Yi-Qi-Jian-Pi Formula Suppresses RIPK1/RIPK3-Complex-Dependent Necroptosis of Hepatocytes Through ROS Signaling and Attenuates Liver Injury in Vivo and in Vitro

Abstract

Acute-on-chronic liver failure (ACLF) is described as a characteristic of acute jaundice and coagulation dysfunction. Effective treatments for ACLF are unavailable and hence are urgently required. We aimed to define the effect of Yi-Qi-Jian-Pi Formula (YQJPF) on liver injury and further examine the molecular mechanisms. In this study, we established CCl₄-, LPS-, and d-galactosamine (D-Gal)-induced ACLF rat models in vivo and LPS- and D-Gal-induced hepatocyte injury models in vitro. We found that YQJPF significantly ameliorates liver injury in vivo and in vitro that is associated with the regulation of hepatocyte necroptosis. Specifically, YQJPF decreased expression of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3) and pseudokinase mixed lineage kinase domain-like (MLKL) to inhibit the migration of RIPK1 and RIPK3 into necrosome. YQJPF also reduces the expression of inflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α, which were regulated by RIPK3 mediates cell death. RIPK1 depletion was found to enhance the protective effect of YQJPF. Furthermore, we showed that YQJPF significantly downregulates the mitochondrial reactive oxygen species (ROS) production and mitochondrial depolarization, with ROS scavenger, 4-hydroxy-TEMPO treatment recovering impaired RIPK1-mediated necroptosis and reducing the expression of IL-6, IL-8, IL-1β, and TNF-α. In summary, our study revealed the molecular mechanism of protective effect of YQJPF on hepatocyte necroptosis, targeting RIPK1/RIPK3-complex-dependent necroptosis via ROS signaling. Overall, our results provided a novel perspective to indicate the positive role of YQJPF in ACLF.

Keywords: Yi-Qi-Jian-Pi formula; acute-on-chronic liver failure; hepatocytes necroptosis; inflammation; reactive oxygen species.

FIGURE 1

Establishment of a new model of ACLF in rats and the experimental procedures. Rats were randomly divided into the following seven groups (eight rats in each group).

FIGURE 2

The major components in YQJPF were assessed using HPLC. Calycosin-7-O-β-D-glucoside, ferulic acid, hesperidin, and glycyrrhizic acid were found to be in the amounts of 0.3676, 0.6639, 2.442, and 0.4028 mg/ml, respectively.

FIGURE 3

YPJPF alleviated liver injury in vivo. (A) Ratio of liver weight and body weight. (B–D) TBIL, AST, and ALT levels in serum. (E) Representative pictures of liver tissues and photograph of H&E-stained sections (Scale bars, 100 μm). Data are expressed as mean ± SD (n = 8) *vs. control group, *vs. ACLF group; *p < 0.05, **p < 0.01; ^# p < 0.05, ^## p < 0.01.

FIGURE 4

Protective effect of YQJPF on LPS- and D-Gal-treated hepatocytes in vitro. Human LO2 cells were treated with LPS + D-Gal for 4 h and further treated with YQJPF and atractylone at indicated concentrations. (A,B) MTT assay to study LO2 cell viability. (C) Cell morphology assessment. Scale bar, 200 μm. (D–F) ALT, AST, and LDH levels in LO2 cell culture. Data are expressed as mean ± SD (n = 6). *vs. control group, ^#vs. LPS + D-Gal group; *p < 0.05, **p < 0.01; ^# p < 0.05, ^## p < 0.01.

FIGURE 5

YQJPF inhibited hepatocytic cell death, which was not by apoptosis, in LPS- and D-Gal-treated LO2 cells in vitro. Human LO2 cells were treated with LPS + D-Gal for 4 h and further with YQJPF and atractylone. (A) Trypan blue staining to detect cell death. Scale bar, 100 μm. (B) TUUNEL staining to detect cell apoptosis. (C) Western blot analysis of the protein expression of caspases three and eight and cleaved caspases three and eight in the treated human LO2 cells. (D) cell apoptosis assay by flow cytometer in the treated human LO2 cells. (E) Transmission electron microscopy (TEM) of cells. Black arrows represent swollen mitochondria (M), whereas white arrows represent condensed and marginated chromatins in nuclei (N). Data are expressed as mean ± SD (n = 3). *vs. control group, ^#vs. LPS + D-Gal group; *p < 0.05, **p < 0.01; ^# p < 0.05, ^## p < 0.01.

FIGURE 6

YQJPF inhibited necroptosis of LPS- and D-Gal-treated hepatocytes in vitro. Human LO2 cells were treated with LPS + D-Gal for 4 h and further with YQJPF and atractylone. (A–D) Real-time PCR analyses of genes of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-18. (E) Western blot analysis and quantitative analysis of the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-18 (F–I) Expression levels of IL-1β, IL-6, TNF-α, and IL-18 in LO2 were detected by ELISA. Data are expressed as mean ± SD (n = 3). *vs. control group, ^#vs. LPS + D-Gal group; *p < 0.05, **p < 0.01; ^# p < 0.05, ^## p < 0.01.

FIGURE 7

The RIPK1/RIPK3-complex is required for YQJPF to inhibit hepatocyte necroptosis in vitro. Human LO2 cells were treated with LPS + D-Gal for 4 h and further with YQJPF and atractylone. (A,B) Immunofluorescence staining of RIPK1 and RIPK3 in LO2 cells. Scale bar, 50 μm. (C) Western blot analysis and quantitative assessment of RIPK1, MLKL and RIPK3. (D,E) Expression levels of RIPK1 and RIPK3 in LO2 were detected by ELISA. (F) RIPK1 was immunoprecipitated with its antibody and resulted in co-immunoprecipitation of RIPK3. Immunoprecipitation of RIPK3 with its antibody caused co-immunoprecipitation of RIPK1 in LO2 cells. Data are expressed as mean ± SD (n = 3). *vs. control group, ^#vs. LPS + D-Gal group; *p < 0.05, **p < 0.01; ^# p < 0.05, ^## p < 0.01.

FIGURE 8

YQJPF inhibited hepatocyte necroptosis in RIPK1-dependent manner in vitro. Human LO2 cells were treated with LPS + D-Gal for 4 h and further pre-treated with 50 μM Nec-1 for 1 h, followed by YQJPF or atractylone treatment for 24 h. (A–D) Expression levels of IL-1β, IL-6, TNF-α, and IL-18 in LO2 cells were detected by ELISA. (E) Western blot and quantitative analysis of IL-1β, IL-6, TNF-α, and IL-18. (F) RIPK1 was immunoprecipitated with its antibody and resulted in co-immunoprecipitation of RIPK3. Immunoprecipitation of RIPK3 with its antibody caused co-immunoprecipitation of RIPK1 in LO2 cells. Data are expressed as mean ± SD (n = 3). *vs. control group, ^#vs. LPS + D-Gal group; *p < 0.05, **p < 0.01; ^# p < 0.05, ^## p < 0.01.

FIGURE 9

YQJPF inhibited hepatocyte necroptosis through the inhibition of mitochondrial ROS generation and depolarization in vitro. Human LO2 cells were treated with LPS + D-Gal for 4 h and further pre-treated with 50 μM Nec-1 for 1 h, followed by YQJPF or atractylone treatment for 24 h. (A) Mitochondrial superoxide was detected and quantified through immunofluorescence by using MitoSox Red staining. Scale bar, 50 μm. (B) ROS production was assessed and quantified by DCFH-DA staining, Scale bar, 50 μm. (C–E) MDA, SOD, and GSH levels were measured by corresponding test kits. (F) ROS levels were assessed using ROS test kit. (G) Mitochondrial membrane potential was detected by JC-1 staining. Scale bar, 50 μm. (H) Western blot and quantitative analysis of the expression of IL-1β, IL-6, TNF-α, and IL-18. Data are expressed as mean ± SD (n = 3). *vs. control group, ^#vs. LPS + D-Gal group; *p < 0.05, **p < 0.01; ^# p < 0.05, ^## p < 0.01.

FIGURE 10

Disruption of hepatocyte necroptosis, and not apoptosis, was required for the action of YQJPF in vivo. (A–D) MDA, ATP, GSH, and SOD levels in the rat livers were measured. (E) Western blot and quantitative analysis of cleaved caspase 8, MLKL, RIPK1, and RIPK3 expression in the rat livers. (F) Immunochemical staining and quantitative analysis of caspase 8, MLKL, RIPK1, and RIPK3 in liver tissues. Scale bar, 100 μm. (G–J) Expression levels of IL-1β, IL-6, TNF-α, and IL-18 in the rat livers were detected by ELISA. Data are expressed as mean ± SD (n = 8) *vs. control group, ^#vs. ACLF group; *p < 0.05, **p < 0.01; ^# p < 0.05, ^## p < 0.01.