Hong Lu1, Xin-Yue Zhang2, Yi-Qi Wang3, Xiao-Liang Zheng2, Wen-Min Xing3, Qin Zhang3. 1. School of Pharmacology, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China. Electronic address: 2698669983@qq.com. 2. Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, Zhejiang Province, China. 3. School of Pharmacology, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China.
Abstract
BACKGROUND AND AIMS: The nephrotoxic mechanisms of andrographolide sodium bisulfate (ASB) remain largely unknown. This study attempted to explore the mechanism of ASB-induced nephrotoxicity using human proximal tubular endothelial cells (HK-2). METHODS: For this study HK-2 cells were treated with rising concentrations of ASB. Their survival rate was detected using MTT assay and ultrastructure was observed with electron microscopy. L-Lactate dehydrogenase (LDH) assay was followed by examination of mitochondrial membrane potential (MMP). Reactive oxygen species (ROS) was detected using different methods and apoptosis/autophage related proteins were detected using immunoblotting. RESULTS: We found that ASB inhibited HK-2 cell proliferation and decreased cell survival rate in a time and dose-dependent manner (P<0.05, P<0.01, respectively). With increasing ASB concentration, cell structure was variably damaged and evidence of apoptosis and autophagy were observed. MMP gradually decreased and ROS was induced. The expression of JNK and Beclin-1 increased and activation of the JNK signaling pathway were seen. Apoptosis was induced via the mitochondrial-dependent caspase-3 and caspase-9 pathway, and autophagy related protein Beclin-1 was enhanced by ASB. CONCLUSION: The data show that ASB induces high levels of ROS generation in HK-2 cells and activates JNK signaling. Furthermore, ASB induces cell apoptosis via the caspase-dependent mitochondrial pathway, and induces cellular autophagy, in part by enhancing Beclin-1 protein expression.
BACKGROUND AND AIMS: The nephrotoxic mechanisms of andrographolide sodium bisulfate (ASB) remain largely unknown. This study attempted to explore the mechanism of ASB-induced nephrotoxicity using human proximal tubular endothelial cells (HK-2). METHODS: For this study HK-2 cells were treated with rising concentrations of ASB. Their survival rate was detected using MTT assay and ultrastructure was observed with electron microscopy. L-Lactate dehydrogenase (LDH) assay was followed by examination of mitochondrial membrane potential (MMP). Reactive oxygen species (ROS) was detected using different methods and apoptosis/autophage related proteins were detected using immunoblotting. RESULTS: We found that ASB inhibited HK-2 cell proliferation and decreased cell survival rate in a time and dose-dependent manner (P<0.05, P<0.01, respectively). With increasing ASB concentration, cell structure was variably damaged and evidence of apoptosis and autophagy were observed. MMP gradually decreased and ROS was induced. The expression of JNK and Beclin-1 increased and activation of the JNK signaling pathway were seen. Apoptosis was induced via the mitochondrial-dependent caspase-3 and caspase-9 pathway, and autophagy related protein Beclin-1 was enhanced by ASB. CONCLUSION: The data show that ASB induces high levels of ROS generation in HK-2 cells and activates JNK signaling. Furthermore, ASB induces cell apoptosis via the caspase-dependent mitochondrial pathway, and induces cellular autophagy, in part by enhancing Beclin-1 protein expression.