| Plasticizers,also known as plasticizers,are often used as additives and are widely used in industrial processing and various life consumables.For example,plastic products,building materials,cosmetics and some medical consumables in real life contain a certain amount of plasticizer.DEHP's full name "bis(2-ethyl)hexyl phthalate" is one of the main components of plasticizers.Studies have shown that DEHP can migrate into the environment of water,soil and air through various ways,and then be exposed to the human body.A large number of epidemiological investigations and studies have shown that DEHP,as an environmental endocrine disrupting substance,is often exposed to various potential toxic hazards to human health.On the basis of disease,long-term exposure to DEHP may even accelerate the progression of the disease.Alcoholic fatty liver disease is a chronic disease caused by long-term excessive drinking,which leads to liver lipid metabolism disorder,liver cell fat metabolism decreases,accumulation increases,and inflammatory injury of liver cells.The toxicity of DEHP to the liver may be related to cellular DNA damage,oxidative stress,lipid peroxidation and inflammation.So far,few people have studied the effect of DEHP on alcoholic fatty liver.According to reports,cytochrome P4502E1 and silent information regulator-1 are closely related to the occurrence of alcoholic fatty liver.The results of this study showed that DEHP exposure significantly accelerated hepatic steatosis,inflammation and oxidative stress damage;the related proteins SIRT1,CYP2E1 involved in the regulation of lipid metabolism pathways were affected,and hepatic inflammation and oxidative stress-related pathway protein P-p38 and P-p65 were increased.It is worth noting that the trend observed in the LO-2 cell experiment results is basically consistent with the in vivo results.Overall,studies have shown that DEHP may accelerate or aggravate the progression of alcoholic fatty liver disease in rats through CYP2E1,SIRT1 and p38 MAPK / NF-?B signaling pathways.OBJECTIVE:A model of fatty liver in rats was induced by using alcohol combined with high-fat diet.To study the effect of DEHP on the pathological process of alcoholic fatty liver rats in different periods(2,4 and 8 weeks).And the role of SIRT1,CYP2E1 and p38MAPK/NF-?B signaling pathway in chronic alcohol-induced fatty liver was further explored in vitro using human normal liver cells(LO-2).METHODS:In vivo experiment,we used alcohol gavage combined with specific high-fat diet to induce fatty liver model in SD rats.Based on the induction model,low,medium and high concentrations of DEHP(0.05,5,500 mg/kg)were given.And DEHP 500mg/kg was used as the control group,plus the normal group for a total of six groups.Administering DEHP in the morning and alcohol in the afternoon continued for eight weeks,rats were sacrificed strictly according to animal ethics at two,four,and eight weekends.Liver tissue HE staining,oil red O staining,and MASSON staining were used to observe the effect of DEHP on liver pathological changes of fatty liver rats induced by alcohol in different periods.Calculate the corresponding liver index based on rat liver weight and body weight;the activity of glutamate transaminase(ALT)and aspartate aminotransferase(AST)in serum is measured using a professional kit.The content of triglyceride(TG),malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in liver tissue homogenate were analyzed.Western blot was used to detect the expressions of SIRT1,CYP2E1,P-p38 and P-p65 proteins,and the laser light was used to observe the expression distribution of SIRT1 and CYP2E1 in liver tissues.In vitro,six groups were also set up: normal group,ethanol group(100mmol/L),ethanol+DEHP(6.25,25,100?mol/L)group,and DEHP 100?mol/L control group.After 48 hours,the effect of DEHP on the viability of LO-2 cells was first detected using the CCK-8 kit.Lipid accumulation model was established by co-stimulation with absolute ethanol(100mmol/L)and oleic acid(90?mol/L)for 48 hours.The experiment was divided into: normal group,ethanol group,ethanol+DEHP6.25?mol/L group,ethanol+DEHP 25?mol/L,ethanol+DEHP 100?mol/L,and DEHP100?mol/L control group.The flow cytometer uses DCFH-DA fluorescent probe method to detect the change of reactive oxygen species(ROS)content in LO-2 cells under the influence of DEHP.Western blot was used to detect the expression of SIRT1,CYP2E1,P-p38 and P-p65 proteins.RESULTS:1.Effects of DEHP exposure on liver function indexes and redox balance system in liver tissues of ratsAfter exposure to DEHP,the liver index of rats is significantly increased,and serum ALT and AST activity levels increased significantly in the detection of biochemical indicators.In liver tissue homogenate,the level of triglyceride(TG)is significantly increased in the medium and high-dose DEHP group.Analyze the changes of the oxidative and antioxidant balance system in liver tissues.Detection of liver tissues by SOD and MAD kits revealed that DEHP can significantly inhibit the activity of SOD in the liver of fatty liver rats induced by chronic alcohol and increase the content of MDA in the tissues.It is suggested that DEHP intake promotes liver damage and severe oxidative damage in alcoholic fatty liver rats,which may be related to aggravating liver oxidative stress and breaking the balance of liver oxidation and oxidative defense system.2.Histopathological changes in rats with alcoholic fatty liver after DEHP exposureOn the second and fourth weeks,there was no apparent difference in the fresh liver samples of each group.In the eighth week,the liver color of the samples was dark red after DEHP exposure.H&E staining of each group showed that there was no obvious abnormality in the other groups except for the slight inflammatory cell infiltration near the blood vessels in the high-dose group of alcohol combined with DEHP in the second week.In the fourth week of HE results,a small amount of lymphocyte infiltration and slight inflammation were associated with the alcohol group and the low and middle dose of DEHP group,while a few fat vacuoles appeared in the cytoplasm of the high-dose DEHP group,and the inflammation and infiltration were obvious.The results of HE and oil red O staining at the eighth week showed that long-term exposure to DEHP may aggravate liver lipid accumulation,inflammatory damage,and cell necrosis in chronic alcohol-induced fatty liver rats.Analysis of Masson collagen staining revealed that burr-like collagen fibers infiltrate around the blood vessels in the alcohol+DEHP 500mg/kg group,with mild to moderate fibrosis symptoms.It is suggested that DEHP exposure can accelerate the pathological process of alcoholic fatty liver rats and even aggravate the development of disease.3.Western blot analysis of the effects of DEHP on SIRT1,CYP2E1,P-p65,P-p38 protein expression in liver tissuesCompared with the model group,the expression levels of CYP2E1,P-p38,and P-p65 proteins were up-regulated in high-dose treatment,especially at high doses in DEHP.SIRT1 protein expression was significantly inhibited in the DEHP500mg/kg+alcohol group.In addition,compared with the normal group,the levels of CYP2E1,SIRT1,P-p38 and P-p65 in the alcohol group and the DEHP control group were significantly changed.It is suggested that DEHP may accelerate or aggravate alcoholic fat by affecting CYP2E1,SIRT1 and P38MAPK/NF-?B signaling pathway.4.Immunofluorescence detection of SIRT1,CYP2E1 protein expression and distribution in rat liver tissues under the influence of DEHPLaser confocal microscopy was used to capture the expression and distribution of CYP2E1 and SIRT1 proteins in liver tissues.The results showed that DEHP can significantly promote the expression of CYP2E1 protein in liver tissues of alcoholic fatty liver rats and inhibit the expression of SIRT1,of which alcohol+DEHP500mg/kg group is most obvious.5.Effect of DEHP on LO-2 activity and ROS levels in cells stimulated by absolute ethanolIn results,ethanol can significantly affect the viability of LO-2 cells.With the increase of DEHP exposure concentration,cell viability decreased,and the cytotoxicity of DEHP gradually appeared.It is implied that DEHP can inhibit cell proliferation induced by ethanol.The DCFH-DA fluorescence probe method was used to detect the ROS content by flow cytometry.Medium to high concentration of DEHP can increase the content of reactive oxygen species in cells.6.Effects of DEHP on the content of CYP2E1,SIRT1,P-p38 and P-p65 proteins in LO-2 cells stimulated by anhydrous ethanolThe data shown that the overall trend of in vitro experiments is basically consistent with the results of animal experiments.DEHP can significantly promote the expression of CYP2E1,P-p38 and P-p65 proteins and inhibit the SIRT1 protein content.Among them,ethanol combined with DEHP 100?M/L was the most obvious.7.Effect of DEHP on LO-2 cell lipid accumulation in vitroThe results showed that after oil red O staining,red lipids were observed to aggregate around the cell membrane under an inverted microscope.As DEHP exposure increased,lipid accumulation near the membrane also increased.In the DEHP high-dose group,red lipid deposition was more extensive,with the highest lipid content and the most severe.CONCLUSIONS1.Through the experimental research on the 2nd,4th and 8th weeks,we found that long-term ingestion of medium and high concentrations of DEHP can significantly promote or aggravate the occurrence and development of alcoholic fatty liver in rats.2.DEHP exposure significantly increased liver function damage in alcoholic fatty liver rats.3.DEHP exposure can significantly promote the accumulation of lipids in liver cells,increase the production of reactive oxygen species in liver cells,disrupt the balance of liver oxidation and antioxidant systems,and continuously increase liver oxidative damage.4.Combined with the in vivo and in vitro experimental results,it is suggested that the aggravating effect of DEHP on alcoholic fatty liver may be related to the increase of CYP2E1,p38MAPK/NF-?B levels and inhibition of SIRT1 signaling pathway. |
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