Journal: Toxicon : official journal of the International Society on Toxinology

Article Title: The DHCR7 is the key target of lipotoxic liver injury caused by matrine through abnormal activation of the cholesterol synthesis pathway.

doi: 10.1016/j.toxicon.2025.108366

Figure Lengend Snippet: Fig. 8. Verifying the mechanisms behind matrine-induced zebrafish liver injury based on the steroid biosynthesis signaling pathway. (A) Dose-toxicity curve of AY 9944 on zebrafish juveniles; (B) Detection results of the total cholesterol content; (C) Statistics of the fluorescence intensity area of CZ16 transgenic liver fluorescent zebrafish; (D–E) Liver morphology and fluorescence area of CZ16 fluorescent zebrafish under white light and fluorescence; (F) Oil Red Staining results for zebrafish liver; (G) H&E staining results for the zebrafish liver; (H) Statistical results of the liver color threshold for zebrafish oil red staining; (I–J) Results of RT-qPCR gene expression after treatment with the AY 9944 inhibitor. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The other materials were sourced as follows: Dimethyl sulfoxide (DMSO) (Biorigin, BN35879-100 ml, China); 4 % immunohistochemistry fixative (Lablead, P4500, China); ALT kit (Nanjing Jiancheng Biotechnology Research Institute Co., Ltd, C009-2-1, China); AST kit (Nanjing Jiancheng Biotechnology Research Institute Co., Ltd, C010-21, China); total cholesterol (TC) content kit (Applygen, E1015-50, China); Oil Red O Staining Kit (Beyotime, C0157S, China); AY 9944 (MCE, HY-107420, America); RPMI-1640 medium (Procell, PM150110, China); fetal bovine serum (FBS) (Procell, 164210, China); penicillinstreptomycin Solution (100x) (Procell, PB180120, China); 1 × phosphate buffered saline (PBS) (Procell, PB180327, China); cell counting kit-8 solution (CCK8) (beyotime, C0038, China); Hoechst 33342 kit (100X) (Beyotime, C1027, China); Calcein AM Cell Activity Detection Kit (Beyotime, C2012-0.5 ml, China); Microplate Reader(PerkinElmer, SPECTROstar Nano, Germany); 3D Live Cell Laser Confocal Imaging Analysis System (PerkinElmer, UltraVIEW VoX, UK); Slice scanner (Zhiyue Medical Technology Co., Ltd, WS-10, China).

Techniques: Fluorescence, Transgenic Assay, Staining, Quantitative RT-PCR, Gene Expression

Journal: PLoS Pathogens

Article Title: Enzymatic chokepoints and synergistic drug targets in the sterol biosynthesis pathway of Naegleria fowleri

doi: 10.1371/journal.ppat.1007245

Figure Lengend Snippet: Sterol flux in N . fowleri .

Article Snippet: AY9944 and abafungin were from Santa Cruz Biotechnology (SC-202965 and SC-474844); tamoxifen was from Sigma (T5648), isavuconazole was from StruChem (SC-98350), fluvoxamine (J90043), fluoxetine (C845), citalopram (K750) and dextromethorphan (X3585) were from AK Scientific.

Techniques:

Journal: PLoS Pathogens

Article Title: Enzymatic chokepoints and synergistic drug targets in the sterol biosynthesis pathway of Naegleria fowleri

doi: 10.1371/journal.ppat.1007245

Figure Lengend Snippet: Gas chromatography of the total sterol fractions derived from N . fowleri trophozoites treated with ( A ) 0.5% DMSO, ( B ) 0.2 μM posaconazole, ( C ) 5.4 μM epiminolanosterol, and ( D ) 5.6 μM AY9944. Peaks are labeled with numbers corresponding to metabolites listed in . Insert in ( C ) shows deconvolution of the major peak resulting from an overlap of three different sterols: 6 , m/z = 349; 7 , m/z = 363 and 13 , m/z = 400. The color scheme is the same as in : CYP51 inhibitor (shown in green), SMT inhibitor (in blue) and ERG2 inhibitor (in dark red).

Article Snippet: AY9944 and abafungin were from Santa Cruz Biotechnology (SC-202965 and SC-474844); tamoxifen was from Sigma (T5648), isavuconazole was from StruChem (SC-98350), fluvoxamine (J90043), fluoxetine (C845), citalopram (K750) and dextromethorphan (X3585) were from AK Scientific.

Techniques: Gas Chromatography, Derivative Assay, Labeling

Journal: PLoS Pathogens

Article Title: Enzymatic chokepoints and synergistic drug targets in the sterol biosynthesis pathway of Naegleria fowleri

doi: 10.1371/journal.ppat.1007245

Figure Lengend Snippet: Dose-response curves are shown for ( A ) SMT inhibitors: abafungin, 25-azacycloartenol and 24,25-epiminolanosterol, ( B ) ERG2 inhibitors: tamoxifen and AY9944, ( C ) CYP51 inhibitor isavuconazole.

Article Snippet: AY9944 and abafungin were from Santa Cruz Biotechnology (SC-202965 and SC-474844); tamoxifen was from Sigma (T5648), isavuconazole was from StruChem (SC-98350), fluvoxamine (J90043), fluoxetine (C845), citalopram (K750) and dextromethorphan (X3585) were from AK Scientific.

Techniques:

Journal: PLoS Pathogens

Article Title: Enzymatic chokepoints and synergistic drug targets in the sterol biosynthesis pathway of Naegleria fowleri

doi: 10.1371/journal.ppat.1007245

Figure Lengend Snippet: Targeting N . fowleri with inhibitors of known MOA.

Article Snippet: AY9944 and abafungin were from Santa Cruz Biotechnology (SC-202965 and SC-474844); tamoxifen was from Sigma (T5648), isavuconazole was from StruChem (SC-98350), fluvoxamine (J90043), fluoxetine (C845), citalopram (K750) and dextromethorphan (X3585) were from AK Scientific.

Techniques:

Journal: British journal of pharmacology

Article Title: Pharmacological characterization of SAGE-718, a novel positive allosteric modulator of N-methyl-d-aspartate receptors.

doi: 10.1111/bph.16235

Figure Lengend Snippet: FIGURE 7 SAGE-718 effects on locomotor activity and sharp wave discharges in the AY9944 model. (a) Representative traces of AY9944-induced sharp wave discharges (SWD). (b) 24(S)-hydroxycholesterol (24(S)-HC) was positively correlated with the number of sharp wave discharges. AY9944 administration increased the number (c) and duration (d) of sharp waves observed on electroencephalogram (EEG) in male rats. SAGE-718 attenuated both the total number and duration of sharp wave discharges at 1 mgkg1. Pooled group includes both 3 and 10 mgkg1 cohorts because brain Cmax values for each dose group were nearly equal. (e) 24(S)-HC was positively correlated with locomotor activity (LMA). AY9944 significantly increased both distance travelled (f) and rearing behaviour (g) in female rats. SAGE-718 attenuated AY9944-induced changes in locomotor activity at 3 and 20 mgkg1. (h) Exposures measured from test animals in the locomotor activity and EEG experiments. Data shown are means ± SEM from the number of animals as follows: for the SWD assays (in c and d): AY9944, n = 9; SAGE-718 (1mg/kg), n = 10; SAGE-718 (pooled), n = 20. For the assays of locomotor activity (in f and g): Vehicle n = 23; AY9944, n = 12; SAGE-718 (3mg/kg), n = 13; SAGE-718 (20mg/kg), n = 14. *P < 0.05 significantly different from AY9944.

Article Snippet: AY9944 dihydrochloride was acquired from Tocris (Minneapolis, MN, USA).

Techniques: Activity Assay

Journal: British journal of pharmacology

Article Title: Pharmacological characterization of SAGE-718, a novel positive allosteric modulator of N-methyl-d-aspartate receptors.

doi: 10.1111/bph.16235

Figure Lengend Snippet: FIGURE 9 Pharmacodynamic profile of SAGE-718. Exposuregram summarizing effects of SAGE-718 on pharmacodynamic and safety studies. No effect—grey; SAGE-718 significantly accelerated return to baseline electroencephalogram (EEG) gamma after ketamine, in rats— orange (solid—significant effect; gradient—pharmacokinetic/pharmacodynamic trend); SAGE-718 reduced sharp wave discharge (SWD) in AY9944-treated rats—yellow; SAGE-718 reduced AY9944-induced hyperactivity—blue; SAGE-718 improved social behaviour in a subchronic phencyclidine (PCP) model in rats—green. Minimally effective brain concentration in the PCP social interaction (SI) model was taken from the high end of the extrapolated concentration range due to drug measurements being below the level of quantification. No effect on MEA synchronicity at any concentration tested or in the pentylenetetrazole (PTZ) seizure model in mice up to a brain exposure of 336 ngg1. GluN1/GluN2A EC50 as measured with manual patch clamp (EC50 = 46 nM or 21 ngg1; Figure 1c). As a reference, GABAA receptor α1β2γ2 EC50 = 570 nM or 260 ngg1. Estimated brain exposure at rat no-observed-adverse-effect level (NOAEL) was 1574.4 ngg1 and was calculated by multiplying the plasma Cmax following 7 mgkg1day1 in male rats (839 ngml1) by the calculated Cmax B:P ratio (B:P = 1.88) in the rat pharmacokinetic study. LMA, locomotor activity.

Article Snippet: AY9944 dihydrochloride was acquired from Tocris (Minneapolis, MN, USA).

Techniques: Concentration Assay, Patch Clamp, Clinical Proteomics, Activity Assay

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Correlation between DHCR7 expression and macrophage polarization in MASH. ( a ) Relative expression of cholesterol biosynthesis and efflux genes in primary macrophages after ox-LDL (100 µg/ mL) or ox-LDL combined with statin treating for 24 h. ( b ) Expression levels of cholesterol biosynthesis and efflux genes in primary macrophages polarized to M1 with LPS (100 ng/mL) or M2 with IL-4 (20 ng/mL) for 24 h. ( c-d ) Western blot analysis for DHCR7 protein in M1 and M2 macrophages, with densitometry. ( e ) Immunofluorescence staining for DHCR7 in the polarized-macrophages, 200× magnification. ( f-g ) Multiple immunofluorescence co-localization for DHCR7 and CD11B on livers of NCD or CDAHFD mice. ( h-i ) DHCR7 protein and mRNA levels in total liver and primary macrophages isolated from livers of mice on control and CDAHFD. ( j-k ) Immunofluorescence multi-labeling of liver sections from NC and MASH patients for DHCR7 and macrophage marker CD68 to determine the proportion of DHCR7 + CD68 + cells among total CD68 + cells. Data are presented as mean ± SD, with n = 3 per group. * P < 0.05, * * P < 0.01, *** P < 0.001, and **** P < 0.0001 between groups

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Isolation, Control, Labeling, Marker

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: The modulatory effect of DHCR7 on macrophage polarization and inflammatory response. ( a ) Relative protein levels of DHCR7 and M2 markers (CD206 and ARG-1) after silencing DHCR7, with densitometry. ( b ) Relative mRNA levels of M1 markers ( Inos , Cd32 ) and inflammatory cytokines ( Tnf-a , Il-6 , Il-1b ) after silencing DHCR7. ( c ) RAW264.7 cells were treated with AY9944 (2.5, 5 or 10 µM) for 24 h to inhibit DHCR7. Relative protein levels of DHCR7, with densitometry. ( d ) Relative mRNA levels of M1 markers ( Inos , Cd32 , Cd16 ) and inflammatory cytokines ( Il6 and Tnfa ) following AY9944 treatment for 24 h. ( e ) CD86 immunofluorescence in AY9944-treated RAW264.7 macrophages, 200× magnification. ( f ) Relative mRNA levels of M1/M2 markers following AY9944 treatment, with densitometry. ( g ) Relative protein quantification after overexpressing DHCR7, with densitometry. ( h ) Relative mRNA levels of M1 markers ( Inos , Cd32 ) and inflammatory cytokines ( Il6 , Il1b , Tnfa ) after overexpressing DHCR7. ( i ) Relative protein levels of M1/M2 markers in LPS-treated RAW264.7 cells overexpressing DHCR7, with densitometry. Data are presented as mean ± SD. * P < 0.05, * * P < 0.01, *** P < 0.001, and **** P < 0.0001 between groups

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Myeloid-macrophage-specific DHCR7 deficiency exacerbates MASH progression. Wildtype (WT) and myeloid-specific DHCR7 knockout (DHCR7 cKO) mice were fed with a normal control diet (CON) or a CDAHFD for 10 weeks ( n = 7). ( a ) Body weight progression, ( b ) liver/body weight ratio, and ( c-d ) serum ALT and AST levels were detected. ( e-f ) Hepatic TG and TC levels were measured. ( g ) ELISA quantification of liver IL-1β and IL-6 levels. ( h ) Histological analysis of liver sections is presented through H&E, ORO staining, and immunohistochemistry for F4/80 + and CD11b + cells (200× magnification). ( i ) NAS score. ( j-k ) Relative protein levels of CD86, ARG-1, CD206 were determined, with densitometry. ( l ) Flow cytometry of primary macrophage in cKO mice and WT with different diets. ( m ) Flow cytometry of TREM2 in macrophages. ( n ) Predominantly of the M1 type in macrophages, including quantification of both the percentage of M1 macrophages and M1 macrophage counts per liver weight. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 between group

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Knock-Out, Control, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: DHCR7 manipulation affects PI3K phosphorylation in macrophages. ( a ) PCA analysis and ( b ) KEGG pathway analysis using RNA sequencing in liver tissues from WT and DHCR7 cKO mice under control diet. ( c ) PCA analysis and ( d ) KEGG pathway analysis using RNA sequencing in liver tissues from NCD and CDAHFD-fed MASH mice. ( e-f ) Protein levels of PI3K and phosphorylated PI3K (P-PI3K) in NCD- and CDAHFD-fed WT and DHCR7 cKO mice. ( g , j ) Protein levels of PI3K, P-PI3K, and DHCR7 in RAW264.7 cells treated with ox-LDL (100 µg/mL) and a co-treatment of ox-LDL (100 µg/mL) and simvastatin (5 µM) for 24 h. ( h , k ) Protein levels of PI3K and P-PI3K in RAW264.7 cells treated with different concentrations of the DHCR7 inhibitor AY9944 (2.5, 5, and 10 µM) for 24 h. ( i , l ) Protein levels of PI3K and P-PI3K in RAW264.7 cells overexpressing DHCR7 and subsequently treated with ox-LDL (100 µg/mL) for 24 h. Data are presented as mean ± SD, with n = 3 per group. * P < 0.05, ** P < 0.01, and *** P < 0.001 between groups

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: RNA Sequencing Assay, Control

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: The DHCR7-PI3K axis in macrophage polarization and inflammatory response. ( a ) RAW264.7 cells were co-treated with LPS (100 µg/mL) with PI3K activator 740 Y-P (5 and 10 µM) for 24 h. Relative mRNA levels of M1 markers ( Inos , Cd32 and Cd16 ) and the inflammatory cytokine ( Il1b and Il6 ) was assessed. RAW264.7 cells were treated with 10 µM AY9944 alone or alongside 5 or 10 µM 740 Y-P for 24 h. ( b ) Relative mRNA levels of M1 markers ( Inos , Cd32 , Cd16 ) and the inflammatory cytokine ( Tnfa and Il-6 ) was assessed. ( c-d ) Relative protein levels of the M1 marker CD86 and M2 markers CD206 and ARG-1 were measured. ( e ) RAW264.7 cells were treated with PI3K inhibitor LY294002 at 2.5, 5, and 10 µM for 24 h, IL-4, or a combination of both for 24 h. Relative mRNA levels of M1 markers ( Cd32 and Cd16 ) and the inflammatory cytokine ( Il-1b and Tnfa) . ( f ) Relative mRNA levels of Mrc-1 were assessed after treating with IL-4 (20 ng/mL) and IL-4 combined with 2.5, 5, 10 µM LY294002 for 24 h. ( g ) RAW264.7 cells were treated with 10 µM AY9944 alone or in combination with 5 or 10 µM 740 Y-P for 24 h, relative mRNA levels of M1 markers ( Cd32 and Cd16 ) and the inflammatory cytokine ( Tnfa and Il-6 ) was assessed. ( h-i ) Relative protein levels of the M1 marker CD86 and M2 markers ARG-1. Data are presented as mean ± SD, with n = 3 per group. * P < 0.05, * * P < 0.01, *** P < 0.001, and **** P < 0.0001 between groups

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Marker

Journal: Journal of Translational Medicine

Article Title: Cholesterol overload in macrophages drives metabolic dysfunction-associated steatohepatitis via inhibiting 7-dehydrocholesterol reductase in mice

doi: 10.1186/s12967-024-05905-1

Figure Lengend Snippet: Schematic illustrating how macrophage cholesterol overload drives inflammation via DHCR7-PI3K axis inhibition, contributing to MASH progression

Article Snippet: For DHCR7-PI3K pathway studies, cells were treated with DHCR7 inhibitor AY9944 (A8658, APExBio, USA) and PI3K inhibitor LY294003 (HY-10108, MCE, USA), or PI3K agonist 740 Y-P (HY-P0175, MCE, USA).

Techniques: Inhibition