Runyu Yang1, Changqin Song1, Jiaxi Chen1, Lvqi Zhou1, Xiubo Jiang1, Xiaomei Cao2, Yang Sun3, Qi Zhang4. 1. School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China. 2. Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China. 3. School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China; Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China. Electronic address: sunyangsunny@sina.com. 4. School of Pharmaceutical Sciences, Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China; Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China. Electronic address: zhangqi@njtech.edu.cn.
Abstract
BACKGROUND: Limonin, a bioactive compound from citrus plants, exerts antioxidant activities, however its therapeutic potential in acetaminophen (APAP)-induced hepatotoxicity remains unclear. PURPOSE: Our study aims to investigate the protective effect of limonin on APAP-induced hepatotoxicity and illuminate the underlying mechanisms. STUDY: design In vitro, we chose L-02 cells to establish in vitro APAP-induced liver injury model. L-02 cells were treated with APAP (7.5 mM) for 24 h after pre-incubation with limonin (10, 25, 50 μM) or NAC (250 μM) for 2 h. In vivo, we used C57BL/6 mice as an in vivo APAP-induced liver injury model. C57BL/6 mice with pre-treatment of limonin (40, 80 mg/kg) or NAC (150 mg/kg) for 1 h, were given with a single dose of APAP (300 mg/kg). METHODS: After pre-incubation with limonin (10, 25, 50 μM) for 2 h, L-02 cells were treated with APAP (7.5 mM) for 24 h.The experiments in vitro included MTT assay, Annexin V/PI staining, measurement of reactive oxygen species (ROS), quantitative real-time PCR analysis, Western blot analysis, immunofluorescence microscopy and analysis of LDH activity. Transfection of Nrf2 or Sirt1 siRNA was also conducted in vitro. In vivo, C57BL/6 mice with pre-treatment of limonin (40, 80 mg/kg) or NAC (150 mg/kg) for 1 h, were given with a single dose of APAP (300 mg/kg). Mice were sacrificed at 4, 12 h after APAP poisoning, and analysis of ALT and AST in serum, GSH level in liver tissues, liver histological observation and immunohistochemistry were performed. RESULTS: Limonin increased the cell viability and alleviated APAP-induced apoptosis in hepatocytes. Limonin also inhibited APAP-induced mitochondrial-mediated apoptosis by decreasing the ratio of Bax/Bcl-2, recovery of mitochondrial membrane potential (MMP), inhibiting ROS production and cleavage of caspase-3 in L-02 cells. Moreover, limonin induced activation of Nrf2 and increased protein expression and mRNA levels of its downstream targets, including HO-1, NQO1 and GCLC/GCLM. The inhibition of limonin on apoptosis and promotion on Nrf2 antioxidative pathway were lessened after the application of Nrf2 siRNA. In addition, limonin inhibited NF-κB transcriptional activation, NF-κB-regulated genes and protein expression of inflammatory related proteins iNOS and COX2. Furthermore, limonin increased the protein expression of Sirt1. Sirt1 siRNA transfection confirmed that limonin activated Nrf2 antioxidative pathway and inhibited NF-κB inflammatory response by upregulating Sirt1. Finally, we established APAP-induced liver injury in vivo and demonstrated that limonin alleviated APAP-induced hepatotoxicity by activating Nrf2 antioxidative signals and inhibiting NF-κB inflammatory response via upregulating Sirt1. CONCLUSION: In summary, this study documented that limonin mitigated APAP-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1, and demonstrated that limonin had therapeutic promise in APAP-induced liver injury.
BACKGROUND:Limonin, a bioactive compound from citrus plants, exerts antioxidant activities, however its therapeutic potential in acetaminophen (APAP)-induced hepatotoxicity remains unclear. PURPOSE: Our study aims to investigate the protective effect of limonin on APAP-induced hepatotoxicity and illuminate the underlying mechanisms. STUDY: design In vitro, we chose L-02 cells to establish in vitro APAP-induced liver injury model. L-02 cells were treated with APAP (7.5 mM) for 24 h after pre-incubation with limonin (10, 25, 50 μM) or NAC (250 μM) for 2 h. In vivo, we used C57BL/6 mice as an in vivo APAP-induced liver injury model. C57BL/6 mice with pre-treatment of limonin (40, 80 mg/kg) or NAC (150 mg/kg) for 1 h, were given with a single dose of APAP (300 mg/kg). METHODS: After pre-incubation with limonin (10, 25, 50 μM) for 2 h, L-02 cells were treated with APAP (7.5 mM) for 24 h.The experiments in vitro included MTT assay, Annexin V/PI staining, measurement of reactive oxygen species (ROS), quantitative real-time PCR analysis, Western blot analysis, immunofluorescence microscopy and analysis of LDH activity. Transfection of Nrf2 or Sirt1 siRNA was also conducted in vitro. In vivo, C57BL/6 mice with pre-treatment of limonin (40, 80 mg/kg) or NAC (150 mg/kg) for 1 h, were given with a single dose of APAP (300 mg/kg). Mice were sacrificed at 4, 12 h after APAP poisoning, and analysis of ALT and AST in serum, GSH level in liver tissues, liver histological observation and immunohistochemistry were performed. RESULTS:Limonin increased the cell viability and alleviated APAP-induced apoptosis in hepatocytes. Limonin also inhibited APAP-induced mitochondrial-mediated apoptosis by decreasing the ratio of Bax/Bcl-2, recovery of mitochondrial membrane potential (MMP), inhibiting ROS production and cleavage of caspase-3 in L-02 cells. Moreover, limonin induced activation of Nrf2 and increased protein expression and mRNA levels of its downstream targets, including HO-1, NQO1 and GCLC/GCLM. The inhibition of limonin on apoptosis and promotion on Nrf2 antioxidative pathway were lessened after the application of Nrf2 siRNA. In addition, limonin inhibited NF-κB transcriptional activation, NF-κB-regulated genes and protein expression of inflammatory related proteins iNOS and COX2. Furthermore, limonin increased the protein expression of Sirt1. Sirt1 siRNA transfection confirmed that limonin activated Nrf2 antioxidative pathway and inhibited NF-κB inflammatory response by upregulating Sirt1. Finally, we established APAP-induced liver injury in vivo and demonstrated that limonin alleviated APAP-induced hepatotoxicity by activating Nrf2 antioxidative signals and inhibiting NF-κB inflammatory response via upregulating Sirt1. CONCLUSION: In summary, this study documented that limonin mitigated APAP-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1, and demonstrated that limonin had therapeutic promise in APAP-induced liver injury.