Journal: British Journal of Pharmacology

Article Title: Dual inhibition of cannabinoid CB 1 receptor and inducible NOS attenuates obesity‐induced chronic kidney disease

doi: 10.1111/bph.14849

Figure Lengend Snippet: MRI‐1867 reverses the fatty acid‐induced reduction in adiponectin signalling. Mice on standard diet (STD) or high‐fat diet (HFD) for 18 weeks were treated with vehicle (Veh) or MRI‐1867 (3 mg·kg −1 ) orally for 28 days. (a–c) MRI‐1867 restored the HFD‐induced reduction in the mRNA renal expression of adiponectin, and Adipo2 but not Adipo1 receptors. Similarly, exposing HK‐2 cells to O:P (0.5 mM, 2:1, respectively) resulted in reduced (d–f) mRNA and protein expression of adiponectin as well as reduced mRNA levels of (g) Adipo1 and (h) Adipo2 receptors. Pretreatment of the cells with MRI‐1867 (100 ng·ml −1 ) completely normalized these changes. (i) A proposed mechanism for the dual blockade of CB 1 receptors and inducible NOS by MRI‐1867 in reversing obesity‐induced chronic kidney disease (CKD) is shown. RQ, relative quantitation. In vivo data represent the mean ± SEM from 8 to 14 mice per group. * P < .05, significantly different from animals on STD; # P < .05, significantly different from animals on the same diet. In vitro data represent the mean ± SEM from five independent experiments. * P < .05, significantly different from Veh‐treated cells under normal conditions; # P < .05, significantly different from Veh‐treated cells under O:P conditions. Data were analysed by one‐way ANOVA, followed by a Bonferroni post hoc test

Article Snippet: HK‐2 cells were transfected with CRISPR‐CAS9 vector containing an sgRNA sequence to target all human isoforms of CNR1 (Genecopeia™, Cat# CS‐HCP263432‐CG01‐01‐B) using Lipofectamine 3000 (Invitrogen, Cat# L3000‐001) and selected with hygromycin (200‐μM; Sigma‐Aldrich, Cat# H3274).

Techniques: Expressing, Quantitation Assay, In Vivo, In Vitro

Journal: Journal of Natural Medicines

Article Title: Allicin ameliorates sepsis-induced acute kidney injury through Nrf2/HO-1 signaling pathway

doi: 10.1007/s11418-023-01745-3

Figure Lengend Snippet: Allicin inhibited the inflammatory response and oxidative stress, and restored mitochondrial function in HK2 cells primed with lipopolysaccharide (LPS). a CCK-8 analysis of the viability of HK2 cells treated with allicin. b CCK-8 analysis of the viability of HK2 cells with LPS and allicin treatments. c Protein levels of IL-6, IL-1β, TNF-α and MCP-1 in HK2 cells with LPS and allicin treatments. d Levels of ROS and MDA, as well as activities of SOD and GSH-Px in LPS-primed HK2 cells after allicin treatment. e Levels of cytochrome c in the mitochondria and cytoplasm of HK2 cells treated with LPS and allicin. f Analysis of JC-1-labeled mitochondrial membrane potential changes by flow cytometry in HK2 cells, and statistical analysis of JC-1 green monomer. g Detection of cell apoptosis by flow cytometry in HK2 cells with LPS and allicin treatment. All data were expressed as mean ± SD. “ns”: no significance, ** p < 0.01, and *** p < 0.001, n = 3 in each group, and the number of technical replicates is 3

Article Snippet: HK2 cell line was purchased from Procell Life Science & Technology (Wuhan, China), and it was cultured in MEM (Solarbio, Beijing, China) supplemented with 10% fetal bovine serum (Tianhang, Zhejiang, China) in an incubator at 37 °C with 5% CO 2 .

Techniques: CCK-8 Assay, Labeling, Membrane, Flow Cytometry

Journal: Journal of Natural Medicines

Article Title: Allicin ameliorates sepsis-induced acute kidney injury through Nrf2/HO-1 signaling pathway

doi: 10.1007/s11418-023-01745-3

Figure Lengend Snippet: Allicin inhibited the cytotoxicity of LPS in HK2 cells by promoting the Nrf2/HO-1 signaling pathway. a Protein levels of cleaved caspase-3, cleaved caspase-9, Bax, and Bcl-2 in HK2 cells after allicin and LPS treatment. b Protein levels of Nrf2 and HO-1 in HK2 cells treated with allicin and LPS. All data were expressed as mean ± SD. c The accumulation of NRF2 in the nucleus was detected by immunofluorescence, bar = 50 μm. * p < 0.05, ** p < 0.01, and *** p < 0.001, n = 3 in each group, and the number of technical replicates is 3

Article Snippet: HK2 cell line was purchased from Procell Life Science & Technology (Wuhan, China), and it was cultured in MEM (Solarbio, Beijing, China) supplemented with 10% fetal bovine serum (Tianhang, Zhejiang, China) in an incubator at 37 °C with 5% CO 2 .

Techniques: Immunofluorescence

Journal: Journal of Natural Medicines

Article Title: Allicin ameliorates sepsis-induced acute kidney injury through Nrf2/HO-1 signaling pathway

doi: 10.1007/s11418-023-01745-3

Figure Lengend Snippet: Validation of the promotive effect of allicin on Nrf2/HO-1 signaling pathway. a Levels of TNF-α, IL-1β and ROS as well as the activity of GSH-Px in HK2 cells with allicin and ML385 treatment. b Analysis of JC-1-labeled mitochondrial membrane potential by flow cytometry in LPS and allicin and ML385 treated HK2 cells. c Evaluation of cell apoptosis by flow cytometry in HK2 cells with LPS and allicin and ML385 treatments. All data were expressed as mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001, it was compared with LPS + allicin-20 group, n = 3 in each group, and the number of technical replicates is 3

Article Snippet: HK2 cell line was purchased from Procell Life Science & Technology (Wuhan, China), and it was cultured in MEM (Solarbio, Beijing, China) supplemented with 10% fetal bovine serum (Tianhang, Zhejiang, China) in an incubator at 37 °C with 5% CO 2 .

Techniques: Biomarker Discovery, Activity Assay, Labeling, Membrane, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the IRE1/JNK pathway in renal tubular epithelial cells attenuates ferroptosis in acute kidney injury

doi: 10.3389/fphar.2022.927641

Figure Lengend Snippet: Primer sequences used in this study (qRT-PCR).

Article Snippet: IRE1α or JNK1 knock-down HK-2 cell lines (stable depletion) were generated by the Obio Technology Corp. Ltd. (Shanghai, China), as described in our previous study ( ).

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the IRE1/JNK pathway in renal tubular epithelial cells attenuates ferroptosis in acute kidney injury

doi: 10.3389/fphar.2022.927641

Figure Lengend Snippet: Renal artery ischemia-reperfusion leads to renal insufficiency and ER stress in renal tubules of mice. Male C57BL/6J mice underwent sham operation or bilateral renal artery clamping for 27 min before reperfusion (A) Changes in serum creatinine (Scr). When the ischemia-reperfusion (I/R) groups were compared at 12, 24, 48, and 72 h after I/R, the expression was highest at 12 h ( p < 0.0001) (B) Changes in blood urea nitrogen (BUN). When the I/R groups were compared at 12, 24, 48, and 72 h after I/R, the expression was highest at 12 h ( p < 0.001) (C) PAS staining showed lesions in renal tubular tissue. Scale bar = 100 μm (D) Transmission electron microscopy was used to observe the ER of mice renal tubules after I/R (E) The Paller scores of each group in PAS staining, the score was the highest at 48 h ( p < 0.0001) (F) The analysis of the width of ER in tubular epithelial cells (TEC) of renal tissue, detected by electron microscopy (G) Western blotting detection of PERK, ATF6, p-IRE1α, p-JNK, and CHOP in the renal tissue samples, β-actin used as an internal control (H) Protein synthesis of ER stress production in western blotting. Due to the death of mice, N = 5 for each group. Statistical significance is denoted as *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 compared with the sham group at the same time point.

Article Snippet: IRE1α or JNK1 knock-down HK-2 cell lines (stable depletion) were generated by the Obio Technology Corp. Ltd. (Shanghai, China), as described in our previous study ( ).

Techniques: Expressing, Staining, Transmission Assay, Electron Microscopy, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the IRE1/JNK pathway in renal tubular epithelial cells attenuates ferroptosis in acute kidney injury

doi: 10.3389/fphar.2022.927641

Figure Lengend Snippet: Detection of hypoxia/reoxygenation-induced ER stress in HK-2 cells at 6, 12, and 24 h after hypoxic for 4 h (A) Transmission electron microscopy was used to observe the ER of HK-2 cells (B) The analysis of the width of ER in HK-2 cells, detected by electron microscopy (C) Representative western blotting of ER stress markers (D) Protein synthesis of ER stress production in western blotting (E) mRNA expressions of IRE1α, JNK1, and CHOP in qRT-PCR. When the hypoxia/reoxygenation (H/R) groups were compared at 6, 12, and 24 h after H/R, the expression was highest at 12 h ( p < 0.0001). Statistical significance is denoted as *: p < 0.05, **: p < 0.01, ****: p < 0.0001 compared with the control group at the same time point.

Article Snippet: IRE1α or JNK1 knock-down HK-2 cell lines (stable depletion) were generated by the Obio Technology Corp. Ltd. (Shanghai, China), as described in our previous study ( ).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Quantitative RT-PCR, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the IRE1/JNK pathway in renal tubular epithelial cells attenuates ferroptosis in acute kidney injury

doi: 10.3389/fphar.2022.927641

Figure Lengend Snippet: Depletion of IRE1 or JNK reduces ER stress in mice after I/R and HK-2 cells after H/R (A) Comparison of p-IRE1α, p-JNK, and CHOP expression in the renal tissue of the I/R, I/R + Irestatin 9389, and I/R + SP 600125 group at 48 h after I/R (B) Protein synthesis of p-IRE1α, p-JNK, and CHOP in western blotting in the renal tissue. Due to the death of mice, N = 5 for each group (C) Representative western blotting of ER stress markers in the HK-2 cells depleted of IRE1α and infected of vehicle at 12 h after H/R, with β-actin used as an internal control (D) Protein synthesis of ER markers in western blotting of the HK-2 cells depleted of IRE1α (E) Representative western blotting of ER stress markers in the HK-2 cells depleted of JNK1 and infected of vehicle at 12 h after H/R (F) Protein synthesis of ER markers in western blotting of the HK-2 cells depleted of JNK1 (G) mRNA expressions of IRE1α, JNK1, and CHOP in the HK-2 cells depleted of IRE1α using qRT-PCR (H) mRNA expressions of IRE1α, JNK1, and CHOP in the HK-2 cells depleted of JNK1 using qRT-PCR. Statistical significance is denoted as *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, NS: p > 0.05 in two groups.

Article Snippet: IRE1α or JNK1 knock-down HK-2 cell lines (stable depletion) were generated by the Obio Technology Corp. Ltd. (Shanghai, China), as described in our previous study ( ).

Techniques: Comparison, Expressing, Western Blot, Infection, Control, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the IRE1/JNK pathway in renal tubular epithelial cells attenuates ferroptosis in acute kidney injury

doi: 10.3389/fphar.2022.927641

Figure Lengend Snippet: Depletion of IRE1 or JNK reduces ferroptosis in mice after I/R and HK-2 cells after H/R. Western blotting detection (A) and protein synthesis (B) of GXP4 and 4-HNE in renal tissues of mice at 48 h after I/R. Due to the death of mice, N = 5 for each group. Western blotting was used to detect the expression of GXP4 and 4-HNE in HK-2 cells depleted of IRE1α (C) or JNK (D) at 12 h after H/R, β-actin used as an internal control. Protein synthesis of GXP4 and 4-HNE in western blotting in HK-2 cells depleted of IRE1α (E) or JNK (F) at 12 h after H/R (G) Flow cytometry detection of ROS produced by HK-2 cells treated with shRNA IRE1α or shRNA JNK1 (H) Analysis of ROS in flow cytometry in HK-2 cells at 12 h after H/R (I) mRNA expression of GXP4 in HK-2 cells treated with shRNA IRE1α or shRNA JNK1 detected by qRT-PCR. Statistical significance is denoted as *: p < 0.05, **: p < 0.01, ****: p < 0.0001 in two groups.

Article Snippet: IRE1α or JNK1 knock-down HK-2 cell lines (stable depletion) were generated by the Obio Technology Corp. Ltd. (Shanghai, China), as described in our previous study ( ).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Produced, shRNA, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Inhibition of the IRE1/JNK pathway in renal tubular epithelial cells attenuates ferroptosis in acute kidney injury

doi: 10.3389/fphar.2022.927641

Figure Lengend Snippet: Inhibition of ferroptosis attenuates IRE1/JNK pathway in mice after I/R and HK-2 cells after H/R. Western blotting detection (A) and protein synthesis (B) of p-IRE1α, p-JNK, and CHOP in mice at 48 h after I/R. Due to the death of mice, N = 5 for each group. Western blotting detection (C) and protein synthesis (D) of p-IRE1α, p-JNK, and CHOP in HK-2 cells at 12 h after H/R. Statistical significance is denoted as *: p < 0.05, **: p < 0.01 in two groups.

Article Snippet: IRE1α or JNK1 knock-down HK-2 cell lines (stable depletion) were generated by the Obio Technology Corp. Ltd. (Shanghai, China), as described in our previous study ( ).

Techniques: Inhibition, Western Blot