Rock1与Rock2亚型，以此探究肝脏上皮细胞ROCK的功能。我们采用0.1% DDC饲料诱导肝纤维化疾病模型，以研究ROCK在肝细胞损伤应答中的作用。对RNA测序数据开展差异基因表达谱分析，分析分组包括：野生型（WT）vs仅敲除Rock1（R1KO）、野生型（WT）vs仅敲除Rock2（R2KO），以及野生型（WT）vs经DDC饲料处理的双敲除小鼠（DKO）。将单基因敲除对应的同窝野生型样本（R1WT与R2WT）合并为一个WT组用于分析。由于DKO小鼠会进入濒死状态，因此按2周与4周两个时间点收集样本；但最终将所有样本合并为WT与DKO两个组进行分析。