Protective Effects And Mechanism Of Hepatopoietin Cn On Alcoholic Liver Injury

Oxidative stress is an important machenism by which ethanol causes liver injury. Alleviation of ethanol-induced oxidative stress in hepatocyte is of great meaning in the prevention and therapeutics of liver diseases. Hepatopoietin Cn （HPPCn）, a novel human hepatic growth factor found by our laboratory, exihibited the activity specificly in the stimulation of liver regeneration and liver-derived cells proliferation. Previous study showed that HPPCn could attenuate liver fiborosis caused by CCl4 in rat. In the present study, we analyzed the impact of HPPCn on ethanol-induced oxidative stress in liver and the potential mechanism.The recombinant human HPPCn （rhHPPCn） was produced by applying high cell density cultivation of E. coli. engineering strain BL21（DE3）/pET-24a（+）-HPPCn. The disovled oxygen feedback control was used during the fermentation process. The expression of target protein was induced by IPTG （isopropylβ-D-1-thiogalactopyranoside, IPTG）. The yield was about 480 g dry cell weigh and the amount of rhHPPCn in the form of inclusion bodies was 51 g. After solubilazation and renaturation, the refolded rhHPPCn was purified by Q-XL anion-exchange chromatograghy and the yield was 19.2 g. The recombinant adenovirus carrying HPPCn gene, Ad-SP-HPPCn was constructed using AdEasyTM adenovirus construction system. The signal peptide sequence was added to guarantee the extracellular secretion of HPPCn. The Ad-SP-HPPCn was amplified in HEK293 cells and purified by CsCl density gradient ultracentrifugation. The tite of Ad-SP-HPPCn was up to 1.6×10¹¹ pfu/ml.The effects of rhHPPCn on the ethanol-induced oxidative stress were analyzed in vitro using human normal liver cell line L02. The results showed that coincubation with 100 mM ethanol for 12 h caused the enhanced released of GOT （glutamic oxaloacetic transaminase, GOT）; accumulation of ROS （reactive oxygen species,ROS）; and destruction of anti-oxidative defence in cells, including reducing of SOD （superoxide dismutase, SOD） activity and decrease of reduced GSH （reduced glutathione hormone, GSH） content. The pretreatment of rhHPPCn significantly alleviated the cell injury caused by ethanol, proved by the reduced GOT release. The oxidative stress in L02 cells was also attenuated by rhHPPCn, which was showed by the maintained SOD and GSH level and the restored ROS scavenging activity. Moreover, the activation of two survival pathways, ERK and SPK, by rhHPPCn indicated that the protective effects of HPPCn dependent at least partly on these pathways.In vivo study was conducted by ethanol intragastric administration in KM mice. Acute ethanol exposure caused significant pathological changes in mice liver and increased GPT level （glutamate-pyruvate transaminase, GPT） in serum. The TG （triglyeride, TG） content both in liver and in serum was elevated by ethanol treatment. The formation of MDA （malondialdehyde, MDA） and 4-HNE （4-Hydroxynonenal, 4-HNE） protein adducts in liver also increased after ethanol administrationl. These damages were correlated with the increase in the CYP450 2E1 expression and decrease in the SOD activity and GSH content induced by ethanol. Pretreatment with rhHPPCn or Ad-SP-HPPCn attenuated the ethanol-induced oxidative stress and liver injury significantly.