Forsythiaside A inhibits the progression of intrahepatic cholangiocarcinoma partially via suppressing oxidative stress-induced hepatic stellate cell activation and macrophage polarization.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a highly malignant tumor. Forsythiaside A (FA) has been proven to have a significant anti-hepatic stellate cells (HSCs) activation effect. However, its specific mechanism of action against ICC remains unclear. METHODS: The subcutaneous tumor-bearing model of ICC in C57BL/6 mice was established, and the animals were randomly divided into 5 groups (n = 24/group). The control group was injected with normal saline, and the H₂O₂ group was injected with normal saline containing 40 µg of H₂O₂. The low/medium/high-dose FA groups were respectively injected with normal saline containing 40 µg H₂O₂+20/40/80 mg/kg FA, respectively. HSCs were isolated from the liver tissue of the mice, and primary tumor-associated macrophages (TAMs) were isolated from tumor tissues. Cell proliferation was detected by CCK-8. Protein expression was analyzed by Western blot. The ROS level was detected by DCFH-DA staining. Mitochondrial membrane potential was detected by flow cytometry combined with JC-1 staining. The activity of mitochondrial ATP synthesis was detected by luciferase luminescence method. Macrophages and endothelial cells were simultaneously labeled by immunohistochemical double staining to evaluate the density of the tumor metastasis microenvironment. The expression of Mena invasion (MenaINV) was detected by immunofluorescence staining. In addition, the mechanism of action of FA in the treatment of ICC was analyzed through network pharmacology. RESULTS: FA inhibited the proliferation of HSCs induced by H₂O₂ and the expression levels of activation markers Wnt5a, α-SMA, and COL1A1 in a dose-dependent manner. FA attenuated the mitochondrial dysfunction of HSCs induced by H₂O, and simultaneously upregulated the expressions of PGC1α, TFAM, MFN2, and OPA1 while reducing the expression of DRP1. H₂O₂ promoted the expression of M2 polarization markers CD200R and CCL22 in macrophages, and increased the density of TMEM and the rate of MenaINV positive cells. FA intervention reversed these effects in a dose-dependent manner. Additionally, FA repressed the activation of TGF-β/Smad pathway, PI3K/Akt pathway and HIF-α pathway in HSCs. CONCLUSION: FA exerts anti-ICC effects, at least partially, by inhibiting oxidative stress-induced HSCs activation and macrophage M2 polarization, providing a new potential strategy for ICC treatment.