Taurine attenuates activation of hepatic stellate cells by inhibiting autophagy and inducing ferroptosis

Abstract

Background: Liver fibrosis is a compensatory response during the tissue repair process in chronic liver injury, and finally leads to liver cirrhosis or even hepatocellular carcinoma. The pathogenesis of hepatic fibrosis is associated with the progressive accumulation of activated hepatic stellate cells (HSCs), which can transdifferentiate into myofibroblasts to produce an excess of the extracellular matrix (ECM). Myofibroblasts are the main source of the excessive ECM responsible for hepatic fibrosis. Therefore, activated hepatic stellate cells (aHSCs), the principal ECM producing cells in the injured liver, are a promising therapeutic target for the treatment of hepatic fibrosis.

Aim: To explore the effect of taurine on aHSC proliferation and the mechanisms involved.

Methods: Human HSCs (LX-2) were randomly divided into five groups: Normal control group, platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) treated group, and low, medium, and high dosage of taurine (10 mmol/L, 50 mmol/L, and 100 mmol/L, respectively) with PDGF-BB (20 ng/mL) treated group. Cell Counting Kit-8 method was performed to evaluate the effect of taurine on the viability of aHSCs. Enzyme-linked immunosorbent assay was used to estimate the effect of taurine on the levels of reactive oxygen species (ROS), malondialdehyde, glutathione, and iron concentration. Transmission electron microscopy was applied to observe the effect of taurine on the autophagosomes and ferroptosis features in aHSCs. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the effect of taurine on the expression of α-SMA, Collagen I, Fibronectin 1, LC3B, ATG5, Beclin 1, PTGS2, SLC7A11, and p62.

Results: Taurine promoted the death of aHSCs and reduced the deposition of the ECM. Treatment with taurine could alleviate autophagy in HSCs to inhibit their activation, by decreasing autophagosome formation, downregulating LC3B and Beclin 1 protein expression, and upregulating p62 protein expression. Meanwhile, treatment with taurine triggered ferroptosis and ferritinophagy to eliminate aHSCs characterized by iron overload, lipid ROS accumulation, glutathione depletion, and lipid peroxidation. Furthermore, bioinformatics analysis demonstrated that taurine had a direct targeting effect on nuclear receptor coactivator 4, exhibiting the best average binding affinity of -20.99 kcal/mol.

Conclusion: Taurine exerts therapeutic effects on liver fibrosis via mechanisms that involve inhibition of autophagy and trigger of ferroptosis and ferritinophagy in HSCs to eliminate aHSCs.

Keywords: Autophagy; Ferroptosis; Hepatic stellate cells; Molecular docking; Taurine.

Figure 1

Effects of taurine on extracellular matrix. A: Detection of effect of taurine on the viability of hepatic stellate cells by Cell Counting Kit-8 method; B: Detection of the effect of taurine on mRNA expression of fibronectin 1, collagen I, and α-SMA by reverse transcription-polymerase chain reaction; C: Detection of effect of taurine on protein expression of fibronectin 1, collagen Ι, and α-SMA by Western blot. Data are expressed as the mean ± SD. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^cP < 0.05 vs platelet-derived growth factor-BB (PDGF-BB), ^dP < 0.01 vs PDGF-BB. PDGF-BB: Platelet-derived growth factor-BB.

Figure 2

Taurine inhibits autophagy in hepatic stellate cells. A: Effect of taurine on expression of autophagy-related molecules; B: Taurine decreases the number of autophagosomes. Yellow arrows indicate monolayer or bilayer autophagosomes. Bar = 1 μm. Data are expressed as the mean ± SD. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^cP < 0.05 vs platelet-derived growth factor-BB (PDGF-BB), ^dP < 0.01 vs PDGF-BB. PDGF-BB: Platelet-derived growth factor-BB.

Figure 3

Taurine induces ferroptosis in hepatic stellate cells. A: Effect of taurine on expression of ferroptosis-related molecules; B: Effect of taurine on levels of glutathione and malondialdehyde; C: Effect of taurine on reactive oxygen species; D: Effect of taurine on mitochondrial structure of hepatic stellate cells. Red arrows indicate mitochondria. Bar = 1 μm; E: Taurine induces iron deposition in hepatic stellate cell. Data are expressed as the mean ± SD. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^cP < 0.05 vs platelet-derived growth factor-BB (PDGF-BB), ^dP < 0.01 vs PDGF-BB. PDGF-BB: Platelet-derived growth factor-BB; GSH: Glutathione; MDA: Malondialdehyde.

Figure 4

Molecular docking results of taurine and ferritin heavy chain 1 and nuclear receptor coactivator 4, respectively. FTH1: Ferritin heavy chain 1; NCOA4: Nuclear receptor coactivator 4.

Figure 5

Mechanisms associated with taurine-mediated death and inactivation of hepatic stellate cells in liver fibrosis. A: Inhibition of autophagy; B: Activation of ferritinophagy; C: Induction of ferroptosis. FTH1: Ferritin heavy chain 1; NCOA4: Nuclear receptor coactivator 4; ROS: Reactive oxygen species; HSC: Hepatic stellate cell; GSH: Glutathione.