Fig 1: Effect of IB/SB/SM on pro-fibrotic gene expression, antioxidant activity, and ALT concentration in AML12 cells. (A-C) Expression of pro-fibrotic genes fibronectin (Fn1), smooth muscle actin (Acta2) and collagen I (Col1a1) after 24 h treatment with TGF-β1 (10 ng/mL) and IB/SB/SM (7.8–31.3 µg/mL) in AML12 cells. (D) Western blot analysis of fibronectin expression in AML12 cells stimulated for 24 h with TGF-β1 (10 ng/mL) and IB/SB/SM at concentration 31.3 µg/mL. Representative western blot bands are shown below the graphs. (E) DPPH antioxidant activity assay. The ability of IB/SB/SM to scavenge DPPH radicals was evaluated at various concentrations (0–500 µg/mL). (F) Determination of alanine aminotransferase (ALT) concentration in the supernatants of AML 12 cells stimulated with TGF-β1 (10 ng/mL) in the presence of the tested compounds (IB, SB, SM) at different concentrations. All results are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0,001 (for western blot: vs. TGF-β1 control)
Fig 2: MDEVs alleviate SALI by promoting Tregs differentiation and inhibiting M1 polarization synchronously(A and B) HE stains (A, scale bars: 100 μm) and Suzuki’s score (B) in mice liver during SALI with/without WT/Roquin-1san/san MEDVs injection (n = 6).(C) serum ALT and AST in SALI mice with/without WT/Roquin-1san/san MEDVs MDEVs injection (n = 6).(D and E) Flow cytometry and quantitation of M1 møs and Tregs in mice liver during SALI with/without MEDVs injection (n = 6).(F–I) Serum TNF-α (f)/TGF-β (g)/IL-6 (h)/IL-10 (i) in SALI mice with/without MDEVs injection (n = 6). p < 0.01∗, p < 0.01 ∗∗, p < 0.001 ∗∗∗, p < 0.0001 ∗∗∗∗. The data are expressed as the mean ± SD. The Student’s t test was used for comparison between the two groups.
Fig 3: VV-ΔTK-4L administration did not produce obvious hepatoxicity in therapeutic tumor models.A: Schematic diagram of the hepatotoxicity assay for α4–1BB mAb and VV-ΔTK-4L treatment in a pancreatic mouse model. B–E: C57BL/6 mice were intraperitoneally injected with 1 × 106 of Pan02 cells and treated with agonist α4–1BB mAb, rat IgG2a isotype control, PBS, VV-ΔTK-4L, or VV-ΔTK 9 days post tumor inoculation. The treated mice and healthy mice (n = 5/group) were sacrificed 21 days post tumor inoculation to harvest their livers, which were then photographed (B), weighed (C), and underwent blood collection to measure ALT (D) and AST (E) levels in sera. Liver tissues were collected to perform RT-qPCR to analyze 4–1BBL expression (F). *P < 0.05; **P < 0.01. ns: not significant.
Fig 4: CePA regulates metabolic disorders in vivo. (a and b) Photographs of mice and mouse livers fed a normal or HFCFG diet with or without CePA for 16 weeks. Scale bar, 1 cm. (c and d) Body weight growth curve (c) and liver weight (d) of mice in the indicated groups. n = 3. (e) Enzyme-linked immunosorbent assay (ELISA) results of serum concentrations of ALT (left) and AST (right) in mice fed a normal or HFCFG diet with or without CePA. (f) Total cholesterol concentrations and blood glucose of mice in the indicated groups. (g) H&E staining of mice in the Ctrl, CePA, HFCFG, and HFCFG + CePA groups. Scale bar, 100 µm. (h) NAS of liver sections and quantitative analysis results of balloon, inflammation, and fibrosis levels of mouse liver sections in the indicated groups. (i) Oil Red O staining images in Ctrl, CePA, HFCFG, and HFCFG + CePA mice. (j) Oil Red O area in the indicated groups. (k) Masson staining of mice in Ctrl, CePA, HFCFG, and HFCFG + CePA groups. Scale bar, 100 µm. (l) Masson area in the indicated groups. (m) Sirius Red staining of mice in Ctrl, CePA, HFCFG, and HFCFG + CePA groups. Scale bar, 100 µm. (n) Sirius Red area in the indicated groups. (o–q) Relative mRNA expression of lipid metabolism-related genes srebp1 (o), acc (p), and fasn (q) in the indicated groups. (r and s) mRNA levels of fibrosis-related markers α-sma and timp in the indicated groups. (t) Western blot to detect the expression of p-mTOR and p-P70(S6K) after HFCFG feeding. Scale bar, 100 μm. The data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 for HFCFG vs. Ctrl or HFCFG + CePA vs. HFCFG; ns indicates not statistically significant.
Fig 5: LCN2 overexpression suppressed LPS-induced oxidative stress and ferroptosis of hepatocytes by inhibiting PTGS2 expression. An in vitro sepsis model was established by stimulating hepatocytes with LPS and then LCN2 was overexpressed in the cell model. The following experiments are carried out. (A) LCN2 mRNA levels were assessed by qRT-PCR in control hepatocytes and LPS-treated hepatocytes before and after LCN2 overexpression. (B and C) Western blot and quantitative analysis of LCN2 protein levels in control hepatocytes and LPS-treated hepatocytes before and after LCN2 overexpression. ALT (D) and AST (E) levels were assessed by ELISA in supernatant of the control hepatocytes and LPS-treated hepatocytes before and after LCN2 overexpression. (F) ROS levels were measured by ELISA in the control hepatocytes and LPS-treated hepatocytes before and after LCN2 overexpression. (G and H) MDA and GSH levels were assessed by ELISA in control hepatocytes and LPS-treated hepatocytes before and after LCN2 overexpression. (I) PTGS2 mRNA (I) and protein (J and K) levels in control hepatocytes and LPS-treated hepatocytes before and after LCN2 overexpression were assessed by qRT-PCR and western blot assays, respectively. Data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.
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