Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Discovery of Isoginkgetin as a naturally-occurring ACLY inhibitor. (A) Schematic diagram of molecular autodocking. Briefly, we conducted virtual molecular docking based on the structure of the ACLY protein to screen for potential natural small molecules that interact with ACLY from a natural product library comprising 3,200 compounds. (B) The predicted affinity results of A. (C) The inhibitory effect of the top candidates on ACLY enzyme activity was evaluated in vitro using the ADP-Glo assay (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. Compared with compound 0 µM. (D) Chemical structure of ISOGK. (E) The cell viability in primary hepatocytes from C57BL/6J mouse treated with dose-dependent ISOGK detected by CCK-8 assay (n = 4). One-way ANOVA followed by Bonferroni's post hoc test. Compared with ISOGK 0 µM. (F) The cell viability in HepG2 cell line treated with dose-dependent ISOGK detected by CCK-8 assay (n = 3, technical replicates). One-way ANOVA followed by Bonferroni's post hoc test. Compared with ISOGK 0 µM. (G) Representative images of Nile Red staining in primary hepatocytes isolated from mice fed a high fat diet for 16 weeks which are treated with vehicle or indicated concentrations ISOGK for 12 h. (H) Quantitative analysis of G (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Activity Assay, In Vitro, Glo Assay, CCK-8 Assay, Staining, Isolation

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin attenuates atherosclerosis in male and female Apoe -/- mice. (A) Scheme of study design. Male and female Apoe -/- mice were induced by high cholesterol diet for 7 weeks. Animals then received ISOGK treatment (20 mg/kg/day) for another 8 weeks. (B) The body weights were shown after ISOGK treatment for 8 weeks (n = 10 or 11). Two-tailed Student's t test. (C) Serum ALT, AST levels of Apoe -/- mice treated with or without ISOGK (n = 10 or 11). Two-tailed Student's t test. (D) Representative images of Oil Red O staining of en face aortas from male and female Apoe -/- mice fed high cholesterol diet. (E) Quantification of en face plaque areas as a percentage of the area in D (n = 10 or 11). Two-tailed Student's t test. (F) The representative images of Oil Red O (upper) staining and H&E (lower) staining of the aortic sinus from male and female Apoe -/- mice fed high cholesterol diet. Arrow indicates necrotic core area. (G) Quantitative analysis of F (n = 6 or 7). Two-tailed Student's t test. The data are means ± SEM, n.s., not significant, ** P < 0.01, *** P < 0.001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Two Tailed Test, Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin modulates lipid metabolism in Apoe -/- mouse liver. (A) Scheme of sample processing. (B) Volcano plot showing differential expression genes (adjusted P < 0.05, | log2 (Fold change) | > 1). (C) Heat map displaying the indicated gene expression in liver tissues from Apoe -/- mice with or without ISOGK (n = 4). (D) GO enrichment analysis of transcriptomes from Apoe -/- mice liver samples. (E) KEGG pathway enrichment analysis of transcriptomes from Apoe -/- mice liver samples. (F) GSEA analysis of transcriptomes from Apoe -/- mice liver samples. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Expressing, Gene Expression

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin displays lipid-lowering effects in hyperlipidemia mice and hamsters. (A) Serum levels of TC, TG, LDL-C, HDL-C, and hepatic levels of TC, TG from male and female Apoe -/- mice fed high cholesterol diet 7 weeks and administrated ISOGK 8 weeks (n = 10 or 11). Two-tailed Student's t test. (B) The distribution of TC and TG in pooled plasma samples from the indicated female Apoe -/- mice. (C) Study design of the hamster's experiment. (D) Plasma levels of TC, TG, LDL-C, HDL-C, and hepatic levels of TC, TG in the indicated hamster's group after treatment ISOGK or vehicle for 4 weeks (n = 8 - 10). One-way ANOVA followed by Bonferroni's post hoc test. (E) The distribution of TC and TG in pooled plasma samples from the indicated hamsters. (F) Representative images of Oil Red O staining in liver sections from the indicated groups. (G) Quantitative analysis of F (n = 8). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Two Tailed Test, Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin ameliorates hyperlipidemia and atherosclerosis in Ldlr -/- hamsters. (A) Scheme of study design. Briefly, 8-week-old male Ldlr -/- hamsters were induced by high cholesterol diet for 2 weeks and then administrated ISOGK (2 mg/kg/day and 5 mg/kg/day) treatment for another 6 weeks. (B) Hepatic levels of TC, TG in the indicated Ldlr -/- hamster's group after treatment ISOGK or vehicle for 8 weeks (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (C) Serum TC, TG, HDL-C and non-HDL-C levels in the indicated Ldlr -/- hamster's group after treatment ISOGK or vehicle for 6 weeks (n = 7). One-way ANOVA followed by Bonferroni's post hoc test. (D) Representative images of Oil Red O staining of en face aortas in indicated Ldlr -/- hamsters group. (E) Quantitative analysis of D (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (F) Representative images of Oil Red O staining in Ldlr -/- hamsters' aortic sinus and representative images of Nile Red staining in Ldlr -/- hamsters' liver tissues. (G-H) Quantitative analysis of F (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Staining

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: Isoginkgetin inhibits ACLY activity in vitro and in vivo. (A) Primary hepatocytes from C57BL/6J mice were cultured with vehicle or 5 µM ISOGK for 4 h in the presence of [1,2- 14 C]-acetate. The hepatic lipogenesis was calculated (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. (B) Dose-dependent inhibition of purified, recombinant ACLY after incubation with ISOGK, the activity at control was defined as 100%. (C) ACLY enzyme activities were measured from the liver extracts of male and female Apoe -/- mice fed high cholesterol diet 7 weeks and administrated ISOGK 8 weeks (n = 10 or 11). Two-tailed Student's t test. (D). ACLY enzyme activities were measured from the liver extracts of hamster after treatment ISOGK or vehicle for 4 weeks (n = 10). One-way ANOVA followed by Bonferroni's post hoc test. (E) ACLY enzyme activities were measured from the liver extracts of Ldlr -/- hamster after treatment with or without ISOGK for 8 weeks (n = 6 or 7). One-way ANOVA followed by Bonferroni's post hoc test. (F) Molecular docking has predicted the binding sites of ISOGK with ACLY. (G) The interaction of ACLY with ISOGK was measured by surface plasmon resonance. (H) CETSA analyzed the thermal stabilization of ACLY with isoginkgetin in AML12 cell lysates. (I) Quantitative analysis of H (n = 3). Two-way ANOVA with Dunnett's T3 post hoc analysis. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Activity Assay, In Vitro, In Vivo, Cell Culture, Inhibition, Purification, Recombinant, Incubation, Control, Two Tailed Test, Binding Assay, SPR Assay

Journal: Theranostics

Article Title: A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY

doi: 10.7150/thno.105782

Figure Lengend Snippet: The anti-atherosclerotic and lipid-lowering effects of isoginkgetin in Apoe -/- mice were ACLY-dependent. (A) Scheme of study design. Briefly, male Apoe -/- mice were induced by high cholesterol diet for 7 weeks and administrated GalNAc-siAcly (3 mg/kg/month). Animals then received ISOGK (20 mg/kg/day) treatment for another 8 weeks. (B) Immunoblotting analysis and quantification of ACLY protein in liver and kidney tissues from indicated groups (n = 3). One-way ANOVA followed by Bonferroni's post hoc test. (C) Representative images of Oil Red O staining of en face aortas from male Apoe -/- mice fed high cholesterol diet. (D) Representative images of ORO staining of the aortic sinus from male Apoe -/- mice fed high cholesterol diet. (E) Representative images of H&E staining of the aortic sinus from male Apoe -/- mice fed High cholesterol diet. (F) Quantitative analysis of C-E (n = 6 - 8). One-way ANOVA followed by Bonferroni's post hoc test. (G) Plasma levels of TC, TG, LDL-C and HDL-C in the indicated groups (n = 6 - 8). One-way ANOVA followed by Bonferroni's post hoc test. The data are means ± SEM, n.s., not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: C57BL/6J mice had a standard chow diet for 4 weeks with concurrent treatment with ISOGK (HY-N2117, MedChemExpress, Piscataway, NJ, USA).

Techniques: Western Blot, Staining