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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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The AL-1-activated ERK1/2 and AKT1 signaling pathways were involved in the anti-H2O2-induced cell cytotoxicity of AL-1.(A) The phosphorylation levels of ERK1/2 and AKT1 were upregulated by AL-1 in a dose-dependent manner, with total ERK1/2 and AKT1 levels remaining unchanged. (B) ROS decrease by NAC treatment blocked AL-1-induced ATK1 and ERK1/2 activation. The AL-1-pretreated cells were further treated with NAC prior to H2O2 exposure, p-ERK1/2, ERK1/2, p-AKT and AKT levels were analyzed by Western blotting. (C) AL-1 increased the phosphorylation of ERK1/2 and AKT1 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. (D) The anti-H2O2-induced cell death of AL-1 was mainly regulated by AL-1-activated ERK1/2 and AKT1. The cells were pretreated by combining 0.1 μM AL-1 with PI3K inhibitor wortmannin (250 nM) or the ERK1/2 inhibitor PD-98059 (25 μM) for 1 h, prior to the exposure to 400 μM H2O2 for 4 h. The number of apoptotic cells was analyzed by flow cytometer. The inhibition of ERK1/2 and AKT1 blocked the protective effect of AL-1 on H2O2-induced RIN-mβ cell death. (E) AL-1 upregulated the expression of antioxidant proteins Prx5 and Trx1 by activating AKT1 and ERK1/2 signaling pathways. (F) The protective effects of low dose H2O2 against high dose H2O2-induced cell death were also attenuated by AKT or ERK inhibition. The low dose H2O2-pretreated cells were further treated with AKT inhibitor wortmannin or ERK1/2 inhibitor PD-98059 prior to 400 μM H2O2 exposure, the cell viability was determined by flow cytometer.
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pone-0063656-g006: The AL-1-activated ERK1/2 and AKT1 signaling pathways were involved in the anti-H2O2-induced cell cytotoxicity of AL-1.(A) The phosphorylation levels of ERK1/2 and AKT1 were upregulated by AL-1 in a dose-dependent manner, with total ERK1/2 and AKT1 levels remaining unchanged. (B) ROS decrease by NAC treatment blocked AL-1-induced ATK1 and ERK1/2 activation. The AL-1-pretreated cells were further treated with NAC prior to H2O2 exposure, p-ERK1/2, ERK1/2, p-AKT and AKT levels were analyzed by Western blotting. (C) AL-1 increased the phosphorylation of ERK1/2 and AKT1 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. (D) The anti-H2O2-induced cell death of AL-1 was mainly regulated by AL-1-activated ERK1/2 and AKT1. The cells were pretreated by combining 0.1 μM AL-1 with PI3K inhibitor wortmannin (250 nM) or the ERK1/2 inhibitor PD-98059 (25 μM) for 1 h, prior to the exposure to 400 μM H2O2 for 4 h. The number of apoptotic cells was analyzed by flow cytometer. The inhibition of ERK1/2 and AKT1 blocked the protective effect of AL-1 on H2O2-induced RIN-mβ cell death. (E) AL-1 upregulated the expression of antioxidant proteins Prx5 and Trx1 by activating AKT1 and ERK1/2 signaling pathways. (F) The protective effects of low dose H2O2 against high dose H2O2-induced cell death were also attenuated by AKT or ERK inhibition. The low dose H2O2-pretreated cells were further treated with AKT inhibitor wortmannin or ERK1/2 inhibitor PD-98059 prior to 400 μM H2O2 exposure, the cell viability was determined by flow cytometer.

Mentions: The phosphorylation level at specific sites of protein kinases, including AKT1 and ERK1/2, represents their activity [29]. In order to validate that AL-1 could activate AKT1 and ERK1/2 signaling pathways by generating ROS, the phosphorylation and protein levels of AKT1 and ERK1/2 were analyzed in RIN-mβ cells treated with different concentrations of AL-1. As shown in Figure 6A, we found that the phosphorylation of ERK1/2 at Thr-202/Tyr-204 and AKT1 at Ser-473 was upregulated by AL-1 in a dose-dependent manner, with their total protein expression levels remaining unchanged. When AL-1-stimulated ROS generation was inhibited by antioxidant NAC, the AL-1-upregulated AKT1 and ERK1/2 phosphorylation was blocked (Fig. 6B). When AL-1-upregulated NADPH oxidase was inhibited by DP1, the activity of AKT1 and ERK1/2 was also repressed (Fig. 6C), suggesting that AL-1 activated ATK1 and ERK1/2 signaling pathways via NADPH oxidase-mediated ROS generation. The anti-H2O2-induced cell death of AL-1 was blocked when AL-1-activated ERK1/2 and AKT1 were inhibited by their inhibitors PD-98059 and wortmannin, respectively (Fig. 6D).


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)

The AL-1-activated ERK1/2 and AKT1 signaling pathways were involved in the anti-H2O2-induced cell cytotoxicity of AL-1.(A) The phosphorylation levels of ERK1/2 and AKT1 were upregulated by AL-1 in a dose-dependent manner, with total ERK1/2 and AKT1 levels remaining unchanged. (B) ROS decrease by NAC treatment blocked AL-1-induced ATK1 and ERK1/2 activation. The AL-1-pretreated cells were further treated with NAC prior to H2O2 exposure, p-ERK1/2, ERK1/2, p-AKT and AKT levels were analyzed by Western blotting. (C) AL-1 increased the phosphorylation of ERK1/2 and AKT1 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. (D) The anti-H2O2-induced cell death of AL-1 was mainly regulated by AL-1-activated ERK1/2 and AKT1. The cells were pretreated by combining 0.1 μM AL-1 with PI3K inhibitor wortmannin (250 nM) or the ERK1/2 inhibitor PD-98059 (25 μM) for 1 h, prior to the exposure to 400 μM H2O2 for 4 h. The number of apoptotic cells was analyzed by flow cytometer. The inhibition of ERK1/2 and AKT1 blocked the protective effect of AL-1 on H2O2-induced RIN-mβ cell death. (E) AL-1 upregulated the expression of antioxidant proteins Prx5 and Trx1 by activating AKT1 and ERK1/2 signaling pathways. (F) The protective effects of low dose H2O2 against high dose H2O2-induced cell death were also attenuated by AKT or ERK inhibition. The low dose H2O2-pretreated cells were further treated with AKT inhibitor wortmannin or ERK1/2 inhibitor PD-98059 prior to 400 μM H2O2 exposure, the cell viability was determined by flow cytometer.
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pone-0063656-g006: The AL-1-activated ERK1/2 and AKT1 signaling pathways were involved in the anti-H2O2-induced cell cytotoxicity of AL-1.(A) The phosphorylation levels of ERK1/2 and AKT1 were upregulated by AL-1 in a dose-dependent manner, with total ERK1/2 and AKT1 levels remaining unchanged. (B) ROS decrease by NAC treatment blocked AL-1-induced ATK1 and ERK1/2 activation. The AL-1-pretreated cells were further treated with NAC prior to H2O2 exposure, p-ERK1/2, ERK1/2, p-AKT and AKT levels were analyzed by Western blotting. (C) AL-1 increased the phosphorylation of ERK1/2 and AKT1 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. (D) The anti-H2O2-induced cell death of AL-1 was mainly regulated by AL-1-activated ERK1/2 and AKT1. The cells were pretreated by combining 0.1 μM AL-1 with PI3K inhibitor wortmannin (250 nM) or the ERK1/2 inhibitor PD-98059 (25 μM) for 1 h, prior to the exposure to 400 μM H2O2 for 4 h. The number of apoptotic cells was analyzed by flow cytometer. The inhibition of ERK1/2 and AKT1 blocked the protective effect of AL-1 on H2O2-induced RIN-mβ cell death. (E) AL-1 upregulated the expression of antioxidant proteins Prx5 and Trx1 by activating AKT1 and ERK1/2 signaling pathways. (F) The protective effects of low dose H2O2 against high dose H2O2-induced cell death were also attenuated by AKT or ERK inhibition. The low dose H2O2-pretreated cells were further treated with AKT inhibitor wortmannin or ERK1/2 inhibitor PD-98059 prior to 400 μM H2O2 exposure, the cell viability was determined by flow cytometer.
Mentions: The phosphorylation level at specific sites of protein kinases, including AKT1 and ERK1/2, represents their activity [29]. In order to validate that AL-1 could activate AKT1 and ERK1/2 signaling pathways by generating ROS, the phosphorylation and protein levels of AKT1 and ERK1/2 were analyzed in RIN-mβ cells treated with different concentrations of AL-1. As shown in Figure 6A, we found that the phosphorylation of ERK1/2 at Thr-202/Tyr-204 and AKT1 at Ser-473 was upregulated by AL-1 in a dose-dependent manner, with their total protein expression levels remaining unchanged. When AL-1-stimulated ROS generation was inhibited by antioxidant NAC, the AL-1-upregulated AKT1 and ERK1/2 phosphorylation was blocked (Fig. 6B). When AL-1-upregulated NADPH oxidase was inhibited by DP1, the activity of AKT1 and ERK1/2 was also repressed (Fig. 6C), suggesting that AL-1 activated ATK1 and ERK1/2 signaling pathways via NADPH oxidase-mediated ROS generation. The anti-H2O2-induced cell death of AL-1 was blocked when AL-1-activated ERK1/2 and AKT1 were inhibited by their inhibitors PD-98059 and wortmannin, respectively (Fig. 6D).

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