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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 upregulated NADPH oxidase, accompanying with the increases of ROS and the protein expression of antioxidation proteins Trx1, Prx5, HO-1, SOD1 and SOD2.(A) Antioxidation proteins Trx1, Prx5, HO-1, SOD1 and SOD2 were upregulated by AL-1 in a dose-dependent manner. (B) ROS level in RIN-mβ cells was increased by AL-1 in a dose-dependent manner. (C, D) Low dose H2O2 pretreatment also attenuated high dose H2O2-induced cell death. The cells were treated with the different concentration H2O2 (0, 5, 15, 30 μM) for 12 h prior to 400 μM H2O2 exposure for 4 h, the protective effects of AL-1 on H2O2-induced cell death were analyzed by MTT assay (C) and flow cytometer (D). (E) The NADPH oxidase expression was upregulated by AL-1 in a dose-dependent manner. (F) AL-1 stimulated ROS generation by upregulating NADPH oxidase. The RIN-mβ cells were pretreated with 0.1 μM AL-1 for 30 min, and then exposed to 10 μM NADPH oxidase inhibitor DPI for 30 min. The inhibition of NADPH oxidase blocked the AL-1-induced generation of ROS.
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pone-0063656-g005: AL-1 upregulated NADPH oxidase, accompanying with the increases of ROS and the protein expression of antioxidation proteins Trx1, Prx5, HO-1, SOD1 and SOD2.(A) Antioxidation proteins Trx1, Prx5, HO-1, SOD1 and SOD2 were upregulated by AL-1 in a dose-dependent manner. (B) ROS level in RIN-mβ cells was increased by AL-1 in a dose-dependent manner. (C, D) Low dose H2O2 pretreatment also attenuated high dose H2O2-induced cell death. The cells were treated with the different concentration H2O2 (0, 5, 15, 30 μM) for 12 h prior to 400 μM H2O2 exposure for 4 h, the protective effects of AL-1 on H2O2-induced cell death were analyzed by MTT assay (C) and flow cytometer (D). (E) The NADPH oxidase expression was upregulated by AL-1 in a dose-dependent manner. (F) AL-1 stimulated ROS generation by upregulating NADPH oxidase. The RIN-mβ cells were pretreated with 0.1 μM AL-1 for 30 min, and then exposed to 10 μM NADPH oxidase inhibitor DPI for 30 min. The inhibition of NADPH oxidase blocked the AL-1-induced generation of ROS.

Mentions: To validate whether AL-1 attenuated H2O2-induced cell death by regulating the expression of antioxidant proteins, some important antioxidant proteins including Trx 1, Prx 5, HO-1, SOD1 and SOD2 were selected for the expression analysis by Western blotting. As shown in Figure 5A, AL-1 upregulated the expression of antioxidant proteins Trx1, Prx 5, HO-1, SOD1 and SOD2 in a dose-dependent manner.


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 upregulated NADPH oxidase, accompanying with the increases of ROS and the protein expression of antioxidation proteins Trx1, Prx5, HO-1, SOD1 and SOD2.(A) Antioxidation proteins Trx1, Prx5, HO-1, SOD1 and SOD2 were upregulated by AL-1 in a dose-dependent manner. (B) ROS level in RIN-mβ cells was increased by AL-1 in a dose-dependent manner. (C, D) Low dose H2O2 pretreatment also attenuated high dose H2O2-induced cell death. The cells were treated with the different concentration H2O2 (0, 5, 15, 30 μM) for 12 h prior to 400 μM H2O2 exposure for 4 h, the protective effects of AL-1 on H2O2-induced cell death were analyzed by MTT assay (C) and flow cytometer (D). (E) The NADPH oxidase expression was upregulated by AL-1 in a dose-dependent manner. (F) AL-1 stimulated ROS generation by upregulating NADPH oxidase. The RIN-mβ cells were pretreated with 0.1 μM AL-1 for 30 min, and then exposed to 10 μM NADPH oxidase inhibitor DPI for 30 min. The inhibition of NADPH oxidase blocked the AL-1-induced generation of ROS.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672203&req=5

pone-0063656-g005: AL-1 upregulated NADPH oxidase, accompanying with the increases of ROS and the protein expression of antioxidation proteins Trx1, Prx5, HO-1, SOD1 and SOD2.(A) Antioxidation proteins Trx1, Prx5, HO-1, SOD1 and SOD2 were upregulated by AL-1 in a dose-dependent manner. (B) ROS level in RIN-mβ cells was increased by AL-1 in a dose-dependent manner. (C, D) Low dose H2O2 pretreatment also attenuated high dose H2O2-induced cell death. The cells were treated with the different concentration H2O2 (0, 5, 15, 30 μM) for 12 h prior to 400 μM H2O2 exposure for 4 h, the protective effects of AL-1 on H2O2-induced cell death were analyzed by MTT assay (C) and flow cytometer (D). (E) The NADPH oxidase expression was upregulated by AL-1 in a dose-dependent manner. (F) AL-1 stimulated ROS generation by upregulating NADPH oxidase. The RIN-mβ cells were pretreated with 0.1 μM AL-1 for 30 min, and then exposed to 10 μM NADPH oxidase inhibitor DPI for 30 min. The inhibition of NADPH oxidase blocked the AL-1-induced generation of ROS.
Mentions: To validate whether AL-1 attenuated H2O2-induced cell death by regulating the expression of antioxidant proteins, some important antioxidant proteins including Trx 1, Prx 5, HO-1, SOD1 and SOD2 were selected for the expression analysis by Western blotting. As shown in Figure 5A, AL-1 upregulated the expression of antioxidant proteins Trx1, Prx 5, HO-1, SOD1 and SOD2 in a dose-dependent manner.

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