Journal: Journal of Inflammation Research

Article Title: Glycochenodeoxycholic acid induces the release of IL-1beta from L02 cells via the NLRP3/caspase-1/GSDMD pathway to activate LX2 cells

doi: 10.2147/JIR.S532042

Figure Lengend Snippet: GCDCA activates the NLRP3 inflammasome in hepatocytes. ( A ) Dose-dependent effects of GCDCA (25–400 μM, 24 h) on protein levels effects of NLRP3, ASC, pro-caspase-1, caspase-1 P20, pro-IL-1β and GAPDH. ( B ) Time-dependent effects of these proteins in L02 cells treated with 200 μM GCDCA. ( C and D ) Synergistic priming by LPS (1 μg/mL, 4 h pre-treatment): GCDCA (50–400 μM, 24 h) ( C ) or time-dependent (12–72 h) ( D ) enhancement of NLRP3, pro-caspase-1, and pro-IL-1β expression in LPS-primed cells. ( E ) Comparative analysis of NLRP3 inflammasome activationin L02 cells treated with GCDCA (200 μM, 24 h) with/without LPS co-stimulation. ( F ) Essential role of NLRP3 in GCDCA-induced pyroptosis. L02 cells were pretreated with/without LPS (1 μg/mL, 4 h) and MCC950 (10 μM, 2 h) prior to GCDCA (200 μM, 24 h) exposure. Whole cell lysates were examined by Western Blot analysis. Molecular weight markers (kDa) are indicated. n ≥ 3 for each group. (LG200: LPS+200 μM GCDCA; G200: GCDCA 200 μM).

Article Snippet: L02 and LX2 cells were synchronized in serum-free medium for 12 h. L02 cells were exposed to graded GCDCA concentrations (25–400 μM, HY-N2334, MCE) for 24 h or 200 μM GCDCA for 6–72 h to establish dose/time-response profiles.

Techniques: Expressing, Western Blot, Molecular Weight

Journal: Journal of Inflammation Research

Article Title: Glycochenodeoxycholic acid induces the release of IL-1beta from L02 cells via the NLRP3/caspase-1/GSDMD pathway to activate LX2 cells

doi: 10.2147/JIR.S532042

Figure Lengend Snippet: GCDCA induces GSDMD-mediated pyroptosis in hepatocytes. ( A and B ) Time- and dose-dependent LDH release in LPS-primed L02 cells treated with GCDCA: ( A ) Dose-response (50–400 μM GCDCA, 24 h); ( B ) Time-course (200 μM GCDCA, 12–72 h). ( C ) Dose-dependent GSDMD proteolysis in LPS-primed cells treated with GCDCA (50–400 μM, 24 h); ( D ) Time-dependent GSDMD proteolysis (200 μM GCDCA, 12–72 h). GAPDH serves as loading control. ( E ) Validation of GSDMD knockdown efficiency in L02 cells. ( F ) LDH release in lentiviruses-transfected L02 cells treated with GCDCA (200 μM, 24 h). ( G ) Representative flow plots showing Annexin V/PI staining in lentiviruses-transfected L02 cells. ( H ) Flow cytometry quantification of pyroptotic cells (Annexin V⁺/PI⁺). n ≥ 3 for each group. a P < 0.005, b P < 0.05, c P < 0.0001.

Article Snippet: L02 and LX2 cells were synchronized in serum-free medium for 12 h. L02 cells were exposed to graded GCDCA concentrations (25–400 μM, HY-N2334, MCE) for 24 h or 200 μM GCDCA for 6–72 h to establish dose/time-response profiles.

Techniques: Control, Biomarker Discovery, Knockdown, Transfection, Staining, Flow Cytometry

Journal: Journal of Inflammation Research

Article Title: Glycochenodeoxycholic acid induces the release of IL-1beta from L02 cells via the NLRP3/caspase-1/GSDMD pathway to activate LX2 cells

doi: 10.2147/JIR.S532042

Figure Lengend Snippet: GCDCA triggers caspase-1-dependent pyroptosis in hepatocytes. ( A and B ) Caspase-1/4 activity in LPS-primed L02 cells treated with GCDCA. ( C and D ) Flow cytometry quantification of pyroptotic cells (Annexin V⁺/PI⁺) in L02 cells with caspase-1 inhibitor Ac-YVAD-cmk (AC, 50 μM) followed by LPS + GCDCA (200 μM, 24 h). ( E ) LDH release in L02 cells with AC pretreatment. (F)Western blot analysis of pyroptosis-related proteins in L02 cells with AC pretreatment. n ≥ 3 for each group. a P < 0.005, b P < 0.0001.

Article Snippet: L02 and LX2 cells were synchronized in serum-free medium for 12 h. L02 cells were exposed to graded GCDCA concentrations (25–400 μM, HY-N2334, MCE) for 24 h or 200 μM GCDCA for 6–72 h to establish dose/time-response profiles.

Techniques: Activity Assay, Flow Cytometry, Western Blot

Journal: Journal of Inflammation Research

Article Title: Glycochenodeoxycholic acid induces the release of IL-1beta from L02 cells via the NLRP3/caspase-1/GSDMD pathway to activate LX2 cells

doi: 10.2147/JIR.S532042

Figure Lengend Snippet: GCDCA drives IL-1β secretion through GSDMD-dependent activation. ( A ) Dose-dependent IL-1β secretion in LPS-primed L02 cells treated with GCDCA (50–400 μM, 24 h). ( B )Time-dependent IL-1β secretion (200 μM GCDCA, 12–72 h). ( C ) Western blot analysis of GSDMD-NT and GSDMD in L02 cells and mature IL-1β in cell supernatant. ( D ) ELISA quantification of extracellular IL-1β. n ≥ 3 for each group. a P < 0.01.

Article Snippet: L02 and LX2 cells were synchronized in serum-free medium for 12 h. L02 cells were exposed to graded GCDCA concentrations (25–400 μM, HY-N2334, MCE) for 24 h or 200 μM GCDCA for 6–72 h to establish dose/time-response profiles.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: Glycochenodeoxycholic acid induces the release of IL-1beta from L02 cells via the NLRP3/caspase-1/GSDMD pathway to activate LX2 cells

doi: 10.2147/JIR.S532042

Figure Lengend Snippet: GCDCA-mediated hepatocyte IL-1β secretion activates HSCs. ( A ) Western blot analysis ofα-SMA and Collagen-I in LX2 cells treated with conditioned medium (CM) from GCDCA/LPS-stimulated L02 hepatocytes. ( B ) The protein levels of α-SMA and Collagen-I in LX2 cells with treatment of IL-1 receptor antagonist (IL-1RA, 200 ng/mL) upon CM exposure. ( C and D ) Representative images and quantification of LX2 migration upon CM exposure detected by wound healing assay. ( E ) CCK-8 assay of LX2 proliferation by CM treatment. n ≥ 3 for each group. a P < 0.05.

Article Snippet: L02 and LX2 cells were synchronized in serum-free medium for 12 h. L02 cells were exposed to graded GCDCA concentrations (25–400 μM, HY-N2334, MCE) for 24 h or 200 μM GCDCA for 6–72 h to establish dose/time-response profiles.

Techniques: Western Blot, Migration, Wound Healing Assay, CCK-8 Assay