Research on the regulation of ferroptosis in hepatic stellate cells line LX2 by recombinant cytoglobin
Intracellular overexpression of cytoglobin(Cygb)has been shown to reduce extracellular matrix deposition and promote liver fibrosis recovery,but its mechanism is not yet clear.This study constructed and expressed a fusion protein(TAT-Cygb)of cell penetrating peptide TAT and Cygb,to investigate the effect of fusion protein TAT-Cygb on regulating hepatic stellate cells(HSCs)ferroptosis.Cultured human hepatic stellate cells line(LX2)were treated with TAT-Cygb and erastin in vitro,respectively.The effects of ferroptosis phenotype in LX2 cells induced by TAT-Cygb,including cell viability,cell morphology,iron ion(Fe2+)content,lipid peroxidation product levels,and antioxidant system indicators,were investigated using trypan blue staining,transmission electron microscopy,Prussian blue staining,and reagent kits detection.After co-treatment with TAT-Cygb and ferrostain-1,the levels of Fe2+,reactive oxygen species(ROS),malondialdehyde(MDA),4-hydroxynonenal(4-HNE),nicotinamide adenine dinucleotide phosphate(NADPH)and glutathione(GSH)were measured by reagent kits.The protein expression levels of alpha smooth actin(α-SMA),collagen Ⅰ and fibronectin were detected by Western blot,and the protein expression level of epidermal growth factor receptor(EGFR)and desmin relevant to fibrosis were observed by immunofluorescence.The results showed that TAT-Cygb could significantly reduce the viability of LX2 cells and trigger events relevant to ferroptosis,including promoting intracellular Fe2+accumulation,and inducing mitochondrial morphological changes,and intensifying lipid peroxidation products accumulation,and decreasing the level of antioxidant indexes,which played a similar role as erastin;Fer-1 significantly weakened the increase in Fe2+,ROS,MDA,4-HNE levels induced by TAT-Cygb,as well as the decrease in NADPH and GSH levels,while also weakening the TAT-Cygb-induced over-expression levels ofα-SMA,collagen Ⅰ and fibronectin,and TAT-Cygb-induced under-expression levels of EGFR and desmin.This cellular level study indicated that TAT-Cygb can induce ferroptosis of activated HSCs.This study revealed the potential mechanism of TAT-Cygb anti-liver fibrosis,and provided the experimental basis for further research on the molecular mechanism of TAT-Cygb realizing biological function by regulating the ferroptosis pathway.
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