Network pharmacology and transcriptomic profiling elucidate the therapeutic effects of Ranunculus ternatus Thunb on liver fibrosis via MK3-NF-κB inhibition.

Activation of hepatic stellate cells (HSCs) is critical in the progression of liver fibrosis and is a promising target for anti-hepatic fibrosis drug development. Moreover, effective pharmacological interventions targeting this pathomechanism are scarce. Our study confirms the therapeutic value of ß-sitosterol, a major constituent of Ranunculus ternatus Thunb, in hepatic fibrosis and identifies its underlying mechanisms. After treatment with ß-sitosterol, CCL4-induced hepatic fibrosis was reversed in mice, while inflammatory and hepatic fibrosis indices were improved. Meanwhile, we explored the molecular mechanism of ß-sitosterol treatment for hepatic fibrosis and, based on RNA-seq results, found that the ameliorative effect of ß-sitosterol on hepatic fibrosis was associated with the MK3 and NF-κB signalling pathways. MK3, an important kinase in the MAPK pathway, plays a role in transmitting upstream and downstream signals, whereas the NF-κB signalling pathway has been shown to be associated with HSC activation. We verified the interaction between MK3 and IκB in HSC cells using endogenous Co-IP, whereas ß-sitosterol reduced the binding of MK3 to IκB and the activation of the NF-κB signalling pathway. Our findings reveal the mechanism of ß-sitosterol in the treatment of liver fibrosis, suggesting that ß-sitosterol may be a promising drug for the treatment of liver fibrosis and deserves further investigation.

Catalpol ameliorates liver fibrosis via inhibiting aerobic glycolysis by EphA2/FAK/Src signaling pathway.

BACKGROUND: Hepatic fibrosis is a pathological process in a variety of acute or chronic liver injuries. Catalpol (CAT), an iridoid glycoside found in Rehmannia glutinosa, has several pharmacological properties, including anti-inflammatory, antidiabetic and anti-fibrotic effects. Nevertheless, there is currently no report on whether CAT regulates the aerobic glycolysis of hepatic stellate cells (HSCs) to inhibit liver fibrosis. OBJECTIVE: This study aimed to investigate the protective effects of CAT on hepatic fibrosis and elucidate its underlying mechanisms. METHODS: To explore whether CAT improved liver fibrosis in vivo and in vitro, hepatic fibrosis was induced to mice by intraperitoneally injecting carbon tetrachloride (CCl4). Additionally, LX-2 cells were stimulated with transforming growth factor-ß (TGF-ß) to simulate fibrosis in vitro. Serum markers of liver injury were examined by using an automatic biochemical analyzer. Histopathological staining, Immunofluorescence (IF) staining, Western blot (WB) analysis, co-immunoprecipitation (Co-IP), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), etc. were employed to identify the targeting between CAT and EphA2 and detect the expression of aerobic glycolysis related proteins, fiber markers and signaling pathways that are responsible for CAT's anti-fibrotic effects of CAT. RESULTS: Results showed that CAT significantly inhibited hepatic injury, fibrogenesis and inflammation in mice treated with CCl4. This was demonstrated by the enhancement of fibrosis markers, liver function indices, and histopathology. In addition, CAT significantly inhibited the activation of HSCs in TGF-ß-induced LX-2 cells, as indicated by decreased proliferation, migration, and expression of collagen I and a-SMA. The study results also suggested that CAT may exert anti-fibrotic effects by inhibiting glycolysis in activated HSCs and in CCl4-treated mice. Mechanistically, CAT directly targets Ephrin type-A receptor 2 (EphA2) to reduce binding with focal adhesion kinases (FAK) and significantly inhibits the FAK/Src pathway. In addition, the pharmacological inhibition of EphA2 cannot further increase the therapeutic effects of CAT on liver fibrosis in vivo and in vitro. CONCLUSION: The study findings generally demonstrated that CAT presented a novel therapeutic method to treat hepatic fibrosis; this method which inhibits the aerobic glycolysis of activated HSCs through the EphA2/FAK/Src signaling pathway.

Bile acids induce liver fibrosis through the NLRP3 inflammasome pathway and the mechanism of FXR inhibition of NLRP3 activation.

BACKGROUND: Altered patterns of bile acids (BAs) are frequently present in liver fibrosis, and BAs function as signaling molecules to initiate inflammatory responses. Therefore, this study was conducted to uncover the notably altered components of BAs and to explore the pathway of altered BA induced inflammation in the development of liver fibrosis. METHODS: Bile acids were quantified by ultraperformance liquid chromatography coupled to mass spectrometry (UPLC‒MS/MS). Cell Counting Kit-8 assays were used to determine the proliferative capacity of HSCs. Transwell assays and wound healing assays were used to determine the migratory capacity of LX2 cells. Protein expression was evaluated by western blotting. RESULTS: Plasma bile acid analysis showed higher levels of GCDCA, TCDCA, GCA and TCA in patients with liver fibrosis than in normal controls. The AUC of GCDCA was the highest. Western blotting showed that GCDCA treatment increased the expression of NLRP3-related proteins and collagen1 in vitro and significantly increased LX2 cells proliferation and migration. Furthermore, knockdown of NLRP3 or overexpression of FXR in LX2 cells decreased the expression of the above proteins, and FXR inhibited NLRP3 (ser 295) phosphorylation in vitro and vivo. In vivo, HE, Masson's trichrome, and Sirius Red staining showed that GCDCA increased collagen fibers in the mouse liver, and the expression of NLRP3-related proteins, collagen 1, and α-SMA in the liver increased significantly. However, the knockout of NLRP3 reversed these patterns. CONCLUSION: (1) Primary conjugated bile acids increased in patients with liver fibrosis; (2) GCDCA induce hepatic fibrosis via the NLRP3 inflammasome pathway; (3) FXR inhibits NLRP3 activity by restraining its phosphorylation; (4) knockdown or knockout of NLRP3 may relieve the onset of hepatic fibrosis.

CLDN5 identified as a biomarker for metastasis and immune infiltration in gastric cancer via pan-cancer analysis.

BACKGROUND: CLDN5 protein is essential for the formation of tight junctions in epithelial cells, and has been associated with epithelial-mesenchymal transition. Research has indicated that CLDN5 is associated with tumor metastasis, the tumor microenvironment, and immunotherapy in multiple types of cancer. Also, no comprehensive evaluation of the expression of CLDN5 and immunotherapy signatures through a pan-cancer analysis or immunoassay has been performed. METHODS: We explored CLDN5's differential expression, survival analysis and clinicopathological staging through the TCGA database, and then corroborated the expression of CLDN5 by utilizing the GEO (Gene expression omnibus) database. To analyze CLDN5 KEGG, GO, and Hallmark mutations, as well as TIMER for immune infiltration, GSEA was utilized with ROC curve, mutation, and other factors such as survival, pathological stage, TME, MSI, TMB, immune cell infiltration, and DNA methylation. Immunohistochemistry was used to assess CLDN5 staining in gastric cancer tissues and paracancerous tissues. Visualization was done with R version 4.2.0 (http://www.rproject.org/). RESULTS: According to TCGA database, CLDN5 expression levels differed significantly between cancer and normal tissues, and the GEO database (GSE49051 and GSE 64951) and tissue microarrays confirmed this result. Infiltrating cluster of differentiation 8+ (CD8+) T cells, CD4+ cells, neutrophils, dendritic cells, and macrophages revealed a correlation with CLDN5 expression. DNA methylation, TMB, and MSI are related to CLDN5 expression. Based on the ROC curve analysis, CLDN5 demonstrates outstanding diagnostic effectiveness for gastric cancer and is comparable to CA-199. CONCLUSIONS: The findings suggest that CLDN5 is implicated in the oncogenesis of diverse cancer types, underscoring its potential significance in cancer biology. Notably, CLDN5 could have implications in immune filtration and immune checkpoint inhibitor therapies, however, further research is needed to confirm this.