Journal: Frontiers in Pharmacology

Article Title: Glycyrrhetinic Acid Protects Renal Tubular Cells against Oxidative Injury via Reciprocal Regulation of JNK-Connexin 43-Thioredoxin 1 Signaling

doi: 10.3389/fphar.2021.619567

Figure Lengend Snippet: c-Jun N-terminal kinase (JNK)-mediated oxidative stress induces connexin 43 (Cx43). (A) Induction of JNK phosphorylation and expression of Cx43 by Px-12. NRK-52E cells in 6-well plates were incubated with Px-12 for 0, 1, 3, 6, 9, and 12 h separately. Cellular lysates were then analyzed by western blotting for phosphorylated JNK and Cx43 expression. Densitometric analysis of p -JNK and Cx43 are shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in control). (B) Effects of 18α-glycyrrhetinic acid (Ga) and sp600125, an inhibitor of JNK, on Px12-induced cell injury. NRK-52E cells were pretreated with Ga (20 μM) and sp600125 (20 μM) for 1 h and then challenged with Px-12 for another 24 h. Then, cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) assay. Data are expressed as the percentage of living cells vs. the untreated control (mean ± SD, n = 6). * p < 0.01 vs. the control. ## p < 0.01 vs. Px-12 in control. (C) Influence of sp600125 and Ga on NRK-52E cell apoptosis staining. NRK-52E cells in 96-well plate were pretreated with 18α-Ga and sp600125 for 1 h and then incubated with Px-12 for another 24 h. Then, apoptotic cells were stained via DT-mediated dUTP nick end labeling (TUNEL) and 4,6-diamidino-2-phenylindole (DAPI) (magnification, ×400). The ratio of positive cells is shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in the control).

Article Snippet: The JNK-mitogen-activated protein kinase (MAPK) signaling pathway inhibitor (sp600125, A4604) was obtained from ApexBio Technology China.

Techniques: Expressing, Incubation, Western Blot, Cell Counting, CCK-8 Assay, Staining, End Labeling, TUNEL Assay

Journal: Frontiers in Pharmacology

Article Title: Glycyrrhetinic Acid Protects Renal Tubular Cells against Oxidative Injury via Reciprocal Regulation of JNK-Connexin 43-Thioredoxin 1 Signaling

doi: 10.3389/fphar.2021.619567

Figure Lengend Snippet: Reciprocal modulation of connexin 43 (Cx43) and c-Jun N-terminal kinase (JNK). (A) Effect of 18α-glycyrrhetinic acid (Ga) and sp600125 on JNK phosphorylation and expression of Cx43. NRK-52E cells in six-well plates were pretreated with Ga and sp600125 for 1 h and then challenged with Px-12 for another 1 h. Cellular lysates were then analyzed by western blotting for phosphorylated JNK and Cx43 expression. Densitometric analysis of p -JNK and Cx43 are shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in the control). (B) NRK-52E cells were transfected with either Cx43 siRNA or siRNA control for 24 h. Cellular lysates were analyzed by western blotting for Cx43 expression. Densitometric analysis of Cx43 is shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the siRNA control group). (C) Effect of Cx43 siRNA on JNK phosphorylation and Cx43 expression. The transfected or normal cells were incubated with Px-12 for 1 h separately. Then, cellular lysates were analyzed by western blotting for phosphorylated JNK and Cx43 expression. Densitometric analysis of p -JNK and Cx43 are shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in the siRNA control group). (D) Effect of Cx43 siRNA on cell viability. The transfected or normal cells were incubated with Px-12 for 24 h. Then, cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) assay. Data are expressed as the percentage of living cells vs. the untreated control (mean ± SD, n = 6). * p < 0.01 vs. Px-12 in normal cells. ## p < 0.01 vs. Px12 in the siRNA control group. (E) Effect of Cx43 siRNA on caspase-3 activation. The transfected or normal cells were incubated with Px-12 for 24 h. Then, cellular lysates were analyzed by western blotting for expression of cleaved caspase-3. Densitometric analysis of cleaved caspase-3 and caspase-3 are shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. Px-12 in the siRNA control group).

Article Snippet: The JNK-mitogen-activated protein kinase (MAPK) signaling pathway inhibitor (sp600125, A4604) was obtained from ApexBio Technology China.

Techniques: Expressing, Western Blot, Transfection, Incubation, Cell Counting, CCK-8 Assay, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Glycyrrhetinic Acid Protects Renal Tubular Cells against Oxidative Injury via Reciprocal Regulation of JNK-Connexin 43-Thioredoxin 1 Signaling

doi: 10.3389/fphar.2021.619567

Figure Lengend Snippet: c-Jun N-terminal kinase (JNK)-mediated oxidative stress induces connexin 43 (Cx43). (A) Induction of JNK phosphorylation and expression of Cx43 by Px-12. NRK-52E cells in 6-well plates were incubated with Px-12 for 0, 1, 3, 6, 9, and 12 h separately. Cellular lysates were then analyzed by western blotting for phosphorylated JNK and Cx43 expression. Densitometric analysis of p -JNK and Cx43 are shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in control). (B) Effects of 18α-glycyrrhetinic acid (Ga) and sp600125, an inhibitor of JNK, on Px12-induced cell injury. NRK-52E cells were pretreated with Ga (20 μM) and sp600125 (20 μM) for 1 h and then challenged with Px-12 for another 24 h. Then, cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) assay. Data are expressed as the percentage of living cells vs. the untreated control (mean ± SD, n = 6). * p < 0.01 vs. the control. ## p < 0.01 vs. Px-12 in control. (C) Influence of sp600125 and Ga on NRK-52E cell apoptosis staining. NRK-52E cells in 96-well plate were pretreated with 18α-Ga and sp600125 for 1 h and then incubated with Px-12 for another 24 h. Then, apoptotic cells were stained via DT-mediated dUTP nick end labeling (TUNEL) and 4,6-diamidino-2-phenylindole (DAPI) (magnification, ×400). The ratio of positive cells is shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in the control).

Article Snippet: The JNK-mitogen-activated protein kinase (MAPK) signaling pathway inhibitor (sp600125, A4604) was obtained from ApexBio Technology China.

Techniques: Expressing, Incubation, Western Blot, Cell Counting, CCK-8 Assay, Staining, End Labeling, TUNEL Assay

Journal: Frontiers in Pharmacology

Article Title: Glycyrrhetinic Acid Protects Renal Tubular Cells against Oxidative Injury via Reciprocal Regulation of JNK-Connexin 43-Thioredoxin 1 Signaling

doi: 10.3389/fphar.2021.619567

Figure Lengend Snippet: Reciprocal modulation of connexin 43 (Cx43) and c-Jun N-terminal kinase (JNK). (A) Effect of 18α-glycyrrhetinic acid (Ga) and sp600125 on JNK phosphorylation and expression of Cx43. NRK-52E cells in six-well plates were pretreated with Ga and sp600125 for 1 h and then challenged with Px-12 for another 1 h. Cellular lysates were then analyzed by western blotting for phosphorylated JNK and Cx43 expression. Densitometric analysis of p -JNK and Cx43 are shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in the control). (B) NRK-52E cells were transfected with either Cx43 siRNA or siRNA control for 24 h. Cellular lysates were analyzed by western blotting for Cx43 expression. Densitometric analysis of Cx43 is shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the siRNA control group). (C) Effect of Cx43 siRNA on JNK phosphorylation and Cx43 expression. The transfected or normal cells were incubated with Px-12 for 1 h separately. Then, cellular lysates were analyzed by western blotting for phosphorylated JNK and Cx43 expression. Densitometric analysis of p -JNK and Cx43 are shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in the siRNA control group). (D) Effect of Cx43 siRNA on cell viability. The transfected or normal cells were incubated with Px-12 for 24 h. Then, cell viability was evaluated using a Cell Counting Kit-8 (CCK-8) assay. Data are expressed as the percentage of living cells vs. the untreated control (mean ± SD, n = 6). * p < 0.01 vs. Px-12 in normal cells. ## p < 0.01 vs. Px12 in the siRNA control group. (E) Effect of Cx43 siRNA on caspase-3 activation. The transfected or normal cells were incubated with Px-12 for 24 h. Then, cellular lysates were analyzed by western blotting for expression of cleaved caspase-3. Densitometric analysis of cleaved caspase-3 and caspase-3 are shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. Px-12 in the siRNA control group).

Article Snippet: The JNK-mitogen-activated protein kinase (MAPK) signaling pathway inhibitor (sp600125, A4604) was obtained from ApexBio Technology China.

Techniques: Expressing, Western Blot, Transfection, Incubation, Cell Counting, CCK-8 Assay, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Glycyrrhetinic Acid Protects Renal Tubular Cells against Oxidative Injury via Reciprocal Regulation of JNK-Connexin 43-Thioredoxin 1 Signaling

doi: 10.3389/fphar.2021.619567

Figure Lengend Snippet: Connexin 43 (Cx43) inhibition suppresses oxidative stress by regulating thioredoxin 1. (A) Effect of 18α-glycyrrhetinic acid (Ga) and sp600125 on reactive oxygens species (ROS)/O 2 − production. NRK-52E cells in a 96-well plate were pretreated with Ga and sp600125 separately for 1 h, challenged with Px-12 for another 1 h, and then incubated with a ROS/O 2 − probe for 30 min. Images were captured using an inverted fluorescence microscope (×100). Mean fluorescence intensity is shown at the bottom (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in the control). (B) Effects of Ga on thioredoxin 1. NRK-52E cells in six-well plates were pretreated with Ga for 1 h and then challenged with Px-12 for another 1 h. Then, cellular lysates were analyzed by western blotting for the expression of thioredoxin 1. Densitometric analysis of thioredoxin 1, as determined by ImageJ, is shown on the right (mean ± SD, n = 3; ** p < 0.01 vs. the control, ## p < 0.01 vs. Px-12 in the siRNA control group).

Article Snippet: The JNK-mitogen-activated protein kinase (MAPK) signaling pathway inhibitor (sp600125, A4604) was obtained from ApexBio Technology China.

Techniques: Inhibition, Incubation, Fluorescence, Microscopy, Western Blot, Expressing