Xing-Wang Hu1, Xiang-Min Li2, Ai-Min Wang2, Yong-Ming Fu3, Fang-Jie Zhang2, Feng Zeng2, Li-Ping Cao2, Hui Long2, Ying-Hui Xiong1, Ji Xu2, Jia Li4. 1. Department of Emergency, Xiangya Hospital Central South University, Changsha, 410008, Hunan Province, People's Republic of China. 2. Department of Infectious Diseases/Hunan Provincial Key Laboratory of Viral Hepatitis, Xiangya Hospital Central South University, Changsha, 410008, Hunan Province, People's Republic of China. 3. Scientific Research Center, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, 510080, Guangdong Province, People's Republic of China. 4. Department of Infectious Diseases/Hunan Provincial Key Laboratory of Viral Hepatitis, Xiangya Hospital Central South University, Changsha, 410008, Hunan Province, People's Republic of China. 4010789@csu.edu.cn.
Abstract
BACKGROUND: Acute liver injury is liver cell injury that occurs rapidly in a short period of time. Caffeine has been shown to maintain hepatoprotective effect with an unclear mechanism. Endoplasmic reticulum stress (ERS) has significant effects in acute liver injury. Induction of GRP78 is a hallmark of ERS. Whether or not caffeine's function is related to GRP78 remains to be explored. METHODS: Acute liver injury model was established by LPS-treated L02 cells and in vivo administration of LPS/D-Gal in mice. Caffeine was pre-treated in L02 cells or mice. Gene levels was determined by real-time PCR and western blot. Cell viability was tested by CCK-8 assay and cell apoptosis was tested by flow cytometry. The interaction of GRP78 and NEDD4L was determined by Pull-down and co-immunoprecipitation (Co-IP) assay. The ubiquitination by NEDD4L on GRP78 was validated by in vitro ubiquitination assay. RESULTS: Caffeine protected liver cells against acute injury induced cell apoptosis and ERS both in vitro and in vivo. Suppression of GRP78 could block the LPS-induced cell apoptosis and ERS. NEDD4L was found to interact with GRP78 and ubiquitinate its lysine of 324 site directly. Caffeine treatment induced the expression of NEDD4L, resulting in the ubiquitination and inhibition of GRP78. CONCLUSION: Caffeine mitigated the acute liver injury by stimulating NEDD4L expression, which inhibited GRP78 expression via ubiquitination at its K324 site. Low dose of caffeine could be a promising therapeutic treatment for acute liver injury.
BACKGROUND: Acute liver injury is liver cell injury that occurs rapidly in a short period of time. Caffeine has been shown to maintain hepatoprotective effect with an unclear mechanism. Endoplasmic reticulum stress (ERS) has significant effects in acute liver injury. Induction of GRP78 is a hallmark of ERS. Whether or not caffeine's function is related to GRP78 remains to be explored. METHODS: Acute liver injury model was established by LPS-treated L02 cells and in vivo administration of LPS/D-Gal in mice. Caffeine was pre-treated in L02 cells or mice. Gene levels was determined by real-time PCR and western blot. Cell viability was tested by CCK-8 assay and cell apoptosis was tested by flow cytometry. The interaction of GRP78 and NEDD4L was determined by Pull-down and co-immunoprecipitation (Co-IP) assay. The ubiquitination by NEDD4L on GRP78 was validated by in vitro ubiquitination assay. RESULTS: Caffeine protected liver cells against acute injury induced cell apoptosis and ERS both in vitro and in vivo. Suppression of GRP78 could block the LPS-induced cell apoptosis and ERS. NEDD4L was found to interact with GRP78 and ubiquitinate its lysine of 324 site directly. Caffeine treatment induced the expression of NEDD4L, resulting in the ubiquitination and inhibition of GRP78. CONCLUSION: Caffeine mitigated the acute liver injury by stimulating NEDD4L expression, which inhibited GRP78 expression via ubiquitination at its K324 site. Low dose of caffeine could be a promising therapeutic treatment for acute liver injury.
Authors: Robert Clarke; Ayesha N Shajahan; Yue Wang; John J Tyson; Rebecca B Riggins; Louis M Weiner; William T Bauman; Jianhua Xuan; Bai Zhang; Caroline Facey; Harini Aiyer; Katherine Cook; F Edward Hickman; Iman Tavassoly; Anael Verdugo; Chun Chen; Alan Zwart; Anni Wärri; Leena A Hilakivi-Clarke Journal: Horm Mol Biol Clin Investig Date: 2011-03