Effects Of Dehp On Hepatic Lipid Metabolism Of Rat And The Underlying Mechanism

Di(2-ethylhexyl)phthalate(DEHP)is an environmental endocrine disruptor which is widely used as a plasticizer in medical equipment,children's toys and food packaging.After DEHP is ingested,it can be metabolized into mono-(2-ethylhexyl)phthalate(MEHP)hydrolyzed by digestive enzymes in the liver and intestines,causing toxic effects on the body.In recent years,it has been found that DEHP/MEHP can affect the lipid metabolism and cause obesity.Liver is the most important organ to regulate lipid metabolism in the body.Exposure to DEHP can cause disturbance of lipid metabolism and promote lipid synthesis and accumulation in the liver,but the mechanism is still unclear.This study analyzed the effects of DEHP exposure on rat liver lipid metabolism by administering DEHP to rats and MEHP to BRL-3A hepatocytes,and explored the effects of lipid metabolism-related signaling pathways and the key lipid metabolism genes in the process.Part I Effects of DEHP on liver lipid metabolism in rats and the role of inflammation in the processObjective: To clarify the effects of DEHP on the lipid metabolism in rat liver and MEHP on BRL-3A hepatocytes,and to explore the role of key lipid metabolism factors and inflammation in the effect of DEHP on the lipid metabolism of rat liver.Methods: 1.In vivo experiments A total of 80 SPF Wistar rats with 21-day age(40 males and 40 males)were selected.The pure DEHP was diluted to different concentrations with corn oil.All rats were randomly divided into 4 groups with 20 rats in each group: 0mg/kg/d,5 mg/kg/d,50mg/kg/d,and 500 mg/kg/d.The rats were continuous exposure to DEHP for 8 weeks.The state of the rats was observed daily and the changes in their body weight were recorded.After the last exposure,blood was collected from the heart of the rat,serum was separated and liver tissue was collected.The contents of triglyceride(TG),total cholesterol(TC),low density lipoprotein(LDL)and high density lipoprotein(HDL)in serum of rats were determined by automatic serum biochemical analyzer.The contents of TG and TC in rat liver tissue were determined by ELISA.Hematoxylin-eosin(HE)staining was used to observe the histopathological changes of rat liver.Real-time RCR was used to detect the m RNA expression levels of PPAR?,Fasn,Acox1,a P2 and Pdk4 in rat liver.The protein expression levels of PPAR?,Fasn,Acox1,a P2 and Pdk4 in rat liver were detected by immunohistochemistry and western blot.IBM SPSS 24.0 statistical software was used for data processing and analysis.Oneway analysis of variance was used to compare the m RNA and protein levels of key genes of lipid metabolism after DEHP exposure.Pair comparison between groups was performed by LSD test,and the test level was set at ?=0.05.2.In vitro experiment After giving BRL-3A cells with different doses of MEHP for 24 hours,the CCK-8 method was used to detect the cell survival rate;and the exposure dose was determined according to the results of the CCK-8 experiment.The experiment was divided into: solvent control group(VCon),control group(Con),10 ?M MEHP group,50 ?M MEHP group,100 ?M MEHP group and 200 ?M MEHP group.After exposure for 24 hours,the contents of TG and TC in BRL-3A cells,as well as the levels of inflammatory cytokines IL-8 and IL-1? in cell culture medium were determined by ELISA.Real-time RCR was used to detect the m RNA expression levels of PPAR?,Fasn,Acox1,a P2 and Pdk4 in BRL-3A cells.The protein expression levels of PPAR?,Fasn,Acox1,a P2,Pdk4 and PPAR? in BRL-3A cells were detected by western blot.After 100?g/m L Aspirin treatment,the groups was divided into VCon,Con,Aspirin,100?M MEHP and Aspirin+MEHP.After anti-inflammatory treatment,the levels of TG,TC,IL-1? and IL-8 were determined by ELISA,PPAR? protein levels were detected by western blot.One-way analysis of variance was used to compare the m RNA and protein levels of key genes of lipid metabolism and the levels of inflammatory factors among multiple groups after MEHP treatment.Pair comparison between groups was performed by LSD test,and the test level was set at ?=0.05.Results: 1.In vivo experiments(1)The weight of the rats in the 500 mg/kg/d DEHP exposure group was significant higher than the control group from the third week to the end of exposure(P<0.05).(2)The level of TC in the serum of the 500 mg/kg/d DEHP exposure group was significantly higher than that of the control group and the 5 mg/kg/d group(P<0.05),and the level of HDL in the serum of the 500 mg/kg/d DEHP group was significant higher than the other groups(P<0.05).(3)After DEHP exposure,the liver tissue of rats showed abnormal changes such as hepatocellular edema,loose cytoplasm and hepatocyte cord disorder.In the liver tissue of the 500 mg/kg/d DEHP exposure group,the boundaries of hepatocytes in the liver tissue were completely blurred,and the hepatocytes appeared a small amount of steatosis,the damage of liver was obvious.(4)Compared with the control group,the Fasn,Acox1 and PPAR? m RNA levels in the liver of the DEHP exposure groups increased significantly,and showed a gradual increase with the increase of the DEHP exposure dose(P<0.05);The a P2 m RNA expression level of the 5 mg/kg/d DEHP group was higher than that of the control group(P<0.05),and the 500 mg/kg/d DEHP group was significantly higher than the other three groups(P<0.05).(5)The protein expression level of Fasn in 50 mg/kg/d DEHP group was significantly higher than that of other groups(P<0.05),The Pdk4 protein levels in the livers of all DEHP exposure groups were higher than that in the control group(P<0.05);Compared with the control group,the PPAR? protein levels in all DEHP groups had no significant changes(P>0.05).The Acox1 protein level of the 500 mg/kg/d DEHP group was significantly higher than that of the control group and the 50 mg/kg/d group(P<0.05).The a P2 protein level of the 5 mg/kg/d and 500 mg/kg/d DEHP group was significantly higher than that of the control group(P<0.05).2.In vitro experiment(1)After MEHP exposure,the TG level of 200 ?M MEHP group was significantly higher than that of control group and 10 ?M group(P<0.05),and the TG level of 100?M group was higher than that of 10 ?M group(P<0.05).The TC level of BRL-3A cells in 200 ?M MEHP group was also significantly increased compared with VCon group and Con group(P<0.05).(2)After MEHP exposure,the m RNA expression levels of Fasn,Pdk4 and a P2 in BRL-3A cells gradually increased with the increase of exposure dose(P<0.05).The m RNA expression levels of PPAR? in the 100 ?M MEHP group was higher than that of the control,10 ?M and 50 ?M MEHP groups.The PPAR? m RNA level in the 200 ?M MEHP group was the highest(P<0.05).The expression level of Acox1 m RNA in the 200 ?M MEHP group was the highest among all groups(P<0.05).(3)The protein expression levels of Fasn and Pdk4 in 100 ?M MEHP group were significantly higher than those in control,10 ?M and 50 ?M groups(P<0.05).The protein levels of Acox1 in BRL-3A cells in the 100 ?M MEHP group were significantly higher than those in the control group,10?M and 50 ?M MEHP groups(P<0.05).Fasn,PPAR?,Acox1 and a P2 protein expression levels in BRL-3A cells in the 200 ?M MEHP group were higher than other groups(P<0.05).(4)IL-8 levels in BRL-3A cell culture medium in 100 ?M and 200 ?M groups were significantly increased compared with control group,10 ?M and 50 ?M groups(P<0.05).The level of IL-1? in 50 ?M MEHP group was significantly higher than that in the control group,the level of IL-1? in 100 ?M group was higher than that in the control group,10 ?M group and 50 ?M group,and the level of IL-1? in 200 ?M group was higher than that in other groups(P<0.05).(5)After MEHP exposure,the protein expression level of PPAR? in BRL-3A cells decreased gradually with the increase of exposure dose,and the levels of PPAR? protein in 50 ?M and 100 ?M groups were lower than control group(P<0.05).(6)After 100 ?g/m L Aspirin treatment,the levels of IL-8 and IL-1? in cell culture medium of Aspirin group did not change significantly(P>0.05).The levels of IL-8 and IL-1? in MEHP groups were significantly higher than those in control and Aspirin groups(P<0.05).The levels of IL-8 and IL-1? in MEHP+Aspirin group were significantly lower than that in MEHP group(P<0.05).(7)There was no significant change in the expression of PPAR? protein in Aspirin group compared with the control group(P>0.05).The expression of PPAR? protein in MEHP group was significantly decreased(P<0.05).The level of PPAR? protein in MEHP+Aspirin group was lower than that in control group and Aspirin group(P<0.05).(8)Compared with the control group,there was no significant change in the levels of TG and TC in Aspirin group(P>0.05).The levels of TG and TC in MEHP group were significantly higher than those in control group and Aspirin group(P<0.05).The TG level in MEHP+Aspirin group was higher than that in control group,but significantly lower than that in MEHP group(P<0.05).The level of TC in MEHP+Aspirin group was significantly lower than that in MEHP group(P<0.05).Conclusions: 1.High dose of DEHP can promote obesity in rats,and cause liver tissue morphology disorder and hepatic steatosis.2.DEHP exposure can promote lipid synthesis and accumulation in rat liver and BRL-3A cells.3.MEHP can increase the levels of IL-8 and IL-1? in BRL-3A cells,and decrease the expression of PPAR? protein.4.Inflammation promotes lipid synthesis and accumulation in BRL-3A cells by inhibiting the expression of PPAR?.5.DEHP exposure can up-regulate the expressions of PPAR?,Fasn,Pdk4,Acox1 and a P2 and affect lipid metabolism of rat hepatocytes.Part II The role and mechanism of JAK-STAT signaling pathway in the effects of DEHP on lipid metabolism in rat liverObjective: To investigate the effect of DEHP on the expression of JAK-STAT signaling pathway in rat liver and MEHP on BRL-3A hepatocytes,and the role of JAK-STAT pathway in the effect of MEHP on lipid metabolism of rat hepatocytes and its mechanism.Methods: 1.In vivo experiment The experimental grouping and DEHP exposure are the same as the first part.Realtime PCR was used to detect the m RNA expression levels of JAK2,STAT5 A,STAT5B,TYK2 and STAT1 in rat liver.The protein expression levels of JAK2,STAT5 A,STAT5B,TYK2 and STAT1 in rat liver were detected by western blot.Correlation analysis was used to analyze the correlation of STAT5,STAT1 protein levels and the protein levels of the key gene related to lipid metabolism.The test level was set at ?=0.05.2.In vitro experiment The expression levels of JAK2,STAT5 A and STAT5 B m RNA in normal BRL-3A cells were detected by Real-time PCR.The protein expression levels of JAK2,STAT5 A,STAT5B,P-STAT5 A and P-STAT5 B protein in normal BRL-3A cells were detected by western blot.BRL-3A hepatocytes were stably transfected with STAT5 A overexpressing lentivirus(Lv/sh-STAT5A)and blank vector virus(Lv/sh-NC)respectively,to construct STAT5 A overexpressing cell lines.Real-time PCR and western blot were used to detect the overexpression efficiency of STAT5 A in cells.The experimental groups after lentivirus transfection were Parental control group,blank vector infection group(Lv/sh-NC),overexpressed STAT5 A infection group(Lv/sh-STAT5A),MEHP exposed blank vector infection group(Lv/sh-NC+MEHP)and MEHP exposed overexpressed STAT5 A infection group(Lv/sh-STAT5A+MEHP).The levels of intracellular TG and TC after lentivirus infection were detected by ELISA.The protein expression levels of Fasn,Acox1 and a P2 in cells after lentivirus infection were detected by western blot.Results: 1.In vivo experiment(1)Compared with the control group,the expression level of JAK2 m RNA in the liver of rats was reduced after DEHP exposure,and the level of JAK2 m RNA in the 500mg/kg/d DEHP group was the lowest(P<0.05).DEHP exposure at doses of 50mg/kg/d and 500 mg/kg/d can significantly increase the expression level of STAT5 B m RNA in rat liver(P>0.05).(2)Compared with the control,5 mg/kg/d and 50 mg/kg/d DEHP groups,500mg/kg/d DEHP exposure can significantly reduce the expression of JAK2 protein in the liver of rats(P<0.05).The expression level of P-STAT5 A protein in 500mg/kg/d DEHP group was higher than that of other groups(P<0.05).The protein levels of STAT5 B in 5 mg/kg/d and 500 mg/kg/d DEHP groups were higher than that of the control group(P>0.05).(3)The m RNA level of TYK2 in the 500 mg/kg/d DEHP group was significantly higher than that of the other groups(P>0.05).Compared with the control group,all DEHP exposure groups can significantly increase the STAT1 m RNA levels(P<0.05).(4)The protein levels of TYK2 in 5 mg/kg/d and 500 mg/kg/d DEHP groups were significantly higher than that of the control group(P<0.05).Compared with the control group,the protein expression level of STAT1 in each DEHP groups did not change(P>0.05).The protein levels of P-STAT1 in the 5mg/kg/d and 50 mg/kg/d DEHP groups were higher than that of the control group and the 500 mg/kg/d group(P<0.05).(5)After DEHP exposure,the protein level of STAT5 A in rat liver was significantly positively correlated with the protein level of ap2,the protein expression of P-STAT5 A was also significantly positively correlated with the protein expression levels of Acox1 and Fasn(P<0.05).2.In vitro experiment(1)The m RNA level of JAK2 in 100 ?M and 200 ?M MEHP groups were lower than the other three groups(P<0.05).The m RNA expression of STAT5 A in 100 ?M MEHP group was significantly lower than those in the control and 10 ?M MEHP groups(P<0.05),and the STAT5 A level in 200 ?M MEHP group was lower than other doses Group(P<0.05).The levels of STAT5 B in MEHP-exposed groups were lower than control group(P<0.05).(2)The protein expression levels of JAK2,P-JAK2,STAT5 A,P-STAT5 A,STAT5B and P-STAT5 B in MEHP-exposed groups were significantly lower than those in the control group(P<0.05).The protein levels of STAT5 A,P-STAT5 A,STAT5B and PSTAT5 B in 100 ?M MEHP group were significantly lower than the 10 ?M MEHP group.(3)Lentivirus(Lv/sh-STAT5A)and blank vector virus(Lv/sh-NC)overexpressing STAT5A successfully infected BRL-3A rat liver cells with high infection efficiency.Real-time PCR and western blot results showed that compared with the Parental group,the m RNA and protein expression levels of STAT5 A in BRL-3A cells of Lv/sh-NC group were not significantly changed(P>0.05),but in Lv/sh-STAT5 A group were significantly increased(P<0.05).(4)The levels of TG and TC in Lv/sh-NC group did not change compared with the Parental group(P>0.05).Compared with the Parental group and Lv/sh-NC group,the levels of TG and TC in Lv/sh-STAT5 A group were significantly decreased(P<0.05).The levels of TG and TC in Lv/sh-NC+MEHP group were higher than those in the Parental,Lv/sh-NC and Lv/sh-STAT5 A groups(P<0.05).The levels of TG and TC in Lv/sh-STAT5A+MEHP group were lower than Lv/sh-NC+MEHP group(P<0.05).(5)Compared with the Parental group,the protein levels of Fasn,Acox1 and a P2 in the Lv/sh-NC group did not change(P>0.05).The protein levels of Fasn and a P2 in Lv/sh-STAT5 A group were significantly lower than those in the Parental and Lv/sh-NC groups(P<0.05).The Fasn,Acox1 and a P2 levels in Lv/sh-NC+MEHP group were significantly higher than the Parental,Lv/sh-NC and Lv/sh-STAT5 A group(P<0.05).The protein levels of Fasn,Acox1 and a P2 in Lv/sh-STAT5A+MEHP group were significantly lower than those in Lv/sh-NC+MEHP group(P<0.05).Conclusions: 1.DEHP can affect the m RNA and protein expression of TYK2-STAT1 signaling pathway gene in rat liver.2.DEHP/MEHP can affect the m RNA and protein expression of JAK2-STAT5 signaling pathway gene in rat liver and BRL-3A cells.3.The protein levels of Fasn,Acox1 and a P2 in the liver of rats after DEHP exposure were significantly correlated with the protein expression level of STAT5 A.4.STAT5 A can inhibite lipid accumulation in BRL-3A cells.5.STAT5 A can affect the lipid metabolism of rat hepatocytes caused by MEHP by regulating the expression of the key enzymes Fasn,Acox1 and a P2.Part III The role and mechanism of Notch signaling pathway in the effect of DEHP on lipid metabolism in rat liverObjective: To investigate the effect of DEHP on the expression of Notch signaling pathway in rat liver and MEHP on BRL-3A hepatocytes,and to explore the role and mechanism of Notch signaling pathway in the effect of MEHP on lipid metabolism of rat hepatocytes.Methods: 1.In vivo experiment The experimental grouping and DEHP exposure are the same as the first part.RealTime PCR was used to detect the m RNA expression levels of Notch signaling pathway receptors Notch1,Notch2,Notch3,and Notch4 in rat liver,and Notch signaling pathway ligands Jag1,Jag2,Dll1 and Dll4.Western blot was used to detect Notch1,Notch2,Notch3,Notch4,Jag1,Jag2,Dll1 and Dll4 protein expression levels in rat liver.The correlations of Notch receptors protein levels and lipid levels were analyzed by Pearson test.The test level was set as ?=0.05.2.In vitro experiment BRL-3A hepatocytes culture,experimental grouping and MEHP exposure were the same as the first part.Real-Time PCR was used to detect the m RNA expression levels of Notch signaling pathway receptors Notch1,Notch2,Notch3,and Notch4 in normal BRL-3A cells,and Notch signaling pathway ligands Jag1,Jag2,Dll1 and Dll4;western blot was used to detect Notch1,Notch2,Notch3,Notch4,Jag1,Jag2,Dll1 and Dll4 protein expression levels in BRL-3A cells;immunofluorescence assay was also used to detect the protein expressions of Notch1,Notch2,Notch3 and Notch4 in BRL-3A cells.The expression of Notch signaling pathway in BRL-3A cells was inhibited by 50?M DAPT,and the protein expression level of Hes1,the target gene of Notch signaling pathway,was detected by western blot to determine the inhibition efficiency.The experimental groups after Notch pathway inhibition were divided into solvent control group(VCon),control group(Con),inhibitor group(DAPT),100 ?M MEHP exposure group(MEHP)and MEHP exposure inhibitor group(MEHP+DAPT).The Notch1,Notch2,Notch3,Notch4 and PPAR? protein expression levels were detected by western blot,IL-8,IL-1?,TG and TC levels were detected by ELISA.One-way analysis of variance was used to compare the differences of Notch signaling pathway gene expression,PPAR? protein expression,inflammatory cytokines and lipid levels among all groups after MEHP treatment,and LSD test was used for pairwise comparison.The test level was set as ?=0.05.Results: 1.In vivo experiment(1)After DEHP exposure,the m RNA levels of Jag1,Dll1 and Dll4 in rat liver increased gradually with the increase of exposure dose.The m RNA level of Jag2 in the 500 mg/kg/d group was higher than that in other groups.Compared with the control group,the m RNA levels of Notch1 in all DEHP exposure groups were significantly increased,and the level of Notch1 in 500mg/kg/d group was the highest(P<0.05).The m RNA levels of Notch2 in 50 mg/kg/d and 500 mg/kg/d DEHP groups were significantly higher than that in the control group and 5 mg/kg/d DEHP group(P<0.05).The m RNA levels of Notch3 were increased with the exposure dose of DEHP(P<0.05).Compared with the control group and the 5 mg/kg/d DEHP group,the Notch4 m RNA levels in 50 mg/kg/d and 500 mg/kg/d DEHP groups were obviously increased(P<0.05).(2)The protein levels of Jag1 and Dll1 in 500 mg/kg/d DEHP exposed group were significantly higher than those of the other three groups(P<0.05).The levels of Jag2 in all DEHP treatment groups were higher than those in the control group(P<0.05).After DEHP exposure,the protein level of Dll4 increased gradually with the increase of dose(P<0.05).Compared with the control group,the protein levels of Notch1 in all DEHP groups were significantly increased,among which the protein level of Notch1 in the 500 mg/kg/d DEHP group was the highest(P<0.05).Compared with the control group,the expression of Notch2,Notch3 and Notch4 in all DEHP-exposed groups were increased,and the protein levels of Notch2 and Notch3 in the 50 mg/kg/d and 500mg/kg/d DEHP groups were higher than that in 5 mg/kg/d DEHP group(P<0.05).(3)After DEHP exposure,the protein levels of Notch1,Notch2,Notch3 and Notch4 were significantly positively correlated with the TG level in rat liver(P<0.05).2.In vitro experiment(1)MEHP exposure can significantly increase the expression of Jag1,Jag2,Dll1,and Dll4 m RNA in BRL-3A cells(P<0.05).Compared with the control group,the m RNA expression of Notch1,Notch2,Notch3 and Notch4 in all MEHP groups was significantly increased(P<0.05).(2)The protein levels of Jag1 and Jag2 in 100 ?M MEHP exposure group were significantly higher than that in other dose groups(P<0.05).Compared with the control group,the Dll1 protein levels in 10 ?M and 100 ?M MEHP groups were significantly increased(P<0.05),and the Dll1 protein level in the 100 ?M MEHP group was the highest(P<0.05).Starting from the 50 ?M MEHP group,the Dll4 protein levels increased with the MEHP dose(P<0.05).The protein level of Notch1 in 100 ?M MEHP group was the highest(P<0.05).The levels of Notch2 in 100 ?M and 200 ?M MEHP groups were significantly higher than other groups(P<0.05).The Notch3 protein in 200?M MEHP group was significantly higher than other groups(P<0.05).The Notch4 level in 100 ?M MEHP group was significantly higher than other groups(P<0.05).(3)All four Notch receptors were expressed in BRL-3A hepatocytes,and Notch1 protein level was the highest,Notch1 and Notch2 protein expression levels were significantly higher than Notch3 and Notch4(P<0.05).After MEHP exposure,the levels of all Notch receptor proteins were higher than those in control group,Notch1 protein level was the highest,the protein levels of Notch1 and Notch2 protein levels were significantly higher than Notch3 and Notch4(P<0.05).(4)After using 50 ?M DAPT to inhibit the Notch signaling pathway in BRL-3A cells,the protein levels of Notch1,Notch2,Notch3,and Notch4 in the DAPT group were lower than those in the control group(P<0.05).The protein expression levels of Notch1,Notch2,Notch3,and Notch4 in the MEHP group were significantly higher than that of the control group and DAPT group(P<0.05).The protein levels of Notch1 and Notch2 in the MEHP+DAPT group were significantly lower than those of the MEHP group(P<0.05).Immunofluorescence results showed that the protein levels of Notch1,Notch2 and Notch3 in the DAPT group were lower than those in the control group(P<0.05),and the protein levels of Notch4 did not change significantly(P>0.05). (5)After the inhibition of Notch pathway,the levels of IL-8 and IL-1? in BRL-3A cell culture medium of DAPT group did not change compared with the control group(P>0.05).The levels of IL-8 and IL-1? in MEHP group were higher than the control group and DAPT group(P<0.05).The levels of IL-8 and IL-1? in MEHP+DAPT group were significantly lower than those in MEHP group(P<0.05).(6)After the inhibition of Notch pathway,the protein level of PPAR? in DAPT group was increased compared with the control group,but the difference was not statistically significant(P>0.05).The level of PPAR? protein after MEHP exposure was significantly lower than that in control group and DAPT group(P<0.05).The level of PPAR? protein in MEHP+DAPT group was significantly higher than that in MEHP group(P<0.05).(7)After Notch pathway was inhibited,the levels of TG and TC in DAPT group were significantly decreased compared with the control group(P<0.05).The levels of TG and TC in MEHP group were significantly higher than those in control group and DAPT group(P<0.05).The levels of TG and TC in MEHP+DAPT group were significantly lower than those in MEHP group(P<0.05).Conclusions: 1.Exposure to DEHP can affect the m RNA and protein expression of Notch signaling pathway gene in rat liver.2.Notch receptors protein levels were significantly correlated with TG level in the liver of rats after DEHP exposure.3.Activation of Notch signaling pathway can promote lipid accumulation in BRL-3A hepatocytes.4.Notch signaling pathway can affect lipid metabolism in rat hepatocytes induced by MEHP by causing inflammation.