Objective: To evaluate the effect of isorhamnetin on diabetic kidney disease and elucidate its underlying mechanisms.
Methods: A high glucose-stimulated mouse podocyte (MPC-5) model was employed to evaluate the effects of isorhamnetin on podocyte injury, secretion of inflammatory cytokines, NLRP3 inflammasome activation, pyroptosis, mitochondrial function, cytosolic release of mtDNA, and activation of the cGAS-STING signaling pathway. Nigericin (an NLRP3 activator) and siRNA were used in combination to clarify the regulatory relationships between these pathways. Furthermore, a mouse model of diabetic kidney disease was established and treated with isorhamnetin via intragastric administration. Blood glucose and renal function parameters were measured, renal histopathological changes were examined, and alterations in podocyte marker proteins, inflammasome activation, pyroptosis, mitochondrial damage, and the cGAS-STING pathway in kidney tissues were analyzed.
Results: Isorhamnetin alleviated high glucose-induced abnormalities in podocyte marker proteins and cellular injury. It also inhibited NLRP3 inflammasome activation, thereby attenuating pyroptosis. Furthermore, isorhamnetin stabilized mitochondrial membrane potential, decreased mitochondrial ROS production, suppressed excessive opening of the mitochondrial permeability transition pore, alleviated mitochondrial damage, and reduced cytosolic mtDNA release, which in turn suppressed activation of the cGAS-STING pathway. Knockdown of STING inhibited high glucose-induced NLRP3-dependent pyroptosis. Consistently, isorhamnetin improved renal function in mice with diabetic kidney disease, attenuated glomerulosclerosis and fibrosis, and markedly suppressed mitochondrial damage, mtDNA leakage, cGAS-STING activation, and NLRP3-mediated pyroptosis and inflammatory responses in renal tissues.
Conclusions: Isorhamnetin treatment attenuates diabetic kidney disease by improving mitochondrial homeostasis and inhibiting the cGAS-STING/NLRP3 inflammasome pathway.
Objective: To investigate the modulatory effects of a Morus alba leaf extract (FBCC-EP1619) on melanogenesis using enzymatic, cellular, and zebrafish models.
Methods: Phytochemical profiling was conducted using UPLC-QTOF-MS/MS in negative ion mode. Mushroom tyrosinase inhibition was assessed in vitro. Cytotoxicity and melanin content were measured in B16F10 melanoma cells under basal and α-melanocyte-stimulating hormone (α-MSH-stimulated conditions. An in vivo pigmentation model was established using α-MSH-treated zebrafish larvae, and pigmentation was quantified. Expression of melanogenesis-related genes (mitfa and tyr) was analyzed by RT-qPCR.
Results: FBCC-EP1619 contained diverse phenolic and lipid-derived metabolites. The extract significantly inhibited mushroom tyrosinase activity in a concentration-dependent manner. In B16F10 cells, it did not induce cytotoxicity but increased melanin production. In contrast, in α-MSH-stimulated zebrafish larvae, FBCC-EP1619 attenuated hyperpigmentation and significantly downregulated mitfa and tyr expression, indicating system- and stimulus-dependent regulation.
Conclusions: FBCC-EP1619 differentially modulates melanogenesis depending on the biological system, enhancing basal melanin production in vitro while suppressing α-MSH-induced pigmentation in vivo. These findings provide pharmacological support for the ethnopharmacological relevance of Morus alba leaves and warrant further investigation into the underlying molecular mechanisms.
Objective: To investigate whether the ethyl acetate fraction of Cyperus amuricus (C. amuricus) induces autophagy-mediated apoptosis in hepatocellular carcinoma cells via the AMPK and PI3K/Akt/mTOR signaling pathways.
Methods: In vitro experiments were conducted using HepG2 and Huh7 hepatocellular carcinoma cell lines. The ethyl acetate fraction of C. amuricus was profiled by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry and quantitative high-performance liquid chromatography with diode-array detection. In HepG2 cells, cytotoxicity (MTT assay), apoptosis (Annexin V/PI staining, sub-G1 analysis, and Western blot for cleaved caspase-3, Bax/Bcl-2, and cytochrome c), autophagy (acridine orange staining, LC3-II/LC3-I ratio, Beclin-1, Atg5-Atg12, Atg7, DAPK3, and autophagic flux assays with bafilomycin A1), and signaling pathways (p-AMPK and total AMPKα, and PI3K/Akt/mTOR/p70S6K by Western blot) were assessed at 100, 150 and 200 μg/mL for 3-24 h. The role of autophagy in apoptosis was further evaluated using the autophagy inhibitors 3-methyladenine and bafilomycin A1. Cytotoxicity, apoptosis, and autophagy were also assessed in Huh7 cells at 100-200 μg/mL for 24 h.
Results: The ethyl acetate fraction of C. amuricus significantly reduced viability dose-dependently with an IC50 of 150 μg/mL (HepG2) and 165 μg/mL (Huh7), activated intrinsic apoptosis, and induced time-dependent autophagy with confirmed flux (P < 0.05). The fraction markedly enhanced AMPK phosphorylation while suppressing the PI3K/Akt/mTOR/p70S6K signaling (P < 0.05). Cotreatment with 3-methyladenine reduced apoptosis from 30.9% to 26.0% and decreased the levels of p-AMPKα, p-mTOR, Atg7, and Beclin-1, and increased LC3-H/LC3-I ratio (P < 0.05), while bafilomycin A1 co-treatment reduced apoptosis to 24.3% in HepG2 cells and confirmed autophagic flux as a pro-apoptotic mechanism, collectively establishing a functional role of autophagy in C. amuricus fraction-induced cell death.
Conclusions: The ethyl acetate fraction of C. amuricus induces autophagy-mediated apoptosis via AMPK activation and PI3K/Akt/mTOR inhibition. Therefore, it holds great potential as a phytotherapeutic candidate for hepatocellular carcinoma, which requires further in vivo validation.