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. 2022 May;12(5):2300-2314.
doi: 10.1016/j.apsb.2021.12.007. Epub 2021 Dec 18.

"VSports" Celastrol induces ferroptosis in activated HSCs to ameliorate hepatic fibrosis via targeting peroxiredoxins and HO-1

Piao Luo  1   2 , Dandan Liu  1 , Qian Zhang  1 , Fan Yang  3   4 , Yin-Kwan Wong  1 , Fei Xia  1 , Junzhe Zhang  1 , Jiayun Chen  1 , Ya Tian  1 , Chuanbin Yang  3 , Lingyun Dai  3 , Han-Ming Shen  5 , Jigang Wang  1   2   3   4   6
Affiliations

"VSports app下载" Celastrol induces ferroptosis in activated HSCs to ameliorate hepatic fibrosis via targeting peroxiredoxins and HO-1

Piao Luo et al. Acta Pharm Sin B. 2022 May.

Abstract

Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron- and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases VSports手机版. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species (ROS) and inducing ferroptosis in activated hepatic stellate cells (HSCs). By using activity-based protein profiling (ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay (CETSA), we show that celastrol directly binds to peroxiredoxins (PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6, through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1 (HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2, PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis. .

Keywords: ABPP; ABPP, activity-based protein profiling; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Anti-oxidant; CCl4, carbon tetrachloride; CETSA, cellular thermal shift assay; COL1A1, collagen type I alpha-1; COX-2, cyclooxygenase 2; Cel-P, celastrol-probe; Celastrol; ECM, extracellular matrix; Ferroptosis; GPX4, glutathione peroxidase 4; HCC, hepatocellular carcinoma; HMGB1, high mobility group protein B1; HO-1; HO-1, heme oxygenase 1; HSCs, hepatic stellate cells; Hepatic fibrosis; LPO, lipid peroxidation; PPARγ, peroxisome proliferators-activated receptor γ; PRDXs, peroxiredoxins; Peroxiredoxin; ROS, reactive oxygen species; Reactive oxygen species; VDACs, voltage-dependent anion channels; VIM, vimentin; α-SMA, alpha smooth muscle actin V体育安卓版. .

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"V体育官网入口" Figures

Image 1
Graphical abstract
Figure 1
Figure 1
Celastrol ameliorates liver injury and fibrosis. (A) Tripterygium wilfordii plant and chemical structure of celastrol (Cel). (B) The scheme of animal experiment. The grouping was as follows: Control; Model (CCl4, without Cel treatment); Cel-0.25 (CCl4, 0.25 mg/kg Cel); Cel-0.5 (CCl4, 0.5 mg/kg Cel); Cel-1.0 (CCl4, 1 mg/kg Cel). (C) Representative photographs of liver and its H&E staining (scale bar = 50 μm). (D) The percentage ratios of liver/body weight (mean ± SEM, n = 5) and the effects of Cel on the levels of serum ALT, AST and ALP in mice (mean ± SEM, n = 8; #P < 0.05, ###P < 0.001 vs. Control; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Model). (E) Liver sections with Masson and Sirius Red staining (Scale bar = 50 μm). (F) Immunofluorescence staining of α-SMA (green) in liver tissues (Scale bar = 100 μm). (G) Immunoblotting assay of the levels of α-SMA, VIM and COL1A1 proteins in vivo (mean ± SEM, n = 3; ##P < 0.01, ###P < 0.001 vs. Control; ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Model). P values are calculated by one-way ANOVA followed by the Tukey's test.
Figure 2
Figure 2
Celastrol exerts anti-fibrosis effect by inducing ferroptosis in activated-HSCs. (A) Levels of Fe2+/Fe3+ and LPO after celastrol treatment in human hepatic stellate LX-2 cells (mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01 vs. DMSO). (B) Levels of GSH and H2O2 after celastrol treatment in LX-2 cells (mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01 vs. DMSO). (C) Immunofluorescence staining of intracellular ROS (green) in LX-2 cells (scale bar = 100 μm). (D, E) The effect of DFO and/or NAC on celastrol-induced ferroptosis (as indicated by two ferroptosis-associated markers: Fe2+/Fe3+ and LPO level) in LX-2 cells (mean ± SEM, n = 3; ##P < 0.01, ###P < 0.001 vs. DMSO; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. Cel). (F) The effect of DFO and/or NAC on the anti-fibrosis effect of celastrol, as indicated by the immunoblotting of COL1A1 and α-SMA protein levels, in LX-2 cells. P values are calculated by one-way ANOVA followed by the Tukey's test.
Figure 3
Figure 3
The ABPP in combination with LC–MS/MS approach to profile and identify the targets of celastrol. (A) Chemical structure of celastrol-probe. (B) Inhibition of LX-2 cell proliferation by celastrol (Cel) and celastrol-probe (Cel-P) in a dose-dependent manner (mean ± SEM, n = 3). (C) Immunofluorescence staining of the distribution of Cel-P in LX-2 cells (scale bar = 200 μm). (D) Overall workflow for ABPP profiling of potential celastrol targets. (E) In situ protein labelling with Cel-P in a dose-dependent manner in LX-2 cells. (F) The competition of in situ protein labeling with Cel-P by Cel in LX-2 cells. (G) Bar plot showing the differential enrichment of a few representative target proteins in Cel-P vs. “Compete” (Cel-P + 4 × Cel) group, each point represents one calculated ratio value of Cel-P/Compete (mean ± SD, n = 4).
Figure 4
Figure 4
Celastrol directly binds to PRDX and HO-1 proteins. (A) Pull-down followed by immunoblotting to verify celastrol directly targeting to PRDX or HO-1 proteins in situ. (B) Immunofluorescence staining of PRDX proteins (green) and Cel-P clicked with a red fluorescence dye TAMRA (scale bar = 25 μm). (C) Scheme of CETSA–WB experiment. (D) CETSA–WB experiment to further confirm the interaction between celastrol and PRDX or HO-1 proteins.
Figure 5
Figure 5
Celastrol inhibits the anti-oxidant activities without affecting expression levels of PRDX proteins. (A) Western blot assay of PRDX1, PRDX2, PRDX4 and PRDX6 in LX-2 cells after treatment with Cel and the corresponding densitometry analysis (mean ± SEM, n = 3). (B) Western blot assay of PRDX1, PRDX2, PRDX4 and PRDX6 in CCl4-induced mice treated with or without celastrol and the corresponding densitometry analysis (mean ± SEM, n = 3; #P < 0.05 vs. Control). (C) The peroxidase/anti-oxidant activity of rhPRDXs (30 μmol/L) with or without celastrol (5 μmol/L) in H2O2 reduction assay (mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Cel 0 μmol/L). P values are calculated by unpaired two-tailed t test.
Figure 6
Figure 6
Celastrol binds to the cysteines of PRDX proteins. (A–D) Cel-P labeling of rhPRDX1, rhPRDX2, rhPRDX4 or rhPRDX6 proteins in a dose-dependent manner. (E–H) Cel-P labeling of rhPRDX1, rhPRDX2, rhPRDX4 or rhPRDX6 proteins in the presence or absence of different competitors. (I–L) IAA-yne labeling of rhPRDX1, rhPRDX2, rhPRDX4 or rhPRDX6 proteins in the presence or absence of different competitors (celastrol, Cel; triptolide, TL; or iodoacetamide, IAA).
Figure 7
Figure 7
Celastrol binds to PRDXs at reactive cysteine residues and inhibits their activities. (A) Absorption spectra of celastrol at different concentrations (0–200 μmol/L). (B) Absorption spectra of celastrol (100 μmol/L) after incubation with rhPRDX1, rhPRDX2, rhPRDX4 or rhPRDX6 (15 μmol/L). (C, D) Mutation analysis on cysteine sites of PRDX1 and PRDX2 proteins. WT or mutants recombinant PRDX1 (C) and PRDX2 proteins (D) were incubated with Cel-P for 45 min were incubated with Cel-P for 45 min, clicked with a fluorescent dye, followed by SDS-PAGE analysis and fluorescence scanning.
Figure 8
Figure 8
Induction of ROS and ferroptosis by knockdown of PRDX or HO-1 proteins. (A) The measurement of LPO levels in LX-2 cells after treatment with si-PRDXs, si-HO-1 or the negative control si-NC (mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01 vs. si-NC). (B) The measurement of Fe2+/Fe3+, with the treatments similarly to Fig. 8A (mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01 vs. si-NC). (C) The measurement of GSH levels, with the treatments similarly to Fig. 8A (mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01. si-NC). (D) Immunofluorescence staining of intracellular ROS (green) after treatment with celastrol (1 μmol/L) and/or si-PRDX1, si-PRDX2 (scale bar = 100 μm) and si-PRDX4, si-PRDX6 (scale bar = 50 μm). P values are calculated by one-way ANOVA followed by the Tukey's test.
Figure 9
Figure 9
Schematic summary of the study. (A) Celastrol induces the ferroptosis of activated-HSCs to ameliorate hepatic fibrosis. (B) ABPP strategy discovered that celastrol directly targets to PRDXs and HO-1. (C) Celastrol induces the ferroptosis of activated-HSCs via regulating HO-1, PRDXs and ROS-mediated processes.
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