Nonalcoholic fatty liver disease (NAFLD) is characterized by increased lipid accumulation and excessive deposition of extracellular matrix (ECM) that results in tissue stiffening. The potential interplay between matrix stiffness and hepatocyte lipid accumulation during NAFLD has not been established. Here, an in vitro NAFLD model is developed using chemically defined, engineered hydrogels and human induced pluripotent stem cell-derived hepatic organoids (HOs). Specifically, dynamic covalent chemistry crosslinking, along with transient small molecule competitors, are used to create dynamic stiffening hydrogels that enable the reproducible culture of HOs. Within matrices that mimic the stiffness of healthy to diseased tissue (≈1-6 kPa), lipid droplet accumulation in HOs is triggered by exposure to an NAFLD-associated free fatty acid. These NAFLD model suggests that higher stiffness microenvironments result in increased hepatic lipid droplet accumulation, increased expression of fibrosis markers, and increased metabolic dysregulation. By targeting the ROCK mechanosignaling pathway, the synergy between matrix stiffness and lipid droplet accumulation is disrupted. The in vitro model of NAFLD has the potential to understand the role of mechanosignaling in disease progression and identify new pathways for therapeutic intervention.

KD025(belumosudil), a selective ROCK2 inhibitor, exhibits unique anti-adipogenic activity through inhibition of casein kinase 2 (CK2). This study investigated the dual inhibitory effects of KD025 on metabolism in a diet-induced obese model. C57BL/6 mice on a high fat diet (HFD) were treated with KD025 for 4 weeks, while fasudil (a pan-ROCK inhibitor) and CX-4945 (a CK2-specific inhibitor) served as comparison treatments. KD025 significantly reduced body weight gain without affecting food intake, serum insulin, or fasting blood glucose levels. In contrast, while both CX-4945 and fasudil treatments showed a trend toward weight reduction, these results were not statistically significant. KD025 improved lipid metabolism by significantly lowering LDL cholesterol and triglyceride, although it slightly impaired glucose metabolism, as observed in insulin and glucose tolerance tests. Weight reduction in the KD025- and CX-4945-treated groups was attributed to decreased adipose tissue mass, particularly in inguinal (ingWAT) and epididymal (epiWAT) fat depots. Hematoxylin and eosin (H&E) staining confirmed smaller adipocyte size in these groups. KD025 had no significant effect on serum levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), or monocyte chemoattractant protein-1 (MCP-1) with varied inflammatory responses. Furthermore, KD025 and CX-4945 upregulated adipogenic and browning markers, such as Cebpa, Cidea, and Pparg in the epiWAT, though without significant UCP1 expression. Overall, KD025 effectively reduced weight gain in HFD-fed mice through dual inhibition of CK2 and ROCK2, highlighting its potential as a therapeutic agent for obesity-related conditions.

Abnormal regulation of lncRNA is strongly linked to metabolic dysfunction-associated steatotic liver disease (MASLD). However, the precise molecular mechanisms remain unclear. This study explores the roles of noncoding RNA activated by DNA damage (NORAD)/miR-511-3p/Rho-associated protein kinase 2 (Rock2) axis and the NORAD/ROCK2 interaction in the development of MASLD.

In vitro and in vivo models of MASLD were created using high-fat diet-fed mice and free fatty acid (FFA)-treated hepatocytes. To examine the relationships between NORAD, miR-511-3p, and ROCK2, we employed bioinformatics, luciferase assays, RNA immunoprecipitation, and biotinylated NORAD pull-down assays. MASLD progression was assessed based on food intake, energy expenditure, insulin resistance, hepatic steatosis, inflammation, white fat growth, and liver fibrosis.

NORAD and ROCK2 were upregulated, while miR-511-3p was downregulated in MASLD liver tissues and FFA-treated hepatocytes. Mechanistically, NORAD competitively interacted with miR-511-3p to modulate Rock2 mRNA expression, and directly stabilized ROCK2 protein by abrogating its ubiquitination degradation. Functionally, liver-specific knockdown of NORAD or overexpression of miR-511-3p significantly slowed MASLD progression. Overexpression of NORAD or ROCK2 partially reversed miR-511-3p-induced inhibition of MASLD. Additionally, ROCK2 knockdown attenuated NORAD-induced worsening of MASLD. Moreover, overexpressing NORAD or ROCK2 or interfering miR-511-3p influenced resmetirom treatment to suppress MASLD development. Finally, metabolic changes in liver driven by the NORAD/miR-511-3p/Rock2 axis and NORAD/ROCK2 interaction also influenced white adipose growth, pancreatic β-cell dedifferentiation, and liver fibrosis.

The NORAD/miR-511-3p/Rock2 axis and the NORAD/ROCK2 interaction play critical roles in MASLD progression, identifying potential therapeutic targets for its treatment.

The mechanisms utilized by differentiating B cells to withstand highly damaging conditions generated during severe infections, like the massive hemolysis that accompanies malaria, are poorly understood. Here, we demonstrate that ROCK1 regulates B cell differentiation in hostile environments replete with pathogen-associated molecular patterns (PAMPs) and high levels of heme by controlling 2 key heme-regulated molecules, BACH2 and heme-regulated eIF2α kinase (HRI). ROCK1 phosphorylates BACH2 and protects it from heme-driven degradation. As B cells differentiate, furthermore, ROCK1 restrains their pro-inflammatory potential and helps them handle the heightened stress imparted by the presence of PAMPs and heme by controlling HRI, a key regulator of the integrated stress response and cytosolic proteotoxicity. ROCK1 controls the interplay of HRI with HSP90 and limits the recruitment of HRI and HSP90 to unique p62/SQSTM1 complexes that also contain critical kinases like mTOR complex 1 and TBK1, and proteins involved in RNA metabolism, oxidative damage, and proteostasis like TDP-43. Thus, ROCK1 helps B cells cope with intense pathogen-driven destruction by coordinating the activity of key controllers of B cell differentiation and stress responses. These ROCK1-dependent mechanisms may be widely employed by cells to handle severe environmental stresses, and these findings may be relevant for immune-mediated and age-related neurodegenerative disorders.

Drug repurposing involves the investigation of existing drugs for new indications. It offers a great opportunity to quickly identify a new drug candidate at a lower cost than novel discovery and development. Despite the importance and potential role of drug repurposing, there is no specific definition that healthcare providers and the World Health Organization credit. Unfortunately, many similar and interchangeable concepts are being used in the literature, making it difficult to collect and analyze uniform data on repurposed drugs. This research was conducted based on understanding general criteria for drug repurposing, concentrating on liver diseases. Many drugs have been investigated for their effect on liver diseases even though they were originally approved (or on their way to being approved) for other diseases. Some of the hypotheses for drug repurposing were first captured from the literature and then processed further to test the hypothesis. Recently, with the revolution in bioinformatics techniques, scientists have started to use drug libraries and computer systems that can analyze hundreds of drugs to give a short list of candidates to be analyzed pharmacologically. However, this study revealed that drug repurposing is a potential aid that may help deal with liver diseases. It provides available or under-investigated drugs that could help treat hepatitis, liver cirrhosis, Wilson disease, liver cancer, and fatty liver. However, many further studies are needed to ensure the efficacy of these drugs on a large scale.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent liver disorder with significant health implications. N6-methyladenosine (m6A) methyltransferase is known to exert regulatory functions in liver-related diseases. This study investigates the intricate role of RNA binding motif protein 15 (RBM15) in modulating inflammation and oxidative stress in NAFLD. An NAFLD model was induced in mice (male, C57BL/6J, 72 mice in the sham group) through a high-fat diet for 9 weeks, and hepatocytes were exposed to long chain-free fatty acids. The expression levels of RBM15, ring finger protein 5 (RNF5), and rho-kinase 1 (ROCK1) were assessed. RBM15 expression was intervened (injection of AAV9 virus at week 9 and detection at week 11). Liver damage was evaluated using staining assays, along with assessments of weight changes and lipid levels. Notably, RBM15 (decreased approximately 40%/60%) and RNF5 (decreased approximately 60%/75%) were poorly expressed while ROCK1 (increased approximately 2.5-fold) was highly expressed in liver tissues and cells. RBM15 overexpression mitigated liver damage, inflammation, and oxidative stress in NAFLD mice, resulting in reduced liver-to-body weight ratio (20%) and decreased levels of alanine aminotransferase (54%), aspartate aminotransferase (36%), total cholesterol (30%), and triglycerides (30%), and inhibited inflammation and oxidative stress levels. Mechanistically, RBM15 upregulated RNF5 expression through m6A methylation modification, and RNF5 suppressed ROCK1 protein levels through ubiquitination modification. RNF5 knockdown or ROCK1 overexpression accelerated inflammation and oxidative stress in NAFLD. Taken together, RBM15 upregulated RNF5 expression through m6A methylation modification. RNF5 inhibited ROCK1 expression through ubiquitination modification to mitigate NAFLD.

The pathogenesis of hepatic fibrosis is driven by dysregulated metabolism precipitated by chronic inflammation. Rho-associated coiled-coil-containing protein kinases (ROCKs) have been implicated in these processes, however the ability of selective ROCK2 inhibition to target simultaneously profibrotic, pro-inflammatory and metabolic pathways remains undocumented. Here we show that therapeutic administration of GV101, a selective ROCK2 inhibitor with more than 1000-fold selectivity over ROCK1, attenuates established liver fibrosis induced by thioacetamide (TAA) in combination with high-fat diet in mice. GV101 treatment significantly reduces collagen levels in liver, associated with downregulation of pCofilin, pSTAT3, pAkt, while pSTAT5 and pAMPK levels are increased in tissues of treated mice. In vitro, GV101 inhibits profibrogenic markers expression in fibroblasts, adipogenesis in primary adipocytes and TLR-induced cytokine secretion in innate immune cells via targeting of Akt-mTOR-S6K signaling axis, further uncovering the ROCK2-specific complex mechanism of action and therapeutic potential of highly selective ROCK2 inhibitors in liver fibrosis.

Obesity increases the severity of airway hyperresponsiveness (AHR) in individuals with asthma, but the mechanism is not well elucidated. G-protein coupled receptor 40 (GPR40) has been found to induce airway smooth muscle contraction after activated by long-chain fatty acids (LC-FFAs), suggesting a close correlation between GPR40 and AHR in obese. In this study, C57BL/6 mice were fed a high-fat diet (HFD) to induce obesity with or without ovalbumin (OVA) sensitization, the regulatory effects of GPR40 on AHR, inflammatory cells infiltration, and the expression of Th1/Th2 cytokines were evaluated by using a small-molecule antagonist of GPR40, DC260126. We found that the free fatty acids (FFAs) level and GPR40 expression were greatly elevated in the pulmonary tissues of obese asthmatic mice. DC260126 greatly reduced methacholine-induced AHR, ameliorated pulmonary pathological changes and decreased inflammatory cell infiltration in the airways in obese asthma. In addition, DC260126 could down-regulate the levels of Th2 cytokines (IL-4, IL-5, and IL-13) and pro-inflammatory cytokines (IL-1β, TNF-α), but elevated Th1 cytokine (IFN-γ) expression. In vitro, DC260126 could remarkedly reduce oleic acid (OA)-induced cell proliferation and migration in HASM cells. Mechanistically, the effects that DC260126 alleviated obese asthma was correlated with the down-regulation of GTP-RhoA and Rho-associated coiled-coil-forming protein kinase 1 (ROCK1). Herein, we proved that targeting of GPR40 with its antagonist helped to mitigate multiple parameters of obese asthma effectively.

Obesity and associated complications increasingly jeopardize global health and contribute to the rapidly rising prevalence of type 2 diabetes mellitus and obesity-related diseases. Developing novel methods for the prevention and treatment of excess body adipose tissue expansion can make a significant contribution to public health. Rho kinase is a Rho-associated coiled-coil-containing protein kinase (Rho kinase or ROCK). The ROCK family including ROCK1 and ROCK2 has recently emerged as a potential therapeutic target for the treatment of metabolic disorders. Up-regulated ROCK activity has been involved in the pathogenesis of all aspects of metabolic syndrome including obesity, insulin resistance, dyslipidemia and hypertension. The RhoA/ROCK-mediated actin cytoskeleton dynamics have been implicated in both white and beige adipogenesis. Studies using ROCK pan-inhibitors in animal models of obesity, diabetes, and associated complications have demonstrated beneficial outcomes. Studies via genetically modified animal models further established isoform-specific roles of ROCK in the pathogenesis of metabolic disorders including obesity. However, most reported studies have been focused on ROCK1 activity during the past decade. Due to the progress in developing ROCK2-selective inhibitors in recent years, a growing body of evidence indicates more attention should be devoted towards understanding ROCK2 isoform function in metabolism. Hence, studying individual ROCK isoforms to reveal their specific roles and principal mechanisms in white and beige adipogenesis, insulin sensitivity, energy balancing regulation, and obesity development will facilitate significant breakthroughs for systemic treatment with isoform-selective inhibitors. In this review, we give an overview of ROCK functions in the pathogenesis of obesity and insulin resistance with a particular focus on the current understanding of ROCK isoform signaling in white and beige adipogenesis, obesity and thermogenesis in adipose tissue and other major metabolic organs involved in energy homeostasis regulation.