Journal: Advanced Science

Article Title: Geniposidic Acid Targeting FXR “S332 and H447” Mediated Conformational Change to Upregulate CYPs and miR‐19a‐3p to Ameliorate Drug‐Induced Liver Injury

doi: 10.1002/advs.202409107

Figure Lengend Snippet: GPA activates FXR into the nucleus and up‐regulates CYPs key enzymes to promote bile acid synthesis and efflux. A) Hypotheses for upstream targets that regulate bile acid synthesis and transport. B,C) The mRNA and protein expression of BSEP, CYP7A1, CYP8B1, CYP27A1, and CYP7B1 in L02 hepatocytes induced by GPA treatment with TP ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. D) Schematic representation of potential bile acid receptors that regulate bile acid synthesis and transport. E) mRNA expression of fxr , tgr5 , pxr , vdr , s1pr2 , and their target genes by in L02 hepatocytes induced by GPA treatment with TP ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. F) Protein expression and distribution of hepatic FXR in TP chronic DILI mice treated with GPA. G) Protein expression and distribution of FXR in L02 hepatocytes induced by GPA treatment with TP. H) Oil red O staining with Nile red staining of L02 hepatocytes induced by GPA treatment with TP. I) Immunofluorescence co‐localization of TP‐induced L02 hepatocytes by GPA treatment. J) siFXR interferes with FXR expression in L02 cells. K,L) The mRNA and protein expression of BSEP, CYP7A1, CYP8B1, CYP27A1, and CYP7B1 in siFXR‐L02 hepatocytes induced by GPA treatment with TP ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. M) Oil red O staining with Nile red staining of siFXR‐L02 hepatocytes induced by GPA treatment with TP. Scale bar: 50 µm. All data are presented as means ± SD. Compared with control group, * P < 0.05, ** P < 0.01, *** P < 0.001. Compared with model group, # P < 0.05, ## P < 0.01, ### P < 0.001. All data are via one‐way analysis of variance (ANOVA).

Article Snippet: AML12 (mouse hepatocytes) and L02 (human hepatocytes) cell lines were purchased from Procell (Shanghai, China) and cultured in DMEM/F‐12 and RPMI‐1640 medium respectively supplemented with 10% fetal bovine serum.

Techniques: Expressing, Staining, Immunofluorescence, Control

Journal: Advanced Science

Article Title: Geniposidic Acid Targeting FXR “S332 and H447” Mediated Conformational Change to Upregulate CYPs and miR‐19a‐3p to Ameliorate Drug‐Induced Liver Injury

doi: 10.1002/advs.202409107

Figure Lengend Snippet: GPA targeting of S332 and H447 residues of FXR induces the forming of the active conformation. A) Schematic of the superposition of GPA and GW4064 bound to FXR LBD. B) Schematic of the superposition of GPA and GW4064 bound to the FXR LBD cavity. C) Demonstration of amino acid residues and hydrogen bonding distances for molecular docking of GPA with FXR. D) 2D lash diagram of GPA binding to FXR LBD. E) Conservation analysis of FXR and chromatogram of human FXR LBD wild‐type and mutant histones. F) Heat transfer curves of the wild type and mutant recombinant proteins of human‐derived FXR LBD. G) Heat transfer curves of GPA with human‐derived FXR LBD wild‐type versus mutant recombinant proteins. H) CETSA experiments of GPA with L02 cells overexpressing human FXR FL wild type versus mutant. I) SPR experiments of GPA, GW4064 with human‐derived FXR LBD wild‐type recombinant protein. J) SPR experiments of GPA with human‐derived FXR LBD mutant recombinant protein. K) Alphascreen experiments of GPA and GW4064 with human FXR LBD wild‐type recombinant protein. L,M) Alphascreen experiments of GPA with human FXR LBD wild type versus mutant recombinant protein screening for co‐binding factors ( n = 6). N) SPR experiments of different doses of GW4064 with human‐derived FXR LBD wild‐type recombinant protein recruited to SRC2‐3. O) SPR experiments of different doses of GPA with human‐derived FXR LBD wild‐type and mutant recombinant proteins recruited for SRC2‐3. P) Dual‐luciferase reporter gene experiments of GPA with human‐derived FXR LBD wild type versus mutant recombinant protein ( n = 6). Bsep activation levels were measured by luciferase reporter gene assay, normalized to vehicle group. Q) Summary of the combination of GPA and the wild type/mutant FXR. R) Assumption of GPA activates FXR conformation change. All data are presented as means ± SD. Compared with control group, * P < 0.05, ** P < 0.01, *** P < 0.001. Compared with model group, # P < 0.05, ## P < 0.01, ### P < 0.001. All data are via one‐way analysis of variance (ANOVA).

Article Snippet: AML12 (mouse hepatocytes) and L02 (human hepatocytes) cell lines were purchased from Procell (Shanghai, China) and cultured in DMEM/F‐12 and RPMI‐1640 medium respectively supplemented with 10% fetal bovine serum.

Techniques: Binding Assay, Mutagenesis, Recombinant, Derivative Assay, Amplified Luminescent Proximity Homogenous Assay, Luciferase, Activation Assay, Reporter Gene Assay, Control

Journal: Advanced Science

Article Title: Geniposidic Acid Targeting FXR “S332 and H447” Mediated Conformational Change to Upregulate CYPs and miR‐19a‐3p to Ameliorate Drug‐Induced Liver Injury

doi: 10.1002/advs.202409107

Figure Lengend Snippet: Activation of FXR by GPA upregulates miR19a‐3p binding to LXR 3′UTR involved in cholesterol synthesis. A) Serum FC, HDLC, and LDLC levels in APAP acute DILI mice, TP acute DILI mice, and TP chronic DILI mice ( n = 6). B) Effect of GPA on hepatic shp and ppar mRNA levels in TP‐induced chronic DILI mice ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. C) Schematic diagram of GPA‐targeted FXR regulation of non‐coding RNA‐mediated cholesterol metabolism. D) Wayne diagram of differentially expressed miRNAs. E) Differential miRNA target gene enrichment analysis. F) Differential miRNA target gene GO enrichment bubble diagram. G) miRNA expression in GPA‐treated TP chronic DILI mice ( n = 6). miRNA levels were measured by qPCR, normalized to U6. H) miRNA expression in L02 cells from TP pharmacogenetic liver injury treated with GPA ( n = 6). miRNA levels were measured by qPCR, normalized to U6. I) Hepatic miR‐19a‐3p probe FISH assay in GPA‐treated TP chronic DILI mice. Scale bar: 50 µm. J) Species conservation analysis of miR‐19a‐3p. K) Plasma nuclear expression of miR‐19a‐3p in GPA‐treated TP chronic DILI mice. Plasma miR‐19a‐3p levels were measured by qPCR, normalized to U6. Nuclear miR‐19a‐3p levels were measured by qPCR, normalized to Neat1. L) Plasma‐nuclear expression of miR‐19a‐3p in L02 cells from TP pharmacogenetic liver injury treated with GPA. Plasma miR‐19a‐3p levels were measured by qPCR, normalized to U6. Nuclear miR‐19a‐3p levels were measured by qPCR, normalized to Neat1. M) Assumption of the relationship between GPA activation of FXR and miR‐19a‐3p regulation. N) Expression of miR‐19a‐3p in siFXR‐L02 cells from TP drug‐derived liver injury treated with GPA ( n = 6). miR‐19a‐3p levels were measured by qPCR, normalized to U6. O) Expression of miR‐19a‐3p in GPA‐treated TP drug‐derived liver injury oeFXR‐L02 cells ( n = 6). miR‐19a‐3p levels were measured by qPCR, normalized to U6. P) mRNA expression of fxr in miR‐19a‐3p mimics‐L02 cells of TP DILI treated with GPA ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. Q) mRNA expression of fxr in GPA‐treated TP pharmacogenetic liver injury miR‐19a‐3p inhibitor‐L02 cells ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. R) Protein expression of FXR in GPA‐treated TP drug‐derived liver injury miR‐19a‐3p mimics‐L02 cells. S) Biotin‐labeled miR‐19a‐3p pull‐down assay in L02 and AML‐12 cells ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. All data are presented as means ± SD. Compared with control group, * P < 0.05, ** P < 0.01, *** P < 0.001. Compared with model group, # P < 0.05, ## P < 0.01, ### P < 0.001. All data are via one‐way analysis of variance (ANOVA).

Article Snippet: AML12 (mouse hepatocytes) and L02 (human hepatocytes) cell lines were purchased from Procell (Shanghai, China) and cultured in DMEM/F‐12 and RPMI‐1640 medium respectively supplemented with 10% fetal bovine serum.

Techniques: Activation Assay, Binding Assay, Expressing, Clinical Proteomics, Derivative Assay, Labeling, Pull Down Assay, Control

Journal: Advanced Science

Article Title: Geniposidic Acid Targeting FXR “S332 and H447” Mediated Conformational Change to Upregulate CYPs and miR‐19a‐3p to Ameliorate Drug‐Induced Liver Injury

doi: 10.1002/advs.202409107

Figure Lengend Snippet: GPA targets FXR to activate nuclear miR19a‐3p to inhibit LXR‐mediated cholesterol synthesis. A) Conservation analysis of miR‐19a‐3p and LXR binding sites. B) Sequence of miR‐19a‐3p with LXR 3′UTR binding site in wild type and mutant. C) Luciferase reporter gene assay of miR‐19a‐3p with LXR 3′UTR binding wild type versus mutant ( n = 6). Lxr activation levels were measured by luciferase reporter gene assay, normalized to vehicle group. D) Protein expression of LXR in L02 cells by miR‐19a‐3p mimics or inhibitors. E) mRNA expression of lxr , srebp1c , fasn , scd , srebp1c, fasn, scd, hmgcr, and apob in GPA‐treated TP chronic DILI mice ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. F) mRNA expression of lxr , srebp1c , fasn , scd , srebp1c, fasn, scd, hmgcr, and apob in L02 cells of TP treated with GPA ( n = 6). All mRNA levels were measured by qPCR, normalized to GAPDH. G) CETSA experiments of GPA with L02 cells overexpressing human LXR FL wild type. H) LXR protein expression of GPA or GW4064 with miR‐19a‐3p mimics/inhibitor‐L02 cells of TP DILI treated with GPA. I,J) miR‐19a‐3p mimics or inhibitor on Oil red O staining in L02 cells. K) Schematic representation of GPA targeting FXR to up‐regulate and activate miR‐19a‐3p into the nucleus to inhibit LXR activity and prevent cholesterol synthesis on DILI. Scale bar: 50 µm. All data are presented as means ± SD. Compared with control group, * P < 0.05, ** P < 0.01, *** P < 0.001. Compared with model group, # P < 0.05, ## P < 0.01, ### P < 0.001. All data are via one‐way analysis of variance (ANOVA).

Article Snippet: AML12 (mouse hepatocytes) and L02 (human hepatocytes) cell lines were purchased from Procell (Shanghai, China) and cultured in DMEM/F‐12 and RPMI‐1640 medium respectively supplemented with 10% fetal bovine serum.

Techniques: Binding Assay, Sequencing, Mutagenesis, Luciferase, Reporter Gene Assay, Activation Assay, Expressing, Staining, Activity Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: G protein–coupled estrogen receptor 1 ameliorates nonalcoholic steatohepatitis through targeting AMPK-dependent signaling

doi: 10.1016/j.jbc.2024.105661

Figure Lengend Snippet: GPER1 plays a crucial role in the protective effects of estradiol on lipid accumulation, oxidative stress, and inflammation in L02 cells under metabolic stress. A , representative images of Nile Red staining in L02 cells treated with vehicle or GPER1-specific agonist G1 (100 nM) followed by BSA or PO (palmitic acid and oil acid mixture) stimulation for 12 h (n = 3 independent experiments). Scale bar represents 100 μm. B , triglyceride (TG) and total cholesterol (TC) contents in L02 cells from the indicated group (n = 6). C , relative mRNA levels of factors related to fatty acid metabolism in L02 cells from the indicated group (n = 6). D , representative images of DCFH-DA probe–stained L02 cells in the indicated group (n = 3 independent experiments). Scale bar represents 100 μm. E , representative images of MitoSOX Red probe–stained L02 cells in the indicated group (n = 3 independent experiments). Scale bar represents 100 μm. F , relative mRNA levels of factors related to inflammatory response in L02 cells from the indicated group (n = 6). G , representative images of Nile Red staining of L02 cells challenged by PO and treated with vehicle, 17β-estradiol (E2; 10 nM), GPER1 antagonist G15 (10 μM), or E2 in combination with G15 (n = 3 independent experiments). Scale bar represents 100 μm. H , TG and TC contents in L02 cells from the indicated group (n = 6). I , relative mRNA levels of factors related to fatty acid metabolism in L02 cells from the indicated group (n = 6). J , representative images of DCFH-DA probe–stained L02 cells in the indicated group (n = 3 independent experiments). Scale bar represents 100 μm. K , representative images of MitoSOX Red probe–stained L02 cells in the indicated group (n = 3 independent experiments). Scale bar represents 100 μm. L , relative mRNA levels of factors related to inflammatory response in L02 cells from the indicated group (n = 6). M , immunoblotting analysis of GPER1 protein level in the WT and GPER1 KO L02 cells (n = 3 independent experiments). N , representative images of Nile Red staining in the WT and GPER1 KO L02 cells challenged by PO and cotreated with vehicle or E2 (n = 3 independent experiments). Scale bar represents 100 μm. O , TG and TC contents in L02 cells from the indicated group (n = 6). P , relative mRNA levels of factors related to fatty acid metabolism in L02 cells from the indicated group (n = 6). Q , representative images of DCFH-DA probe–stained L02 cells in the indicated group (n = 3 independent experiments). Scale bar represents 100 μm. R , representative images of MitoSOX Red probe–stained L02 cells in the indicated group (n = 3 independent experiments). Scale bar represents 100 μm. S , relative mRNA levels of factors related to inflammatory response in L02 cells from the indicated group (n = 6). In all statistical plots, data are expressed as the mean ± SD and analyzed by one-way ANOVA with Bonferroni analysis. ∗∗∗ p < 0.001, comparison between the indicated groups; n.s., no significance, p ≥ 0.05, comparison between the indicated groups. The mRNA expression of target genes was normalized to that of Actb. BSA, bovine serum albumin; GPER1, G protein–coupled estrogen receptor 1; PO, palmitic acid-oleic acid mixture.

Article Snippet: The L02 cell lines (human normal hepatocyte) were purchased from the China Center for Type Culture Collection and cultured in Roswell Park Memorial Institute 1640 medium with 10% FBS and 1% penicillin-streptomycin.

Techniques: Staining, Western Blot, Comparison, Expressing

Journal: The Journal of Biological Chemistry

Article Title: G protein–coupled estrogen receptor 1 ameliorates nonalcoholic steatohepatitis through targeting AMPK-dependent signaling

doi: 10.1016/j.jbc.2024.105661

Figure Lengend Snippet: GPER1 mediates the release of cAMP and activates the AMPK signaling pathway. A , cyclic AMP levels in PO (palmitic acid and oil acid mixture)-challenged L02 cells for 30 min after treatment with 10 −10 to 10 −5 M GPER1-specific agonist G1 (n = 4). B , cyclic AMP levels in PO-challenged L02 cells after treatment with G1 (100 nM) for the indicated times (n = 4). C , cyclic AMP levels in WT or GPER1 KO L02 cells challenged by PO and cotreated with vehicle or 17β-estradiol (E2; 10 nM) for the indicated times (n = 3). D , cyclic AMP levels in WT or GPER1 KO HepG2 cells were challenged by PO and cotreated with vehicle or E2 for the indicated times (n = 3). E , immunoblotting analyses of total and phosphorylated AMPKα and ACCα protein levels in primary hepatocytes that isolate from WT female mice challenged by PO and cotreated with vehicle or G1 in the absence or presence of Gαs inhibitor NF449 (10 μM). F , immunoblotting analyses of total and phosphorylated AMPKα and ACCα protein levels in L02 cells ( left ) and primary hepatocytes ( right ) that isolate from WT female mice challenged by PO and cotreated with vehicle or G1 in the absence or presence of AC inhibitor MDL-12330A (20 μM). G , immunoblotting analyses of total and phosphorylated AMPKα and ACCα protein levels in L02 cells ( left ), HepG2 cells ( middle ), and primary hepatocytes ( right ) that isolate from WT female mice challenged by PO and cotreated with vehicle or G1 in the absence or presence of EPAC antagonist ESI-09 (10 μM). H , immunoblotting analyses of total and phosphorylated AMPKα and ACCα protein levels in L02 cells ( left ), HepG2 cells ( middle ), and primary hepatocytes ( right ) that isolate from WT female mice challenged by PO and cotreated with vehicle or G1 in the absence or presence of PKA inhibitor H89 (10 μM). I , immunoblotting analyses of total and phosphorylated AMPKα and ACCα protein levels in L02 cells ( left ) and HepG2 cells ( right ) challenged by PO and cotreated with vehicle or G1 in the absence or presence of PKA catalytic subunit α (PKAc) siRNA. In all statistical plots, data are expressed as the mean ± SD and analyzed by one-way ANOVA with Bonferroni analysis. For ( A ) and ( B ), ∗ p < 0.05, ∗∗∗ p < 0.001, G1-PO group versus Vehicle-PO group. For ( C ) and ( D ), ∗∗∗ p < 0.001, comparison between the indicated groups; n.s., no significance, p ≥ 0.05, comparison between the indicated groups. AC, adenylyl cyclase; AMPK, AMP-activated protein kinase; EPAC, exchange protein activated by cAMP; GPER1, G protein–coupled estrogen receptor 1; PO, palmitic acid-oleic acid mixture.

Article Snippet: The L02 cell lines (human normal hepatocyte) were purchased from the China Center for Type Culture Collection and cultured in Roswell Park Memorial Institute 1640 medium with 10% FBS and 1% penicillin-streptomycin.

Techniques: Western Blot, Comparison

Journal: Journal of Tissue Engineering

Article Title: A new cell-free therapeutic strategy for liver regeneration: Human placental mesenchymal stem cell-derived extracellular vesicles

doi: 10.1177/20417314221132093

Figure Lengend Snippet: hPMSCs-EVs promote liver regeneration in vivo and ameliorates liver injury after PH, and facilitate hepatocyte proliferation in vitro. (a) Liver tissues were collected and followed by H&E staining of the liver postoperative 48 and 72 h. (b) The number of mitotic hepatocytes was counted. (c) After 2 and 3 days post PH, liver tissue sections were performed PCNA staining. (d) The percentages of PCNA-positive cells were analyzed. (e) Serum ALT and AST levels were analyzed to evaluate the liver injury. (f) PKH26-labeled hPMSCs-EVs were colocalized with L02 and AML12 cells. Immunofluorescent images of PKH26 (red) together with DAPI for nuclei (blue). (g) Cell viabilities were measured by CCK8 assay in AML12 and L02 cells treated with hPMSCs-EVs at the concentrations of 0%, 10%, 20%, 30%, and 40% for 24 h. Error bars represent the mean±standard error of the mean (SEM). * p < 0.05, *** p < 0.001. EVs-IN: hPMSCs-EVs treated.

Article Snippet: Human hepatocyte cell line L02 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan China).

Techniques: In Vivo, In Vitro, Staining, Labeling, CCK-8 Assay

Journal: Journal of Tissue Engineering

Article Title: A new cell-free therapeutic strategy for liver regeneration: Human placental mesenchymal stem cell-derived extracellular vesicles

doi: 10.1177/20417314221132093

Figure Lengend Snippet: hPMSCs-EVs circ-RBM23 serve as a sponge for miR-139-5p. (A) High-throughput sequencing was used to obtain the top 14 expression levels of circ-RNA in hPMSCs-EVs, of which circ-RBM23 was highest. (b) Related RNA expression level (RT-qPCR) of the top five circ-RNAs in hPMSCs-EVs. (c) RT-qPCR analysis showed that linear RNA can be digested by RNase R and that β-actin expression decreased significantly while circ-RBM23 was not particularly affected. (d) Sanger sequencing and circPrimer2.0 were used to confirm the characteristics of circ-RBM23 in hPMSCs. (e) Related RNA expression level of the circ-RBM23 in L02 cells treated with circ-RBM23-siRNA for 72 h. (f) Cell viabilities were measured by CCK8 in L02 cells treated with circ-RBM23-siRNA for 72 h. (g) Relative RNA expression level of indicated miRNAs after L02 cells were co-transfected with circ-RBM23-siRNA. (h) Relative luciferase activity detected after L02 cells were co-transfection with miR-139-5p and circ-RBM23 promoter or circ-RBM23-siRNA. (i) RIP experiment using anti-Ago2 or lgG antibodies as immunoprecipitates; circ-RBM23 and miR-139-5p expressions measured by RT-qPCR. Error bars represent the SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human hepatocyte cell line L02 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan China).

Techniques: Next-Generation Sequencing, Expressing, RNA Expression, Quantitative RT-PCR, Sequencing, Transfection, Luciferase, Activity Assay, Cotransfection

Journal: Journal of Tissue Engineering

Article Title: A new cell-free therapeutic strategy for liver regeneration: Human placental mesenchymal stem cell-derived extracellular vesicles

doi: 10.1177/20417314221132093

Figure Lengend Snippet: circ-RBM23 regulates hepatocytes proliferation via the miR-139-5p/RRM2/AKT/mTOR pathway in vivo and in vitro. (a) Western blot analysis and mRNA expression levels of RRM2 and AKT/mTOR-associated protein in murine liver samples with or without hPMSCs-EVs treatment ( n = 10). (b) mRNA and protein levels of AKT/mTOR-associated protein in L02 cells with miR-139-5p mimic or mimic NC (negative control) and miR-139-5p inhibitor or inhibitor NC (negative control). (c) mRNA and protein levels of RRM2 and AKT/mTOR-associated protein after transfection of miR-139-5p mimics or mimic NC in L02 cells with RRM2 overexpression or NC. (d) mRNA and protein levels of RRM2 and AKT/mTOR associated protein after transfection of miR-139-5p mimics or mimic NC in L02 cells with circ-RBM23 overexpression or NC. Western blots were quantified based on at least three replicates. Error bars represent the SEM. * p < 0.05.

Article Snippet: Human hepatocyte cell line L02 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan China).

Techniques: In Vivo, In Vitro, Western Blot, Expressing, Negative Control, Transfection, Over Expression

Journal: Journal of Tissue Engineering

Article Title: A new cell-free therapeutic strategy for liver regeneration: Human placental mesenchymal stem cell-derived extracellular vesicles

doi: 10.1177/20417314221132093

Figure Lengend Snippet: Knockdown of hPMSCs-EVs circ-RBM23 inhibits hepatocyte proliferation. (a) Related expression level (RT-qPCR) of the circ-RBM23 in hPMSCs-EVs treated with circ-RBM23-siRNA for 72 h. (b) mRNA and protein levels of RRM2 and AKT/mTOR associated protein after co-culture with circ-RBM23-siRNA EVs or circ-RBM23-siRNA NS in L02 cells. (c) mRNA and protein levels of RRM2 and AKT/mTOR associated protein after PH with circ-RBM23-siRNA EVs or circ-RBM23-siRNA NS treatment in liver tissues. Western blots were quantified based on at least three replicates. (d) After 2 days post PH, liver tissue sections from hPMSCs-EVs, circ-RBM23-siRNA EVs or control treatment were performed H&E staining and PCNA staining, the number of mitotic hepatocytes and percentages of PCNA-positive cells were counted. Error bars represent the SEM. * p < 0.05, *** p < 0.001. NS siRNA: non-specific siRNA.

Article Snippet: Human hepatocyte cell line L02 was purchased from Procell Life Science & Technology Co., Ltd (Wuhan China).

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Co-Culture Assay, Western Blot, Control, Staining

Journal: Cell metabolism

Article Title: Low-Dose Sorafenib Acts as a Mitochondrial Uncoupler and Ameliorates Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.04.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell Line The human hepatocyte L02 cell line was purchased from the China Center for Type Culture Collection, Wuhan, China.

Techniques: Recombinant, Protease Inhibitor, Western Blot, SYBR Green Assay, Modification, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Cholesterol Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Gene Knockout, Knock-Out, Plasmid Preparation, Software

Journal: Cell metabolism

Article Title: Low-Dose Sorafenib Acts as a Mitochondrial Uncoupler and Ameliorates Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.04.011

Figure Lengend Snippet: (A) Immunoblotting analysis of B-Raf in wild type (WT) and two strains of BRAF knockout (KO) L02 hepatocytes. (B and C) Oil Red O staining (B), and TG and TC contents (C) of WT and two strains of BRAF KO L02 hepatocytes stimulated by palmitic acid and oleic acid (PAOA), together with DMSO or sorafenib. n = 3 replicates. Scale bar, 100 μm. (D) qPCR analyses of pro-inflammatory factors IL-6 and IL-8 mRNA levels in WT and two strains of BRAF KO L02 hepatocytes stimulated by palmitic acid (PA) with DMSO or sorafenib. n = 3 replicates. (E) Immunoblotting analysis of C-Raf in WT and two strains of CRAF KO L02 hepatocytes stimulated by PA and treated with DMSO or sorafenib. (F and G) Oil Red O staining (F), and TG and TC contents (G) of WT and two strains of CRAF KO L02 hepatocytes stimulated by PAOA, together with DMSO or sorafenib. n = 3 replicates. Scale bar, 100 μm. (H) qPCR analyses of IL-6 and IL-8 mRNA levels in WT and two strains of CRAF KO L02 hepatocytes stimulated by PA and treated with DMSO or sorafenib. n = 3 replicates. The mRNA expression levels of target genes were normalized to that of ACTB (β-Actin). The data in (C), (D), (G), and (H) were presented as the means ± SEMs and analyzed by Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S3.

Article Snippet: Cell Line The human hepatocyte L02 cell line was purchased from the China Center for Type Culture Collection, Wuhan, China.

Techniques: Western Blot, Knock-Out, Staining, Expressing

Journal: Cell metabolism

Article Title: Low-Dose Sorafenib Acts as a Mitochondrial Uncoupler and Ameliorates Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.04.011

Figure Lengend Snippet: (A) Scheme (upper) showing the phospho-proteomic analysis of L02 hepatocytes stimulated by PA and treated with DMSO or sorafenib. The top ten enriched signaling pathways are shown (bottom). The gray lines represent −log10 (p value) for the enriched KEGG pathways. The dashed gray line indicates p value = 0.05, whereas the red circles represent the number of DEGs. FC, fold change. (B) Immunoblotting analyses of total and phosphorylated AMPKα, ACC, mTOR, and total SREBP1 proteins in L02 hepatocytes stimulated by PA with indicated concentrations of sorafenib (left, n = 3 replicates) and in the liver tissues of HFHC-fed mice treated with vehicle or 15 mg/kg/2 days sorafenib (right, n = 6 mice per group). (C) H&E and immunohistochemistry staining of serial liver sections from HFHC-fed mice in indicated groups show the inverse zonation of p-AMPKα (Thr172) and p-mTOR (Ser2448). n = 6 mice per group. Scale bar, 100 μm. (D) STRING and PubMed database correlation analyses show that differentially phosphorylated AMPK signaling molecules are correlated to a variety of differentially expressed genes, which are related to inflammation, lipid metabolism, and fibrosis in RNA-seq data obtained on livers of HFHC-fed mice. (E) Immunoblotting analyses of total and phosphorylated AMPKα and ACC proteins in WT and PRKAA1/2 (encoding AMPKα1/α2) double-knockout (DKO) L02 hepatocytes challenged by BSA vehicle (−) or PA (+), with or without sorafenib. n = 3 replicates. (F and G) Oil Red O staining (F), and TG and TC contents (G) of WT and PRKAA1/2 DKO L02 hepatocytes challenged by PAOA with DMSO or sorafenib. n = 3 replicates. Scale bar, 100 μm. (H) qPCR analyses of IL-6 and IL-8 mRNA levels in WT and PRKAA1/2 DKO L02 hepatocytes challenged by BSA or PA and treated with DMSO or sorafenib. The mRNA expression levels of target genes were normalized to that of ACTB (β-Actin). n = 3 replicates. The data in (G) and (H) were presented as the means ± SEMs and analyzed by Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., no significance, p > 0.05. See also Figure S4.

Article Snippet: Cell Line The human hepatocyte L02 cell line was purchased from the China Center for Type Culture Collection, Wuhan, China.

Techniques: Protein-Protein interactions, Western Blot, Immunohistochemistry, Staining, RNA Sequencing, Double Knockout, Expressing

Journal: Cell metabolism

Article Title: Low-Dose Sorafenib Acts as a Mitochondrial Uncoupler and Ameliorates Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.04.011

Figure Lengend Snippet: (A) Immunoblotting analyses of PP2A, PP2C, and total and phosphorylated AMPKα, LKB1, TAK1, and CAMKK2 proteins in L02 hepatocytes (left, n = 3 replicates) stimulated by PA with indicated concentrations of sorafenib, and in the liver tissues (right) of vehicle- or sorafenib-treated mice on HFHC diet (n = 6 mice per group). (B) AMP/ATP and ADP/ATP ratios in mouse primary hepatocytes (upper) stimulated by PA with DMSO or sorafenib (n = 6 mice per group) and in mouse liver tissues treated with vehicle or sorafenib (bottom) (n = 7 mice per group). (C) Representative (upper) and statistical (bottom) results of complex-I-related respiration on digitonin (Dig)-permeabilized primary mouse hepatocytes treated with DMSO or sorafenib. OCR, oxygen consumption rate. P+M+G, complex I substrates pyruvate, malate, and glutamate. Omy, F1Fo ATP synthase inhibitor oligomycin. FCCP, mitochondrial uncoupler. Rot, complex I inhibitor rotenone. n = 3 replicates. (D) Representative (upper) and statistical (bottom) results of complex-II-related respiration on digitonin-permeabilized primary mouse hepatocytes treated with DMSO or 10 μM sorafenib. Rotenone (Rot) is used to pre-inhibit complex I. Suc, complex II substrates succinate. Ama, complex III inhibitor antimycin A. n = 3 replicates. (E) Representative (upper) and statistical (bottom) results of complex-IV-related respiration measured from digitonin-permeabilized primary mouse hepatocytes treated with DMSO or 10 μM sorafenib. As+Tm, ascorbate (As) and N,N,N’,N’-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD). Sodium azide (Azd) is a complex IV inhibitor. n = 3 replicates. (F) Dependence of proton leak rate (measured as the respiration in the presence of oligomycin) on membrane potential in the presence or absence of sorafenib. n = 3 replicates. (G) Statistical results of mitochondrial membrane potential indicator TMRM fluorescence on PA-stimulated primary mouse hepatocytes with or without sorafenib treatment. n = 3 replicates. a.u., arbitrary unit. (H) Energy expenditure of HFHC-fed mice treated with vehicle or sorafenib (15 mg/kg/2 days). n = 6 mice per group. The data in (B), (C), (D), (E), (G), and (H) were presented as the means ± SEMs and analyzed by Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., no significance, p > 0.05. See also Figure S5.

Article Snippet: Cell Line The human hepatocyte L02 cell line was purchased from the China Center for Type Culture Collection, Wuhan, China.

Techniques: Western Blot, Membrane, Fluorescence

Journal: Cell metabolism

Article Title: Low-Dose Sorafenib Acts as a Mitochondrial Uncoupler and Ameliorates Nonalcoholic Steatohepatitis

doi: 10.1016/j.cmet.2020.04.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Cell Line The human hepatocyte L02 cell line was purchased from the China Center for Type Culture Collection, Wuhan, China.

Techniques: Recombinant, Protease Inhibitor, Western Blot, SYBR Green Assay, Modification, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Cholesterol Assay, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Gene Knockout, Knock-Out, Plasmid Preparation, Software