Protective Effects Of Hesperidin On BDE-209 Induced Liver Injury In Mice

The impact of environmental pollution on the ecological environment and human health has attracted people’s attention.Poly Brominated Diphenyl Ethers is a kind of Persistent Organic Pollutants,which are mainly exposed to the human body through diet and cause a variety of toxicities.BDE-209 is the most widely used PBDEs,with the highest detection rate and the toxicology.Due to its persistence,lipophilicity and other characteristics,BDE-209 is mainly stored in animal adipose tissue and liver.Hesperidin（Hsp）is the main flavonoid active component in citrus fruits.Preliminary studies have confirmed that Hsp has a variety of biological activities,including anti-inflammatory,anti-oxidant,anti-obesity,anti-diabetic,immune regulation,etc.At present,there are no studies comparing the effects of BDE-209 on liver damage in obese individuals,and no reports on Hsp intervention.Therefore,in this study,mice were used as experimental subjects and exposed to BDE-209 under high-fat and normal diets to compare the liver toxicity of BDE-209.At the same time,by intervening with the simultaneous supplementation of Hsp and BDE-209,the protective effect and potential mechanism of Hsp on BDE-209-induced liver injury in mice was evaluated,mainly from the three aspects of oxidative damage,endoplasmic reticulum stress and mitochondrial apoptosis.In addition,based on the fact that BDE-209 is mainly found in animals and aquatic foods and coexists with the food additive sodium nitrite,this study also established in vitro experiments to explore the toxicity relationship between BDE-209 and sodium nitrite,and evaluate the protective effect of Hsp in vitro.Our research provides theoretical basis for the mechanism of liver damage induced by BDE-209 at different levels and also has important and practical value for the dietary prevention against BDE-209.It also contributes to the development of high value-added products using Hsp as raw materials.The main research results are as follows:（1）The effects of BDE-209（100 mg/kg）on liver damage in mice fed high-fat or normal diet were compared.The results showed that BDE-209 exposure aggravated liver fat accumulation in mice,reflected by the increment of TG and LDL.Compared with the control group,BDE-209 induced more severe liver function decline and tissue damage under a high-fat diet,ALT and AST are at higher levels,and it also increases the appearance of focal infiltration of inflammatory cells in the liver tissue.Necrotic degeneration and nuclear fragmentation,fatty degeneration of liver tissue,promote TC accumulation.Secondly,BDE-209 had a more significant oxidative stress-inducing ability when taken with high-fat diet together.Compared with the normal diet,the high-fat diet fed mice exposed to BDE-209 had a greater increase in ROS and MDA.The inhibitory effects on antioxidant syetem consisting of SOD,GSH and GST were more obvious.Finally,BDE-209 promoted hepatocyte apoptosis under the two dietary patterns,but it plays a obvious role in the status of obesity.（2）Series of experiments in vivo were conducted to observe the pathological state of the mouse liver,determine the biochemical indicators of serum and liver tissue,and explore the protective effect of Hsp（100 mg/kg）on BDE-209（100mg/kg）-induced liver injury in mice.The results showed that Hsp significantly alleviated the BDE-209-induced hepatocyte apoptosis and liver tissue structure destruction.In addition,Hsp could significantly improve the liver damage induced by BDE-209,inferring from the reducing of serum liver AST,ALT,and ALP activities.At the same time,Hsp enhanced the activity of the antioxidant enzyme system in the body,with the activities of SOD,GSH-Px and CAT significantly increasing,and the content of reactive oxygen species（ROS）and peroxide product MDA significantly decreasing.In addition,Hsp could down-regulate the expression of IL-1β,IL-2,and TNF-a in serum and liver tissue,and increase the levels of anti-inflammatory factors（IL-4 and IL-10）,so effectively alleviate the inflammatory response induced by BDE-209.（3）The study of endoplasmic reticulum and mitochondrial function further clarified the protective mechanism of Hsp.The results showed that in BDE-209treated liver,MAPK（ERK1/2,JNK）pathway phosphoric acid level increased.The expression of endoplasmic reticulum stress marker protein（ATF6,CHOP,p-ire1/ire1and p-perk/perk）raised significantly,and mitochondrial membrane potential（MMP）,mitochondrial ATP content and mitochondrial copy number（mtDNA）decreased significantly.Using transmission electron microscope,it was found that the hepatic mitochondrial membrane in mice exposed to BDE-209 was damaged.The endoplasmic reticulum calcium channels（IP3R,Sigmar1）and calcium ion receptors（CaSR,CaM）were greatly activated.In vitro experiment confirmed that BDE-209（6.25μM）treatment on HepG2 cells increased the level of mitochondrial Ca2+.However,Hsp（160μM）intervention can significantly alleviate the above adverse changes.It was concluded that through alleviating the oxidative stress via inhibiting the phosphorylation of the MAPK pathway,Hsp improved the endoplasmic reticulum stress,so that reduced the excessive transfer of Ca²⁺from the endoplasmic reticulum to the mitochondria to inhibit the mitochondrial intrinsic apoptosis induced by BDE-209 in mice.（4）A Chou-Talalay（CI equivalent）mathematical model was established to evaluate the interaction between BDE-209 and Sodium Nitrite（SN）,and then High Content Analysis（HCA）technology was used to reveal its cytotoxicity mechanism and protection of Hsp.The results showed that the toxicity of the mixture of BDE-209and SN showed a synergistic effect.The pretreatment of Hsp could significantly reverse the accumulation of ROS and the decrease of mitochondrial density and membrane potential,the loss of Ca²⁺homeostasis,the decrease of superoxide dismutase（SOD）activity,the significant increase ofγH2AX fluorescence foci and the number of cell micronuclei induced by the mixture of BDE-209 and SN in HepG2 cells,indicating that Hsp had protection effects on the combination of BDE-209 and sodium nitrite toxicity on disturbance of work and DNA has a protective effect.In summary,BDE-209 exerts more severe liver damage in obese mice.Hsp,as a natural source of edible ingredient,could prevent dietary BDE-209 toxicity and its combined toxicity and relieve the damage on liver,which provided a theoretical basis for the further development of Hsp products.