Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways.

BACKGROUND: G-protein coupled receptors (GPCRs) are recognized as attractive targets for drug therapy. However, it remains poorly understood how GPCRs, except for a few chemokine receptors, regulate the progression of liver fibrosis. Here, we aimed to reveal the role of GPR65, a proton-sensing receptor, in liver fibrosis and to elucidate the underlying mechanism. METHODS: The expression level of GPR65 was evaluated in both human and mouse fibrotic livers. Furthermore, Gpr65-deficient mice were treated with either bile duct ligation (BDL) for 21 d or carbon tetrachloride (CCl4) for 8 weeks to investigate the role of GPR65 in liver fibrosis. A combination of experimental approaches, including Western blotting, quantitative real-time reverse transcription­polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA), confocal microscopy and rescue studies, were used to explore the underlying mechanisms of GPR65's action in liver fibrosis. Additionally, the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated. RESULTS: We found that hepatic macrophages (HMs)-enriched GPR65 was upregulated in both human and mouse fibrotic livers. Moreover, knockout of Gpr65 significantly alleviated BDL- and CCl4-induced liver inflammation, injury and fibrosis in vivo, and mouse bone marrow transplantation (BMT) experiments further demonstrated that the protective effect of Gpr65 knockout is primarily mediated by bone marrow-derived macrophages (BMMs). Additionally, in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited, while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and transforming growth factor-ß (TGF-ß), all of which subsequently promoted the activation of hepatic stellate cells (HSCs) and the damage of hepatocytes (HCs). Mechanistically, GPR65 overexpression, the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-α and IL-6 via the Gαq-Ca2+-JNK/NF-κB pathways, while promoted the expression of TGF-ß through the Gαq-Ca2+-MLK3-MKK7-JNK pathway. Notably, pharmacological GPR65 inhibition retarded the development of inflammation, HCs injury and fibrosis in vivo. CONCLUSIONS: GPR65 is a major regulator that modulates the progression of liver fibrosis. Thus, targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

LncRNA Airn maintains LSEC differentiation to alleviate liver fibrosis via the KLF2-eNOS-sGC pathway.

BACKGROUND: Long noncoding RNAs (lncRNAs) have emerged as important regulators in a variety of human diseases. The dysregulation of liver sinusoidal endothelial cell (LSEC) phenotype is a critical early event in the fibrotic process. However, the biological function of lncRNAs in LSEC still remains unclear. METHODS: The expression level of lncRNA Airn was evaluated in both human fibrotic livers and serums, as well as mouse fibrotic livers. Gain- and loss-of-function experiments were performed to detect the effect of Airn on LSEC differentiation and hepatic stellate cell (HSC) activation in liver fibrosis. Furthermore, RIP, RNA pull-down-immunoblotting, and ChIP experiments were performed to explore the underlying mechanisms of Airn. RESULTS: We have identified Airn was significantly upregulated in liver tissues and LSEC of carbon tetrachloride (CCl4)-induced liver fibrosis mouse model. Moreover, the expression of AIRN in fibrotic human liver tissues and serums was remarkably increased compared with healthy controls. In vivo studies showed that Airn deficiency aggravated CCl4- and bile duct ligation (BDL)-induced liver fibrosis, while Airn over-expression by AAV8 alleviated CCl4-induced liver fibrosis. Furthermore, we revealed that Airn maintained LSEC differentiation in vivo and in vitro. Additionally, Airn inhibited HSC activation indirectly by regulating LSEC differentiation and promoted hepatocyte (HC) proliferation by increasing paracrine secretion of Wnt2a and HGF from LSEC. Mechanistically, Airn interacted with EZH2 to maintain LSEC differentiation through KLF2-eNOS-sGC pathway, thereby maintaining HSC quiescence and promoting HC proliferation. CONCLUSIONS: Our work identified that Airn is beneficial to liver fibrosis by maintaining LSEC differentiation and might be a serum biomarker for liver fibrogenesis.

STARD5 as a potential clinical target of hepatocellular carcinoma.

To reveal whether STARD5 is a potential biomarker for diagnosis and prognosis of HCC. Using gene expression omnibus and the cancer genome atlas (TCGA) to screen differentially expressed genes in HCC and STARD5 was selected by LASSO algorithm. Then, we analyzed the association between STARD5 and clinical characteristics of HCC patients in TCGA and International Cancer Genome Consortium. Meanwhile, the mRNA and protein level of STARD5 was also verified by collecting 87 cases of HCC patients' liver tissues using qRT-PCR and WB. Next, we applied gene set enrichment analysis (GSEA) for pathways analysis of STARD5. Finally, TIMER1.0 and TISIDB were used to explore the correlation of STARD5 with immune cell infiltration. The expression of STARD5 was lower in HCC and negatively correlated with tumor grade (p < 0.05), while high expression of STARD5 suggested a better prognosis for HCC patients (p < 0.01) and it could be an independent prognostic predictor (p < 0.001). Meanwhile, STARD5 also had strong diagnostic accuracy for HCC patients. GSEA revealed that STARD5-related genes were mainly enriched in E2F targets, G2M checkpoint and KRAS signaling. The TIMER1.0 and TISIDB databases found a negative correlation between STARD5 and tumor immune infiltrating cells. STARD5 could be used as a potential target for HCC diagnosis and prognosis.

LncRNA-Airn alleviates acute liver injury by inhibiting hepatocyte apoptosis via the NF-κB signaling pathway.

Acute liver injury is a common and serious syndrome caused by multiple factors and unclear pathogenesis. If the injury persists, liver injury can lead to cirrhosis and liver failure and ultimately results in the development of liver cancer. Emerging evidence has indicated that long noncoding RNAs (lncRNAs) play an important role in the development of liver injury. However, the role of antisense Igf2r RNA (Airn) in acute liver injury and its underlying mechanism remain largely unclear. In this study, we show that Airn is upregulated in liver tissue and primary hepatocytes from an acute liver injury mouse model. Consistently, Airn is also overexpressed in serum samples of patients with acute-on-chronic liver failure and is negatively correlated with the Model for End-Stage Liver Disease (MELD) score. Moreover, gene knockout and rescue assays reveal that Airn alleviates CCl 4-induced liver injury by inhibiting hepatocyte apoptosis and oxidative stress in vivo. Further investigation reveals that Airn decreases H 2O 2-induced hepatocyte apoptosis in vitro. Mechanistically, we reveal that Airn represses CCl 4- and H 2O 2-induced enhancement of phosphorylation of p65 and IκBα, suggesting that Airn inhibits hepatocyte apoptosis by inactivating the NF-κB pathway. In conclusion, our results demonstrate that Airn can alleviate acute liver injury by inhibiting hepatocyte apoptosis via inactivating the NF-κB signaling pathway, and Airn could be a potential biomarker for acute liver injury.

HOTAIRM1 promotes osteogenic differentiation and alleviates osteoclast differentiation by inactivating the NF-κB pathway.

Osteoporosis (OP), one of the most prevalent chronic progressive bone diseases, is caused by deficiency in bone formation by osteoblasts or excessive bone resorption by osteoclasts and subsequently increases the risk of bone fractures. Emerging evidence has indicated that long noncoding RNAs (lncRNAs) play key roles in many biological processes and various disorders. However, the role and mechanism of HOX antisense intergenic RNA myeloid 1 (HOTAIRM1), a myeloid-specific lncRNA, in osteoclast differentiation, osteogenic differentiation, and OP remain unclear. In this study, we found that HOTAIRM1 was upregulated during ossification of ligamentum flavum and osteogenic differentiation, while it was downregulated in osteoclast differentiation and in the bone and serum of human and mouse with OP. Further investigation revealed that silencing Hotairm1 decreased the expression of the osteogenic markers and attenuated osteogenesis. Moreover, forced Hotairm1 expression inhibited the expressions of the osteoclastogenesis markers and alleviated receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation. Mechanically, Hotairm1 repressed the phosphorylation of p65 and inhibitor of κBα (IκBα) and attenuated RANKL-mediated enhancement of phos-p65 and IκBα, suggesting that Hotairm1 inhibits RANKL-induced osteoclastogenesis through the NF-κB pathway. In conclusion, our data identified a crucial role of HOTAIRM1 in OP, providing a proof of this molecule as a potential diagnostic marker and a possible therapeutic target against OP.