Hepatocyte-specific NR5A2 deficiency induces pyroptosis and exacerbates non-alcoholic steatohepatitis by downregulating ALDH1B1 expression

Abstract

Nonalcoholic steatohepatitis (NASH) is a prevalent chronic disease, yet its exact mechanisms and effective treatments remain elusive. Nuclear receptor subfamily 5 group A member 2 (NR5A2), a transcription factor closely associated with cholesterol metabolism in the liver, has been hindered from comprehensive investigation due to the lethality of NR5A2 loss in cell lines and animal models. To elucidate the role of NR5A2 in NASH, we generated hepatocyte-specific knockout mice for Nr5a2 (Nr5a2^HKO) and examined their liver morphology across different age groups under a regular diet. Furthermore, we established cell lines expressing haploid levels of NR5A2 and subsequently reintroduced various isoforms of NR5A2. In the liver of Nr5a2^HKO mice, inflammation and fibrosis spontaneously emerged from an early age, independent of lipid accumulation. Pyroptosis occurred in NR5A2-deficient cell lines, and different isoforms of NR5A2 reversed this form of cell death. Our findings unveiled that inhibition of NR5A2 triggers pyroptosis, a proinflammatory mode of cell death primarily mediated by the activation of the NF-κB pathway induced by reactive oxygen species (ROS). As a transcriptionally regulated molecule of NR5A2, aldehyde dehydrogenase 1 family member B1 (ALDH1B1) participates in pyroptosis through modulation of ROS level. In conclusion, the diverse isoforms of NR5A2 exert hepatoprotective effects against NASH by maintaining a finely tuned balance of ROS, which is contingent upon the activity of ALDH1B1.

Fig. 1. NR5A2 expression decreased in the liver of patients with NASH and mouse models of NASH.

A–C Western blotting (A), statistical analysis (B), and RT-qPCR assay (C) were performed to evaluate the expression of NR5A2 in the livers of NASH patients and control group. N = 6/group, ns not significant. Data are presented as means ± SD. D–F Western blotting (D), statistical analysis (E), and RT-qPCR assay (F) were conducted to assess liver Nr5a2 expression in BKS-db mice and BKS-NC mice. N = 6/group, **P < 0.01, ***P < 0.001. Data are presented as means ± SD. G, H Immunohistochemical staining (G) and RT-qPCR assay (H) were employed to detect Nr5a2 expression in the HFD and HFD + CCL4 induced NASH mouse model. N = 5–6/group, ****P < 0.0001. Data are presented as means ± SD. I RT-qPCR was used to measure NR5A2 mRNA levels in LO2 and AML12 cell lines stimulated with different concentrations of cholesterol for 24 h. N = 6/group, *P < 0.05. Data are presented as means ± SD.

Fig. 2. The hepatic inflammation and fibrosis preceded lipid accumulation in Nr5a2^HKO mice.

A Liver morphology of 32-week-old and 12-week-old Nr5a2^HKO mice and their littermate was examined. B Serum levels of LDL cholesterol and HDL cholesterol were measured in the 32-week-old Nr5a2^HKO mice model. N = 6/group, **P < 0.01, ****P < 0.0001. Data are presented as means ± SD. C, D RT-qPCR analysis was performed to assess the expression of genes related to lipid metabolism (C) (2 technical replicates), as well as inflammation and fibrosis (D), in the liver of 12-week-old Nr5a2^HKO mice model. N = 6/group, *P < 0.05, **P < 0.01, ****P < 0.0001. Data are presented as means ± SD. E The expression levels of Tnf-α and Il-1β were determined by RT-qPCR in the liver of 32-week-old Nr5a2^HKO mice model. N = 6/group, ns, not significant, *P < 0.05. Data are presented as means ± SD. F Liver paraffin sections from 5-week-old and 8-week-old Nr5a2^HKO mice model were stained with HE and sirius red for histological examination. G, H Respiratory exchange ratio was measured in the 12-week-old (G) and 32-week-old (H) Nr5a2^HKO mice model. N = 6/group, data are presented as means ± SD. I Insulin tolerance test (ITT) and glucose tolerance test (GTT) were conducted on the 32-week-old Nr5a2^HKO mice model. N = 6/group, data are presented as means ± SD.

Fig. 3. Pyroptosis was observed in the hepatocytes of Nr5a2^HKO mice, as evidenced by the occurrence of cell death in NR5A2-deficient cell lines.

A A schematic diagram illustrating the design of NR5A2 knockout in LO2 cell line. B RT-qPCR analysis was performed to determine the expression level of NR5A2 in the LO2-NR5A2^1in9397-C4 cell line. LentiV2 refer to the LentiCRISPR-v2 vector. N = 8/group, ****P < 0.0001. Data are presented as means ± SD. C Morphological changes associated with cell death were observed using optical microscopy during the monoclonal cell line cultivation. D Morphological changes associated with cell death were observed using transmission electron microscope (TEM) in the LO2-LentiV2 (control group), LO2-NR5A2^1in9397-C4 and LO2-NR5A2^2ex85 cell lines, Scale bars, 2 μm and 200 nm. E Heatmap representation of genes related to pyroptosis based on RNA-seq data obtained from 5-week-old Nr5a2^HKO mice model, N = 3/group. F Western blotting was conducted to assess the expression levels of pyroptosis molecules in the liver tissue of 5-week-old Nr5a2^HKO mice model. G Immunohistochemical staining of Cleaved-Il-1β in the liver paraffin sections of 12-week-old Nr5a2^HKO mice model. H, I Lactic dehydrogenase (LDH) release of LO2-NR5A2^1in9397-C4 (H), LO2-NR5A2^2ex85 (H), AML12-Nr5a2⁶ⁱⁿ³²⁷ (I) and their control cell lines. N = 3/group, **P < 0.01, ****P < 0.0001. Data are presented as means ± SD. J, K Western blotting analysis was performed to evaluate key molecules involved in the pyroptosis process in the LO2-NR5A2^1in9397 (J), LO2-NR5A2^2ex85 (J) and AML12-Nr5a2⁶ⁱⁿ³²⁷ (K) cell lines, respectively.

Fig. 4. ROS serves as a pivotal factor in pyroptosis activation through NF-κB pathway in the context of NR5A2 deficiency.

A, B Western blotting analysis was performed to detect the expression of pyroptosis-related molecules in the LO2-NR5A2^1in9397 (A) and AML12- Nr5a2⁶ⁱⁿ³²⁷ (B) cell lines after treated with gradient concentrations of inhibitors BAY11-7082 and MCC950. C, D GSEA was conducted to analyze the NF-κB pathway (C) and inflammation (D) using RNA-seq data from 5-week-old Nr5a2^HKO mice model. E KEGG Enrichment Scatterplot of 1285 different expressed genes (DEGs), based on the RNA-seq data from 5-week-old Nr5a2^HKO mice model. F MDA content assay was performed to measure liver and serum levels in the 5-week-old Nr5a2^HKO mice model. N = 3/group, ns, not significant, *P < 0.05. Data are presented as means ± SD. G Serum MDA content assay of 8-week-old Nr5a2^HKO mice model (3 technical replicates). N = 3/group, *P < 0.05. Data are presented as means ± SD. H ROS assay was carried out in the LO2-NR5A2^1in9397-C4 cell line and its negative control. N = 7/group, ****P < 0.0001. Data are presented as means ± SD.

Fig. 5. ALDH1B1 exhibited downregulation in the liver of Nr5a2^HKO mice and was found to modulate the NF-κB signaling pathway.

A Volcano plot. The plot displayed -log10(P value) versus log2(fold change) of 1285 differentially expressed genes (DEGs), with 1055 genes upregulated and 230 genes downregulated in the liver of 5-week-old Nr5a2^HKO mice compared to their littermates. B ROS assay of LO2 cell line overexpressing ALDH1B1. CHO: cholesterol. Cholesterol was used at a concentration of 80 μg/mL for 24 h. pCDH refers to pCDH_EF1a-IRES-puro_BsrG1 vector. N = 3/group, *P < 0.05. Data are presented as means ± SD. C–F Western blotting (C), its statistical analysis (D), RT-qPCR assay (E), and immunohistochemical staining (F) were performed to evaluate Aldh1b1 expression in the liver of 12-week-old Nr5a2^HKO mice model. N = 6/group, **P < 0.01, ****P < 0.0001. Data are presented as means ± SD. G, H Immunofluorescence staining was conducted to examine ALDH1B1 and phosphorylated NF-κB p65 levels in the AML12 (G) and LO2 (H) cell lines overexpressing ALDH1B1. Cholesterol was used at a concentration of 80 μg/mL for 24 h in the LO2 cell line, while it was used at a concentration of 320 μg/mL for 24 h in the AML12 cell line.

Fig. 6. NR5A2 transcriptionally upregulates the expression of ALDH1B1.

A, B Schematic representation of predicted NR5A2 binding sites in the promoter region of human (A) and mouse (B) ALDH1B1, as assessed by the dual luciferase reporter assay. The promoter region spans from −2000 bp to +100 bp relative to the transcriptional start site in human and from −2079 bp to +100 bp in mice. h(m)AP-NBS: NR5A2 binding site within the promoter region of human (mouse) ALDH1B1. (C-D) Evaluation of NR5A2 transcription activity on truncated versions of human (C) and mouse (D) ALDH1B1 promoter regions using a dual-luciferase reporter system. h(m)AP: promoter region of human (mouse) ALDH1B1. 2/3 h(m)AP: The second or third truncation of the promoter region. N = 3/group, ns, not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001. Data are presented as means ± SD. E Schematic representation of NR5A2 binding site mutations in the promoter region of ALDH1B1. F, G Dual-luciferase reporter system employed to analyze the transcriptional activity of NR5A2 on the mutated human (F) and mouse (G) ALDH1B1 promoter region. h(m)AP-mut1/2/3 indicates mutation at the first, second or third predicted binding site was mutant in human (mice). Human: N = 3/group, mouse: N = 6/group, ns not significant, ***P < 0.001, ****P < 0.0001. Data are presented as means ± SD. H Western blotting analysis of ALDH1B1 expression in the LO2-NR5A2^1in9397-C4 and AML12-Nr5a2⁶ⁱⁿ³²⁷ cell lines. I RT-qPCR quantification of ALDH1B1 expression in the LO2-NR5A2^1in9397, LO2-NR5A2^2ex85 and AML12-Nr5a2⁶ⁱⁿ³²⁷ cell lines. LO2: N = 3/group, AML12: N = 4/group, *P < 0.05. Data are presented as means ± SD. J, K RT-qPCR assessment (J) and western blotting analysis (K) of ALDH1B1 expression in the LO2 and AML12 cell lines overexpressing NR5A2 isoform1. LO2: N = 6/group, AML12: N = 3/group, **P < 0.01, ****P < 0.0001. Data are presented as means ± SD.

Fig. 7. ALDH1B1 can ameliorate the activation of pyroptosis, and NR5A2 different isoforms exhibit a similar function.

A, B Western blotting analysis was performed to detect the expression of ALDH1B1 in the livers of NASH patients and control group (A), followed by statistical analysis (B). N = 6/group, data are presented as means ± SD. C The mRNA level of ALDH1B1 was measured in the livers of NASH patients and control group. N = 6/group, data are presented as means ± SD. D Immunohistochemistry staining was conducted to examine the expression of Aldh1b1 in the liver paraffin sections of BKS-db mice and BKS-NC mice. E, F The protein expression of pyroptosis molecules were assessed after overexpressing ALDH1B1 in the NR5A2^1in9397-C4 LO2 cell line (E) and AML12-Nr5a2⁶ⁱⁿ³²⁷ cell line (F). G, H The western blotting analysis was performed to evaluate the expression levels of ALDH1B1 and pyroptosis molecules after replenishment with different isoforms of NR5A2 in the LO2-NR5A2^1in9397-C4 cell line (G) and AML12-Nr5a2⁶ⁱⁿ³²⁷ cell line (H). I, J RT-qPCR was conducted to measure TGF-β mRNA expression in the LO2 cell line (I) and AML12 cell line (J), following haploid knockout or replenishment of NR5A2 with different isoforms. LO2: N = 4/group, AML12: N = 3/group, ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are presented as means ± SD.