Research Of Grape Seed Proanthocyanidin Extract Protects Against Lead-induced Hepatotoxicity

Lead(Pb) is a pervasive toxic heavy metal with many properties, such as softness, malleability, and resistance to corrosion, which is widely applied to modern industry, especially in developing country. Although measures have been used to control Pb pollution on global scale, there are approximately 20,000 tons of Pb are kept out of the atmosphere in the form of soot, which poses a great threat to the health of human and animal. About 33% of absorbed Pb is stored in liver tissue and cause serious damage to the liver. So reducing the Pb accumulation in the body and ameliorating the damage of liver is an urgent issue. Grape seed proanthocyanidin extract(GSPE) is a natural antioxidant, and possess a broad spectrum of biological properties, such as anti-inflammatory, antibacterial, anti-carcinogenic, cardioprotective, and hepatoprotective, and neuroprotective effect. Therefore, the present study was designed to investigate the protective roles of GSPE on Pb-induced liver injury, Pb accumulation in the liver, DNA damage, oxidative stress, and apoptosis in vivo and vitro.Forty Wistar rats were divided into 4 groups as follows: Control group, GSPE group, Pb group, and Pb+GSPE group, which lasted for thirty days. Rats in the control group received vehicle only, rats in the GSPE group received 200 mg/kg per day, rats in the Pb group received 2.5 g/L lead acetate dissolving in the drinking water and rats in the Pb+GSPE group received 200 mg/kg GSPE per day and 2.5 g/L lead acetate dissolving in the drinking. After 30 days, the antioxidant indexes(superoxide dismutase, glutathione, and malondialdehyde), apoptosis, and DNA damage were measured by Reagent Kit method,Alanine aminotransferase(ALT) and Aspartate transaminase(AST) were measured by automatic biochemistry analyzer, antomatic haemacytometer analyzer was used to determine the number of red blood cells(RBC) and the concentration of hemoglobin(Hb), the content of Pb in liver and bone were measured by flame atomic absorption spectrometry, relative protein expression of Nrf2, and apoptosis signal pathway were determined by the method of western blot.Primary hepatocytes from Wistar rats were cultured in vitro. After identification, hepatocytes were incubated with Pb and GSPE. The groups are as followed; Contro group, GSPE(100 mg/L) group, Pb(100 μM) group, and Pb(100 μM) + GSPE(100 mg/L) group, incubating GSPE for 2 h later, adding Pb to incubate for 24 h. Cell viability, LDH released, and ROS generation were measured according to the instruction of kits.The results showed as follows: GSPE could ameliorate Pb-induced the declined number of red blood cells and hemoglobin concentration. ALT, AST, and pathological changes of liver induced by GSPE can be exhibited. Compared with Control group, the level of superoxide dismutase and glutathione were significantly decreased, and the content of malondialdehyde was significantly increased, all these indicators suggested that the organism was under a state of oxidative stress, while, GSPE protected against Pb toxicity through reducing the effects of oxidative stress. The Pb accumulation in liver and bone of rats exposed to Pb was significant elevated, and perfusion of GSPE can lower the content of Pb in bone; meanwhile, the level of apoptosis and DNA damage were decreased by GSPE. Compared with Pb group, related protein expression of Nrf2 signaling pathway, such as Nrf2, Keap 1, NQO1, and HO-1, were significantly elevated in Pb+GSPE group. GSPE suppressed the increased apoptosis-related protein expression of Bcl-2 and Bcl-xl, and decreased the expression of Bax and P53 induced by Pb. GSPE also protected hepatocytes in vitro against Pb toxicity through increasing cell viability and lower cytotoxicity. According to the result of reactive oxygen species(ROS)assay, GSPE decreased oxidative stress induced Pb by inhibiting ROS generation.These findings suggested that GSPE attenuated oxidative stress and apoptosis induced by Pb in the liver by activating Nrf2 signaling pathway. This study will provide theoretical and practical basis for the application of GSPE to control the Pb poisoning of animal.