Effects Of DEHP On Non-alcoholic Fatty Liver In Rats And The Potential Mechanism Involved In Lipid Metabolism

At present, with the continuous improvement of people’s living standard in our country, nonalcoholic fatty liver disease(NAFLD) is more and more common in the crowd. NAFLD is characterized by hepatic steatosis in the absence of a history of significant alcohol use or other known liver disease.It may develop advanced liver diseases such as nonalcoholic steatohepatitis, fibrosis, and even cirrhosis without prevention and treatment. some Researches have shown that many environmental contaminants may affect the pathological process of NAFL, and plays an important role in it.Plasticizers, as one of common environment estrogens, are widely used in all kinds of plastic products. Phthalate esters (PAEs) are the most commonly used, including dioctyl phthalate (DEHP), diisononyl phthalate (DINP), dioctyl phthalate (DNOP), didecyl phthalate (DIDP), etc. DEHP has the largest application and it has great practical significance to study its toxic effects and related mechanism.In recent years, the studies found that DEHP played a role on the liver injury and hepatic lipid metabolism by aggravate oxidative stress and lipid peroxidation, increase the release of inflammatory cytokines. But there is no researches to study the effects and mechanism of DEHP on NAFL.Aims The purpose of this study was to evaluate the effects of DEHP on NAFL in rats and investigate the potential molecular mechanism, and establish vitro model to further explore the regulation of DEHP on lipid metabolism.Methods1. We conducted NAFL model in rats fed with modified high-fat diet and treated with DEHP(0.05、 5、 500mg/kg). After 8 weeks blood samples were collected into tubes and serums were separated. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase(ALT),Interleukin-6(IL-6) and tumor-necrosis factor-α(TNF-α) were measured according to commercial analysis kits. The activity of hepatic SOD and the levels of hepatic MDA and TG were measured by liver homogenate. Paraffin sections of liver were prepared for histological examination. Western blot detected the expression of PPARa and SREBP-1c in the liver. 2. We established vitro model of hepatic lipid accumulation in HepG2 cells in the presence of oleic acid. The vitro model was evaluated by oil red O (ORO) staining. Effects of DEHP on HepG2 cell viability were measured according to CCK-8 assay kits(48h). Western blot detected the expression of PPARa and SREBP-lc.Results1. The harmful effects of DEHP on NAFL in SD ratsPathology examination showed that DEHP can cause significant liver damage, aggravate the degree of liver steatosis and induce mildly to liver fibrosis. The body weight in model group was significantly lower than control group, DEHP had no obvious effects on the weight of NAFL rats, but low, medium and high doses DEHP(0.05、 5、500mg/kg) could increase liver index in different degree. Biological indicator test showed that DEHP could significantly increase the levels of serum ALT and AST. The above results suggested DEHP has obvious aggravating effects on NAFL rats.2. The effects of DEHP on the levels of serum IL-6 and TNF-a in NAFL ratsAfter administrated with the high-fat diet, the levels of serum IL-6 and TNF-a in NAFL rats significantly increased compared with normal group, and high dose DEHP (500mg/kg) could obviously increase the levels of serum IL-6 and TNF-a in NAFL and normal rats. The above results suggested DEHP can aggravate NAFL through increasing the levels of inflammatory factors.3. The effects of DEHP on the lipid metabolism in NAFL ratsCompare with control group, high-fat diet resulted in significant increase in the liver level of TG. Middle and high doses DEHP(5、 500mg/kg) could significantly increase hepatic TG level,but high dose DEHP (500mg/kg) had no effects on hepatic TG level in normal rats. The above results suggested DEHP may aggravate rats NAFL through increasing lipid accumulation.4. The effects of DEHP on the oxidative metabolism in NAFL ratsModel group showed a significant decrease in the activities of SOD and a remarkable increase in the level of MDA compared with control group. After the treatment of DEHP, three doses DEHP(0.05、5、500mg/kg) could obviously increase in the level of MDA and high dose DEHP (500mg/kg) could significantly decrease in the activities of SOD.High dose DEHP (500mg/kg) could also significantly increase in the level of MDA and decrease in the activities of SOD in normal rats.The above results suggested DEHP may aggravate rats NAFL through increasing oxidative stress and lipid peroxidation.5. The effects of DEHP on the expression of PPARa and SREBP-lc in NAFL ratsCompared with control group, the levels of PPARa and SREBP-lc increased significantly in liver of NAFL rats, and increased after the treatment of DEHP. But high dose DEHP (500mg/kg) had no effects on the expression of SREBP-1c in the normal rats. The above results suggested the effects of DEHP on regulating hepatic lipid metabolism in NAFL rats may be associated with activated PPARa and SREBP-lc activity.6. The effects of DEHP on HepG2 cell viabilityCell viability experiment results showed that high concentration DEHP(100μM,200μM) can inhibit the ability of HepG2 cell viability significantly.7. The effects of DEHP on the lipid metabolism in HepG2 cells induced by oleic acidTo further explore how DEHP regulates lipid metabolism, we exposed oleic acid in HepG2 cells to establish vitro model of lipid accumulation. ORO staining results showed that oleic acid group have more lipid droplets, and the treatment of high concentration DEHP(100μM) can increase lipid droplets mildly compared with model group. Western blot measurement results showed that the levels of PPARa and SREBP-lc increased significantly in model group compared with the control. Compared with model group, high concentration DEHP(50,100μM) could significantly increase the protein expression of PPARa, but the expression of SREBP-lc only increased in the high concentration. The above results suggested the effects of DEHP on regulating lipid metabolism in HepG2 cells may be associated with activated PPARa and SREBP-lc activity.Conclusion1. DEHP can cause liver injury in pathology of rats,increase hepatic lipid accumulation. DEHP can aggravate NAFL rats by increasing the serum levels of ALT, AST, IL-6,TNF-a and hepatic levels of TG and MDA, decreasing the activity of hepatic SOD.2. DEHP can significantly increase the hepatic levels of PPARa and SREBP-lc in NAFL rats, but high dose DEHP(500mg/kg) can’t inhance the expressin of SREBP-lc in normal rats.The results suggested the effects of DEHP on regulating hepatic lipid metabolism in NAFL rats may be associated with activated PPARa and SREBP-lc activity.3. The treatment of high concentration DEHP(100μM) can increase lipid droplets in HepG2 cells induced by oleic acid. The results suggested the effects of DEHP on regulating lipid metabolism in HepG2 cells may be associated with activated PPARa and SREBP-lc activity.