| Di-(2-ethylhexyl)phthalate(DEHP)is the most commonly plasticizer used in phthalates.It is widely used in food packaging materials,medical devices,cosmetics and children’s toys.It can be absorbed by human body through water,food and skin directly or indirectly.After DEHP ingestion,it can rapidly metabolize into mono-(2-ethylhexyl)phthalate(MEHP),a hydrolysate of DEHP,in liver and small intestine cells.The toxic effect of DEHP on human is mainly affected by MEHP.As an environmental endocrine disruptor,DEHP exposure can produce toxic effects on reproductive system,nervous system,endocrine system and other tissues and systems.In recent years,the National Health and Nutrition Examination Survey(NHANES)reported that high levels of phthalate metabolites in human urine were significantly associated with increased waist circumference,suggesting that phthalate exposure may be a risk factor for abdominal obesity.Epidemiological and toxicological studies have also shown that DEHP exposure can affect lipid metabolism and induce obesity.MEHP can promote the growth of 3T3-L1 preadipocytes,promote 3T3-L1 cells to differentiate into mature adipocytes and increase intracellular lipid deposition,but its mechanism is still unclear.In this study,3T3-L1 mice preadipocytes were cultured in vitro and exposed to MEHP,a metabolite of DEHP.The effects of MEHP exposure on differentiation and lipid metabolism of 3T3-L1 mice preadipocytes were analyzed.The effects of MEHP exposure on differentiation and lipid metabolism of 3T3-L1 mice preadipocytes and the role and mechanism of lipid metabolism related signaling pathways and key genes were explored,which could be used to provide theoretical basis for prevention and treatment the diseases related to lipid metabolism caused by environmental endocrine disruptors in the population.The diseasesPart Ⅰ Effects of MEHP on Differentiation and Lipid Metabolism in 3T3-L1 CellsObjective: To clarify the effects of MEHP on differentiation and lipid metabolism of 3T3-L1 cells,and explore the role of key genes of lipid metabolism in MEHP on differentiation of 3T3-L1 cells.Methods: 3T3-L1 preadipocytes were cultured in DMEM medium containing 10% calf serum.Firstly,3T3-L1 cells were exposed to the gradient MEHP.MTT assay was used to detect the survival rate of 3T3-L1 preadipocytes to determine the exposure doses and the groups.Exposure doses and the groups: control group(blank control,0 μM MEHP;solvent control group,0.25‰ DMSO)0μM MEHP;low dose group,10 μM MEHP;middle dose group,50 μM MEHP;high dose group,250 μM MEHP.Flow cytometry was used to detect the changes of cell cycle,intracellular reactive oxygen species and mitochondrial membrane potential.Then the differentiation of 3T3-L1 preadipocytes was induced by classical hormone cocktail method,and different doses of MEHP were continuously exposed during the differentiation process.Oil red O staining was used to observe the differentiation of 3T3-L1 preadipocytes;chemiluminescence was used to detect the levels of triglyceride and cholesterol;western blot was used to detect the expression levels of lipid metabolism regulators Acox-1,C/EBPβ,FABP4,FAS,LPL,PDK4 and PPARγ;Real-time PCR was used to detect the expression levels of lipid metabolism regulators Acox-1,C/EBPβ,FABP4,FAS,LPL,PDK4 and PPARγ m RNA.IBM SPSS 24.0 was used for statistical analysis.Results: 1.Compared with the control group,the survival rate of 3T3-L1 cells increased significantly after exposure to 63,125,250,500 μM MEHP for 24 hours(P<0.05).2.After MEHP exposure,the cycle of 3T3-L1 preadipocyte did not change significantly(P>0.05);the content of reactive oxygen species(ROS)in 3T3-L1 cells increased significantly(P<0.05)and the mitochondrial membrane potential decreased significantly(P<0.05)in middle and high dose groups.3.During the induction of differentiation,the volume of 3T3-L1 preadipocytes increased and their morphology became rounded,reflective lipid droplets appeared in the cytoplasm,and the lipid content increased with the increase of MEHP exposure dose(P<0.05)the 3T3-L1 preadipocytes became bigger and rounder and appeared reflective lipid droplets in the cytoplasm and the lipid content increased with the increase of MEHP exposure dose(P<0.05);after differentiation,the content of TG in the middle and high dose groups was significantly higher than that in the control group(P<0.05).4.After differentiation of 3T3-L1 preadipocytes,the expression of Acox-1 in MEHP exposed groups was significantly higher than that in the control group(P< 0.05),and the expression of Acox-1 in middle and high dose groups was significantly higher than that in low dose group(P<0.05);the relative expression of FABP4 and PDK4 in middle dose group was significantly higher than that in the control group,and the relative expression of FABP4 in high dose group was significantly higher than that in the other groups.The relative expression of FAS and LPL in high dose group was significantly higher than that in the other groups(P<0.05);the relative expression of PPARγ in 3T3-L1 cells increased with the increase of MEHP exposure dose(P<0.05).5.After differentiation of 3T3-L1 preadipocytes,the expression of Acox-1 protein in low,middle and high dose groups was significantly higher than that in control group(P<0.05),while the expression of Acox-1 protein in middle and high dose groups was significantly higher than that in low dose groups(P<0.05),and the expression of C/EBPβ protein in middle and high dose MEHP exposure groups was significantly higher than that in control group(P<0.05).The expression level of FABP4 protein in the middle dose group was significantly higher than that in the control group,and the expression level of FABP4 protein in the high dose group was significantly higher than that in the other groups(P<0.05);the expression level of FAS protein in the middle dose MEHP exposure group was significantly higher than that in the other groups(P<0.05);the expression level of LPL and PDK4 protein in the middle dose MEHP exposure group was significantly higher than that in the control group,with significant difference(P<0.05).The expression of PPARγ protein in MEHP exposed group was significantly higher than that in low dose group and control group(P<0.05).Conclusions: 1.MEHP exposure can promote the proliferation of 3T3-L1 preadipocytes.2.MEHP can promote cell differentiation and lipid accumulation of 3T3-L1 preadipocytes.3.MEHP exposure can change the mitochondrial membrane potential and intracellular reactive oxygen species levels of 3T3-L1 preadipocytes,and affect mitochondrial function.4.MEHP exposure can affect the lipid metabolism of 3T3-L1 preadipocytes by up-regulating the expression levels of Acox-1,C/EBPβ,FAS,FABP4,LPL,PDK4 and PPARγ in 3T3-L1 preadipocytes.Part Ⅱ: The effects of MEHP on lipid metabolism-related signaling pathwaysObjective: Clarify the role of JAK-STAT signaling pathway and Notch signaling pathway in the effects of MEHP on differentiation and lipid metabolism of 3T3-L1 preadipocytes.Methods: 3T3-L1 preadipocytes culture,the experimental groups and the detection of the levels of triglyceride and cholesterol were the same as the first part.Real-time-PCR was used to detect the m RNA expression levels of JAK-1,JAK-2,JAK-3,TYK-2,STAT-1,STAT-3,STAT-5a and STAT-5b in JAK-STAT signaling pathway.Western Blot was used to detect the protein expression levels of JAK-1,JAK-2,JAK-3,TYK-2,STAT-1,STAT-3,STAT-5a and STAT-5b in JAK-STAT signaling pathway.The m RNA expression levels of Notch-1,Notch-2,Notch-3,Notch-4 and their ligands Dll-1,Dll-4,Jagged-1 and Jagged-2 in Notch signaling pathway were detected by Real-time-PCR.The protein expression levels of Notch-1,Notch-2,Notch-3,Notch-4 and their ligands Dll-1,Dll-3,Jagged-1 and Jagged-2 in Notch signaling pathway were detected by western Blot method.IBM SPSS 24.0 was used for statistical analysis.Results: 1.After the differentiation,the m RNA expression of JAK-2 and JAK-3 in high dose group was significantly lower than that in the other groups(P<0.05);compared with the control group,the expression of TYK-2 m RNA in exposure groups significantly increased(P<0.05),and the expression of TYK-2 m RNA in middle and high dose groups was significantly higher than that in low dose group(P<0.05);the expression of STAT-1 m RNA in high dose group was significantly higher than that in blank control group(P<0.05);the expression of STAT-3 m RNA in MEHP exposure group was significantly higher than that in the control group(P<0.05);the expression of STAT-3 m RNA in middle and high dose groups was significantly higher than that in low dose group(P<0.05);the expression of STAT-5a m RNA in high dose group was significantly higher than that in the control group and the low dose group(P<0.05).2.After the differentiation,the expression of JAK-1 protein in the high dose group of 3T3-L1 preadipocytes was the highest,which was significantly higher than that in the blank control group(P<0.05);the expression of TYK-2 protein in the MEHP exposure groups was significantly higher than that in the control group(P<0.05);the expression of TYK-2 protein in the middle and high dose groups was significantly higher than that in the low dose group(P<0.05);the expression of STAT-3 and STAT-5a proteins in the MEHP exposure groups was significantly higher than that in the control group(P<0.05);the expression of STAT-3 and STAT-5a proteins in the middle and high dose groups was significantly higher than that in the low dose group(P<0.05);the expression level of STAT-5b protein in the middle and high dose groups significantly increased(P<0.05),and the expression level of STAT-5b protein in the high dose group was the highest(P<0.05).3.Some studies found that the relationship between the expression of genes in JAK-STAT signaling pathway and the level of the triglyceride.After the differentiation,the content of the triglyceride in 3T3-L1 preadipocytes was significantly relative to the expression of TYK-2、STAT-3、STAT-5a m RNA and protein(P<0.05).4.After differentiation,the relative expression of Notch-1 m RNA in 3T3-L1 preadipocytes in middle dose group was significantly higher than that in the control group(P<0.05),while Notch-1 m RNA in high dose group was significantly higher than that in the other groups(P<0.05).The expression levels of Notch-3 and Notch-4 m RNAs in 3T3-L1 preadipocytes of increased with the increase of exposure dose,and the difference was statistically significant compared with the blank control group(P<0.05).Compared with the control group,the expression of Jagged-1 m RNA in the middle dose group was significantly higher than that in the negative group(P<0.05);the expression of Jagged-2 m RNA in the middle and high dose groups was significantly higher than that in the control group(P<0.05);the expression of Jagged-2 m RNA in the high dose group was significantly higher than that in the low dose group(P<0.05);and the relative expression of Dll-4 m RNA in the high dose group was significantly higher than that in the low dose group(P<0.05).5.After the differentiation,the expression of Notch-2 protein in the 3T3-L1 preadipocytes in the high dose group was significantly higher than that in the other groups(P<0.05);the expression of Jagged-1 in the middle dose group was significantly higher than that in the control group(P<0.05);the expression of Jagged-2 protein in the high dose group was significantly higher than that in the control group(P<0.05);the expression of Dll-1 protein in the high dose group was higher than that in the other groups.The expression of Dll-4 protein in low and dose groups increased significantly(P<0.05).6.The results of the relationship between the content of TG in 3T3-L1 preadipocytes and the relative genes and proteins in Notch signaling pathway were illustrated in Table 7.After the differentiation,the content of TG in 3T3-L1 preadipocytes was significantly relative to the m RNA levels of Notch-1 and Jagged-2.(P<0.05)Conclusions:1.MEHP exposure can affect the expression of JAK-STAT signaling pathwayrelated genes and proteins,and affect the differentiation and maturation of 3T3-L1 preadipocytes.2.The content of TG in 3T3-L1 preadipocytes was significantly relative to the expression of TYK-2、STAT-3 and STAT-5a m RNAs and proteins in JAK-STAT signaling pathway after the MEHP exposure.3.MEHP exposure can affect the expression of genes and proteins in Notch signaling pathway and the differentiation and maturation of 3T3-L1 mouse preadipocytes.4.The content of TG in 3T3-L1 preadipocytes was significantly relative to the expression of Notch-1 and Jagged-2 m RNA and proteins in Notch signaling pathway after the MEHP exposure.Part Ⅲ: The role and mechanism of STAT-3 in the effects of MEHP on the differentiation and lipid metabolism of 3T3-L1 preadipocytesObjective: construct a stable transfected STAT-3 silencing ?silenced/silcent cell line of 3T3-L1 preadipocytes;clarify the role of STAT-3 in the JAK-STAT signaling pathway in the effects of MEHP on the differentiation and lipid level of 3T3-L1 preadipocyte,and-explore the mechanism of STAT-3 in the lipid metabolism disorder induced by MEHP.Methods: STAT-3 silent lentivirus(Lv/sh-STAT-3)and non-silent lentivirus(Lv/sh-NC)were used to infect 3T3-L1 preadipocytes,and a stable STAT-3 silent cell line was constructed.Real-time PCR and western blot were used to detect the silenced efficiency of STAT-3 in cells.Normal 3T3-L1 preadipocytes and Lv/sh-NC,Lv/sh-STAT-3 transfected 3T3-L1 preadipocytes were induced to differentiate by classical hormone cocktail method.The experimental groups were parental control group(Parental),0 μM MEHP;non-silent control group(Lv/sh-NC),0 μM MEHP;STAT-3 silent group(Lv/sh-STAT-3),0 μM MEHP;250 μM MEHP exposed STAT-3 silent group(Lv/sh-STAT-3+MEHP)and 250 μM MEHP exposed non-silent control group(Lv/sh-NC+MEHP).Oil red O staining was used to observe the differentiation of 3T3-L1 preadipocytes;chemiluminescence was used to detect the levels of triglyceride and cholesterol;western blot was used to detect the expression levels of lipid metabolism regulating factors Acox-1,C/EBPβ,FABP4,FAS,LPL,PDK4 and PPARγ;Real-time PCR was used to detect the m RNA expression levels of lipid metabolism regulating factors Acox-1,C/EBPβ,FABP4,FAS,LPL,PDK4 and PPARγ.Results: 1.STAT-3 silent lentivirus(Lv/sh-STAT-3)and non-silent lentivirus(Lv/sh-NC)infected the 3T3-L1 preadipocytes and the infection efficiency was higher.2.Compared with the parental group,the expression of STAT-3 m RNA in the Lv/sh-NC group had no significant changes(P>0.05).The expression of STAT-3 m RNAs and proteins in Lv/sh-STAT-3 group was significantly lower than that in Lv/shNC group(P<0.05).3.Compared with the parental group and Lv/sh-NC group,the intracellular TG content of 3T3-L1 cells in Lv/sh-STAT-3 group decreased slightly,with no significant difference(P>0.05);after MEHP exposure,the intracellular TG content in Lv/sh-STAT-3+NC group and Lv/sh-STAT-3+MEHP group increased significantly(P<0.05);after STAT-3 gene silenced,the intracellular TG content in the Lv/sh-STAT-3+MEHP group was significantly lower than that in the Lv/sh-NC +MEHP group(P<0.05).4.Compared with the parental group,the m RNA expression levels of Acox-1,FAS,FABP4,LPL and PDK4 in Lv/sh-NC and Lv/sh-STAT-3 groups did not change significantly(P>0.05).Compared with the non-exposed MEHP group,the expression m RNA levels of Acox-1,FAS,FABP4,LPL and PDK4 in MEHP exposed group increased significantly(P<0.05).5.Compared with the Parental group,the m RNA expression level of C/EBPβ in Lv/sh-NC group did not change significantly(P>0.05),but the m RNA expression level of C/EBPβ gene in Lv/sh-STAT-3 group was significantly lower than that in Parental group and Lv/sh-NC group(P<0.05);after MEHP exposure,the m RNA expression level of C/EBPβ in Lv/sh-NC+MEHP group and Lv/sh-STAT-3+MEHP group increased significantly(P<0.05);compared with Lv/sh-NC group,the m RNA expression level of C/EBPβ in Lv/sh-STAT-3+MEHP group was significantly lower(P<0.05);compared with parental group,the m RNA expression level of PPARγ in Lv/sh-NC group did not change significantly;while the expression level of PPARγ in Lv/sh-STAT-3 group decreased significantly(P<0.05);compared with Lv/sh-NC group,the m RNA expression level of PPARγ in Lv/sh-STAT-3 group decreased significantly(P<0.05).After MEHP exposure,the m RNA expression of PPARγ in Lv/sh-NC+MEHP group and Lv/sh-STAT-3+MEHP group increased significantly(P<0.05),while the m RNA expression of PPARγ in Lv/sh-STAT-3+MEHP group significantly decreased(P<0.05)compared with Lv/sh-NC+MEHP group.6.Compared with the parental group,the protein expression levels of Acox-1,FABP4,FAS and PDK4 in Lv/sh-NC group and Lv/sh-STAT-3 group did not change significantly.After MEHP exposure,the protein expression levels of Acox-1,FABP4,FAS and PDK4 in Lv/sh-NC+MEHP group and Lv/sh-STAT-3+MEHP group increased significantly(P<0.05).Compared with the parental group,the protein expression levels of C/EBPβ and PPARγ in Lv/sh-NC group and Lv/sh-STAT-3 group did not change significantly(P<0.05).After MEHP exposure,the protein expression levels of C/EBPβ and PPARγ in Lv/sh-NC+MEHP group and Lv/sh-STAT-3+MEHP group significantly increased(P<0.05).Conclusions: 1.STAT-3 silencing lentivirus(Lv/sh-STAT-3)was successfully transfected into 3T3-L1 preadipocy to construct a stable STAT-3 silencing cell line.2.STAT-3 can promote lipid accumulation in 3T3-L1 preadipocytes.3.STAT-3 can promote the differentiation and maturation of 3T3-L1 preadipocytes.4.STAT-3 may play a role in lipid metabolism disorders through regulating the expression levels of C/EBPβ and PPARγ m RNA and proteins induced by MEHP. |
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