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Protective effects of andrographolide analogue AL-1 on ROS-induced RIN-mβ cell death by inducing ROS generation.

Yan GR, Zhou HH, Wang Y, Zhong Y, Tan ZL, Wang Y, He QY - PLoS ONE (2013)

Bottom Line: In this work, we used proteomics to identify AL-1-regulated proteins in β-cells and found that 13 of the 71 proteins regulated by AL-1 were closely associated with antioxidation.Functional investigation demonstrated that AL-1 exerted its protective effects on H2O2-induced cell death of β-cells by generating NADPH oxidase-dependent ROS to activate ERK1/2 and AKT1 signaling pathways.To the best of our knowledge, this is the first comprehensive proteomic analysis illustrating a novel molecular mechanism for the protective effects of antioxidants on β-cells from H2O2-induced cell death.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, China. tgryan@jnu.edu.cn

ABSTRACT
Oxidative stress is considered to be a major factor contributing to pathogenesis and progression of many diseases. A novel andrographolide-lipoic acid conjugate (AL-1) could protect pancreatic β-cells from reactive oxygen species (ROS)-induced oxidative injury. However, its protective mechanism is still unclear. In this work, we used proteomics to identify AL-1-regulated proteins in β-cells and found that 13 of the 71 proteins regulated by AL-1 were closely associated with antioxidation. These differential proteins were mainly involved in the ERK1/2 and AKT1 signaling pathways. Functional investigation demonstrated that AL-1 exerted its protective effects on H2O2-induced cell death of β-cells by generating NADPH oxidase-dependent ROS to activate ERK1/2 and AKT1 signaling pathways. As a consequence, the expressions of antioxidant proteins including Trx1, Prx1 and Prx5, and anti-apoptotic proteins including PDCD6IP, prohibitin, galectin-1 and HSP were upregulated. AL-1 probably worked as a "vaccinum" to activate the cellular antioxidant system by inducing the generation of low concentration ROS which then reciprocally protected β-cells from oxidative damage caused by high-level ROS from H2O2. To the best of our knowledge, this is the first comprehensive proteomic analysis illustrating a novel molecular mechanism for the protective effects of antioxidants on β-cells from H2O2-induced cell death.

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AL-1 attenuated H2O2-induced RIN-mβ cell death.(A) Effect of AL-1 on H2O2-induced cell viability. The RIN-mβ cells were pretreated with different concentrations (0.01, 0.1, 1 μM) of AL-1 prior to 400 μM H2O2 exposure for 4 h. The cell viability was measured by MTT assay. (B) Flow cytometric analysis for the AL-1 protection of RIN-mβ cells against H2O2-induced death. The RIN-mβ cells were treated with 0.1 μM AL-1, 400 μM H2O2, or 0.1 μM AL-1 for 1 h prior to 400 μM H2O2. The number of apoptotic cells was measured by flow cytometer.
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pone-0063656-g001: AL-1 attenuated H2O2-induced RIN-mβ cell death.(A) Effect of AL-1 on H2O2-induced cell viability. The RIN-mβ cells were pretreated with different concentrations (0.01, 0.1, 1 μM) of AL-1 prior to 400 μM H2O2 exposure for 4 h. The cell viability was measured by MTT assay. (B) Flow cytometric analysis for the AL-1 protection of RIN-mβ cells against H2O2-induced death. The RIN-mβ cells were treated with 0.1 μM AL-1, 400 μM H2O2, or 0.1 μM AL-1 for 1 h prior to 400 μM H2O2. The number of apoptotic cells was measured by flow cytometer.

Mentions: To determine the protective effects of AL-1 on H2O2-induced cell death, RIN-mβ cells were pretreated with different concentrations (0.01, 0.1, 1 μM) of AL-1 prior to 400 μM H2O2 exposure for 4 h. MTT assay showed that the number of the surviving cells was increased by AL-1 in a dose-dependent manner as compared to the treatment with H2O2 alone, while the AL-1 itself had no effect on the cell death (Fig. 1A). Also the cells were pretreated with 0.1 μM AL-1 for the different time (0, 0.5, 1, 2, 4, 8, 12, 24 h) prior to 400 μM H2O2 exposure for 4 h, MTT assay demonstrated that AL-1 exhibited the protective effect against H2O2-induced cell death when its pretreatment time was less than 8 h (Fig. S1). These observations suggested that AL-1 could attenuate H2O2-induced cell death. To exclude a direct protective effect of AL-1, the cells were co-treated with the different concentrations of AL-1 (0, 0.01, 0.1, 1 μM) plus 400 μM H2O2 for 4 h, our results showed that the cell viability was not significantly different as compared to the treatment with H2O2 alone, suggesting that AL-1 had not direct protective effect on the high dose H2O2-induced cell death (Fig. S2). Hoechst 33258 staining demonstrated massive nuclear condensation, a typical morphology characteristic of apoptotic cells/bodies [19], in cells exposed to H2O2, while the nuclear condensation significantly decreased in AL-1-pretreated cells (Fig. 1B). The protective effects of AL-1 on H2O2-induced cell death were further investigated by flow cytometric analysis. The percentage (18.6%) of cell death in RIN-mβ cells pretreated with 0.1 μM AL-1 for 1 h prior to 400 μM H2O2 exposure for 4 h was substantially lower than that (49.8%) in the control cells treated with 400 μM H2O2 alone for 4 h (Fig. 1C). Taken together, these observations demonstrated that AL-1 could attenuate H2O2-induced cell death in RIN-mβ cells.


Protective effects of andrographolide analogue AL-1 on ROS-induced RIN-mβ cell death by inducing ROS generation.

Yan GR, Zhou HH, Wang Y, Zhong Y, Tan ZL, Wang Y, He QY - PLoS ONE (2013)

AL-1 attenuated H2O2-induced RIN-mβ cell death.(A) Effect of AL-1 on H2O2-induced cell viability. The RIN-mβ cells were pretreated with different concentrations (0.01, 0.1, 1 μM) of AL-1 prior to 400 μM H2O2 exposure for 4 h. The cell viability was measured by MTT assay. (B) Flow cytometric analysis for the AL-1 protection of RIN-mβ cells against H2O2-induced death. The RIN-mβ cells were treated with 0.1 μM AL-1, 400 μM H2O2, or 0.1 μM AL-1 for 1 h prior to 400 μM H2O2. The number of apoptotic cells was measured by flow cytometer.
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pone-0063656-g001: AL-1 attenuated H2O2-induced RIN-mβ cell death.(A) Effect of AL-1 on H2O2-induced cell viability. The RIN-mβ cells were pretreated with different concentrations (0.01, 0.1, 1 μM) of AL-1 prior to 400 μM H2O2 exposure for 4 h. The cell viability was measured by MTT assay. (B) Flow cytometric analysis for the AL-1 protection of RIN-mβ cells against H2O2-induced death. The RIN-mβ cells were treated with 0.1 μM AL-1, 400 μM H2O2, or 0.1 μM AL-1 for 1 h prior to 400 μM H2O2. The number of apoptotic cells was measured by flow cytometer.
Mentions: To determine the protective effects of AL-1 on H2O2-induced cell death, RIN-mβ cells were pretreated with different concentrations (0.01, 0.1, 1 μM) of AL-1 prior to 400 μM H2O2 exposure for 4 h. MTT assay showed that the number of the surviving cells was increased by AL-1 in a dose-dependent manner as compared to the treatment with H2O2 alone, while the AL-1 itself had no effect on the cell death (Fig. 1A). Also the cells were pretreated with 0.1 μM AL-1 for the different time (0, 0.5, 1, 2, 4, 8, 12, 24 h) prior to 400 μM H2O2 exposure for 4 h, MTT assay demonstrated that AL-1 exhibited the protective effect against H2O2-induced cell death when its pretreatment time was less than 8 h (Fig. S1). These observations suggested that AL-1 could attenuate H2O2-induced cell death. To exclude a direct protective effect of AL-1, the cells were co-treated with the different concentrations of AL-1 (0, 0.01, 0.1, 1 μM) plus 400 μM H2O2 for 4 h, our results showed that the cell viability was not significantly different as compared to the treatment with H2O2 alone, suggesting that AL-1 had not direct protective effect on the high dose H2O2-induced cell death (Fig. S2). Hoechst 33258 staining demonstrated massive nuclear condensation, a typical morphology characteristic of apoptotic cells/bodies [19], in cells exposed to H2O2, while the nuclear condensation significantly decreased in AL-1-pretreated cells (Fig. 1B). The protective effects of AL-1 on H2O2-induced cell death were further investigated by flow cytometric analysis. The percentage (18.6%) of cell death in RIN-mβ cells pretreated with 0.1 μM AL-1 for 1 h prior to 400 μM H2O2 exposure for 4 h was substantially lower than that (49.8%) in the control cells treated with 400 μM H2O2 alone for 4 h (Fig. 1C). Taken together, these observations demonstrated that AL-1 could attenuate H2O2-induced cell death in RIN-mβ cells.

Bottom Line: In this work, we used proteomics to identify AL-1-regulated proteins in β-cells and found that 13 of the 71 proteins regulated by AL-1 were closely associated with antioxidation.Functional investigation demonstrated that AL-1 exerted its protective effects on H2O2-induced cell death of β-cells by generating NADPH oxidase-dependent ROS to activate ERK1/2 and AKT1 signaling pathways.To the best of our knowledge, this is the first comprehensive proteomic analysis illustrating a novel molecular mechanism for the protective effects of antioxidants on β-cells from H2O2-induced cell death.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, China. tgryan@jnu.edu.cn

ABSTRACT
Oxidative stress is considered to be a major factor contributing to pathogenesis and progression of many diseases. A novel andrographolide-lipoic acid conjugate (AL-1) could protect pancreatic β-cells from reactive oxygen species (ROS)-induced oxidative injury. However, its protective mechanism is still unclear. In this work, we used proteomics to identify AL-1-regulated proteins in β-cells and found that 13 of the 71 proteins regulated by AL-1 were closely associated with antioxidation. These differential proteins were mainly involved in the ERK1/2 and AKT1 signaling pathways. Functional investigation demonstrated that AL-1 exerted its protective effects on H2O2-induced cell death of β-cells by generating NADPH oxidase-dependent ROS to activate ERK1/2 and AKT1 signaling pathways. As a consequence, the expressions of antioxidant proteins including Trx1, Prx1 and Prx5, and anti-apoptotic proteins including PDCD6IP, prohibitin, galectin-1 and HSP were upregulated. AL-1 probably worked as a "vaccinum" to activate the cellular antioxidant system by inducing the generation of low concentration ROS which then reciprocally protected β-cells from oxidative damage caused by high-level ROS from H2O2. To the best of our knowledge, this is the first comprehensive proteomic analysis illustrating a novel molecular mechanism for the protective effects of antioxidants on β-cells from H2O2-induced cell death.

Show MeSH
Related in: MedlinePlus