Icariside II modulates pulmonary fibrosis via PI3K/Akt/ß-catenin pathway inhibition of M2 macrophage program.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a debilitating interstitial lung disorder characterized by its limited therapeutic interventions. Macrophages, particularly the alternatively activated macrophages (M2 subtype), have been acknowledged for their substantial involvement in the development of pulmonary fibrosis. Hence, targeting macrophages emerges as a plausible therapeutic avenue for IPF. Icariside II (ISE II) is a natural flavonoid glycoside molecule known for its excellent anti-tumor and anti-fibrotic activities. Nevertheless, the impact of ISE II on pulmonary fibrosis and the intricate mechanisms through which it operates have yet to be elucidated. OBJECTIVE: To scrutinize the impact of ISE II on the regulation of M2 macrophage polarization and its inhibitory effect on pulmonary fibrosis, as well as to delve deeper into the underlying mechanisms of its actions. METHODS: The effect of ISE II on proliferation and apoptosis in RAW264.7 cells was assessed through the use of EdU-488 labeling and the Annexin V/PI assay. Flow cytometry, western blot, and qPCR were employed to detect markers associated with the M2 polarization phenotype. The anti-fibrotic effects of ISE II in NIH-3T3 cells were investigated in a co-culture with M2 macrophages. Si-Ctnnb1 and pcDNA3.1(+)-Ctnnb1 plasmid were used to investigate the mechanism of targeted intervention. The murine model of pulmonary fibrosis was induced by intratracheal administration of bleomycin (BLM). Pulmonary function, histopathological manifestations, lung M2 macrophage infiltration, and markers associated with pulmonary fibrosis were evaluated. Furthermore, in vivo transcriptomics analysis was employed to elucidate differentially regulated genes in lung tissues. Immunofluorescence, western blot, and immunohistochemistry were conducted for corresponding validation. RESULTS: Our investigation demonstrated that ISE II effectively inhibited the proliferation of RAW264.7 cells and mitigated the pro-fibrotic characteristics of M2 macrophages, exemplified by the downregulation of CD206, Arg-1, and YM-1, Fizz1, through the inhibition of the PI3K/Akt/ß-catenin signaling pathway. This impact led to the amelioration of myofibroblast activation and the suppression of nuclear translocation of ß-catenin of NIH-3T3 cells in a co-culture. Consequently, it resulted in decreased collagen deposition, reduced infiltration of profibrotic macrophages, and a concurrent restoration of pulmonary function in mice IPF models. Furthermore, our RNA sequencing results showed that ISE II could suppress the expression of genes related to M2 polarization, primarily by inhibiting the PI3K/Akt and ß-catenin signaling pathway. In essence, our findings suggest that ISE II holds potential as an anti-fibrotic agent by orchestrating macrophage polarization. This may have significant implications in clinical practice. CONCLUSION: This study has provided evidence that ISE II exerts a significant anti-fibrotic effect by inhibiting macrophage M2 polarization through the suppression of the PI3K/Akt/ß-catenin signaling pathway. These findings underscore the potential of ISE II as a promising candidate for the development of anti-fibrotic pharmaceuticals in the future.

RNA-seq combined network pharmacology reveals that Fu-Gan-Wan (FGW) inhibits liver fibrosis via NF-κB/CCL2/CCR2 and lipid peroxidation via Nrf2/HMOX1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Liver fibrosis is a serious complication of liver disease characterized by excessive collagen deposition, without effective therapeutic agents in the clinic. Fu-Gan-Wan (FGW) is an empirical formula used for the clinical treatment of hepatitis and cirrhosis. It has been shown to reverse experimental liver fibrosis. However, its corresponding mechanisms remain unclear. AIM OF THE REVIEW: This study aimed to elucidate the key pathways and target genes of FGW in attenuating liver fibrosis. MATERIALS AND METHODS: The therapeutic effects of different doses of FGW on liver fibrosis were investigated using a 2 mL/kg 15% CCl4-induced mouse model. Then, RNA-seq combined with network pharmacology was used to analyze the key biological processes and signaling pathways underlying the anti-liver fibrosis exertion of FGW. These findings were validated in a TGF-ß1-induced model of activation and proliferation of mouse hepatic stellate cell line JS-1. Finally, the key signaling pathways and molecular targets were validated using animal tissues, and the effect of FGW on tissue lipid peroxidation was additionally observed. RESULTS: We found that 19.5 g/kg FGW significantly down-regulated CCl4-induced elevation of hepatic ALT and AST, decreased collagen deposition, and inhibited the expression of pro-fibrotic factors α-SMA, COL1α1, CTGF, TIMP-1, as well as pro-inflammatory factor TGF-ß1. Additionally, FGW at doses of 62.5, 125, and 250 µg/mL dose-dependently blocked JS-1 proliferation, migration, and activation. Furthermore, RNA-seq identified the NF-κB signaling pathway as a key target molecular pathway for FGW against liver fibrosis, and network pharmacology combined with RNA-seq focused on 11 key genes. Significant changes were identified in CCL2 and HMOX1 by tissue RT-PCR, Western blot, and immunohistochemistry. We further demonstrated that FGW significantly attenuated CCl4-induced increases in p-p65, CCL2, CCR2, and HMOX1, while significantly elevating Nrf2. Finally, FGW significantly suppressed the accumulation of lipid peroxidation products MDA and 4-HNE and reconfigured the oxidation-reduction balance, including promoting the increase of antioxidants GPx, GSH, and SOD, and the decrease of peroxidation products ROS and GSSG. CONCLUSIONS: This study demonstrated that FGW exhibits potential in mitigating CCl4-induced hepatic fibrosis, lipid peroxidation, and iron metabolism disorders in mice. This effect may be mediated through the NF-κB/CCL2/CCR2 and Nrf2/HMOX1 pathways.