This study aims to investigate the anticancer effect of Oroxin B (OB) both in vitro and in vivo, and the molecular mechanism involved in microRNA-221 and the PI3K/Akt/PTEN pathway through modulation of apoptosis in Hepatocellular carcinoma (HCC). DEN-induced rats and HepG2 cells based on the microfluidic chip were employed, while the mRNA and protein expression of microRNA-221, PI3K, p-Akt and PTEN were evaluated by RT-PCR and Western blot analysis. Based on Microfluidic Chip and DEN-induced rat model, OB effectively exerts anti-liver cancer effect both in vitro and in vivo, and the expression of miR-221 in OB treated groups was significantly lower than that in the control group (** p < 0.01). The RT-PCR and Western blot results suggested the PI3K mRNA and protein in OB treated groups were both lower than those in control group and indicated the overexpression of PTEN. Therefore, OB effectively exerts anticancer effects by positively regulating the PTEN gene and then inactivating the PI3K/Akt signaling pathway through down-regulating the expression of the microRNA-221, thereby inducing apoptosis of liver cancer cells. This study offers a theoretical evidence for further development and clinical guidance of OB as an anti-tumor agent.

BackgroundAccumulating evidence indicates the fungal fermentation enhanced the health benefits and potential anti-tumor effect of dark tea. However, low bioavailability and unclarified therapeutic mechanisms impeded its deeply application. Plant-derived exosome-like nanoparticles offered a promising option for improving bioavailability and targeted delivery capability.ObjectiveTo obtain dark tea-derived exosome-like nanovesicles and clarify its material basis, investigate the therapeutic effects on hepatocyte carcinoma (HCC) and potential mechanisms.MethodsDark tea-derived exosome-like nanovesicles (DTELNs) were obtained by tangential flow filtration. TEM, NTA, nucleic acid & protein analysis system and LC-MS/MS were used to identify its morphological characteristics and constituent composition. Orthotopic HCC model mice were established and employed for DTELNs’ oral administration. Living imaging, H&E and biochemical assays were used to evaluate therapeutic effects. Untargeted metabolomic, 16S rRNA sequencing and mass cytometry (CyTOF) were used to investigate the alteration of endogenous substances and immune feature. Potential targets were explored by network pharmacology, protein-protein interaction, and verified by western blot. Finally, HCC patient-derived organoids were established to evaluate the clinical applicability of DTELNs.ResultsDTELNs exhibited typical exosomal features, major components including flavonoids, phenolic acids, lipids, amino acids and their derivatives, and miRNAs. DTELNs significantly inhibited tumor growth, modulated gut microbial diversity, recovered the bio-functions of liver-gut axis and enhanced immune responses. DTELNs distinctly inhibited the expressions of p-Akt, NF-κB, bcl-2, cyclin D1, increased the expressions of p-p53, PTEN, Bax, cleaved-caspase 3 and p21. It also exhibited well inhibitory effect on HCC organoids.ConclusionDTELNs demonstrated potent therapeutic efficacy against HCC. The mechanism mainly involved direct regulation of Akt-relevant signalling pathways, rebalance gut microbiota and relevant metabolites, further boost immune anti-tumor responses.