Nonylphenol Induced Rat Obesity And Promotion Of Differentiation Of 3T3-L1 Preadipocytes

The surge in obesity at home and abroad has become an important safety hazard for life and health.Epidemiological investigations and animal experiments have shown that in addition to obesity caused by poor diet and lifestyle habits,environmental endocrine disruptors（EDCs）exposure is associated with obesity.Nonylphenol（NP）,as a typical EDCs,is widely used in the living environment and enters the body through pollution of food and food chains,disrupting endocrine homeostasis.To explore whether NP exposure leads to obesity and its possible role,this study used in vitro and in vivo experiments to investigate the effects of NP exposure on rat fattening and NP on adipocyte differentiation.PartⅠ Study on the effect of NP exposure on rat fatteningObjective:To explore the proliferative effect of long-term exposure to NP on rats.Methods:Forty four-week-old SD rats（150±10）g,half male and half female,were randomly divided into 4 groups,10 in each group.They were:control group（group C）,[low（L）,medium（M）,high（H）]NP dose group,the dose was 0,0.02,0.2,2μg/(kg·day^-1).Detection of Total cholesterol（TC）,Triglyceride（TG）,Hight density lipoprotein cholesterol（HDL-C）,Low density lipoprotein cholesterol（LDL-C）in serum.ELISA method for detecting lipid metabolism protein[Fatty acid synthetase（FAS）,CCAAT enhancer binding protein Alpha（CEBPα）,peroxisome proliferators-activated receptorγ（PPARγ）,Sterrol regulatory element-binding protein factor1（SREBP1）]content in serum.The fat pad of the paragonadal fat pad is weighed to calculate the fat organ coefficient.After HE staining of adipose tissue,pathological changes were observed under microscope and fat cell counts were performed.High performance liquid chromatography（HPLC）was used to detect NP content.An appropriate amount of adipose tissue was extracted from 100 mg of protein/RNA for Western Blot/Quantitative real-time reverse Transcription-Polymerase Chain Reaction（RT-PCR）to detect the expression of lipid metabolism（CEBPα,FAS,SREBP1,PPARγ）and apoptosis[Bcl-2-associated X protein（bax）,Cysteiny1 aspartate specific protein-3（caspase-3）]-related proteins/RNA in adipose tissue.Results:1.In rats with NP exposure 1-17 weeks,the body weight of rats in NP exposure group increased,but there was no statistical difference.From 21 weeks to 26 weeks,body weight showed the C group with the increase of NP dose<L group<M group<H group The trend of change（P<0.05）.2.The weight of fat/the Organ coefficient increased with the increase of NP dose(F_{fat weight}=103.605;and(F_{Organcoefficient}=169.807,P<0.001).3.The NP content in the adipose tissue of each exposure group was higher than that of the control group.The higher the concentration of the drug,the larger the accumulation in the tissue（F=561.353,P<0.001）.4.TC,TG,LDL-C contents in NP group were higher than those in control group(F_TC=3.798,F_TG=14.117,F_LDL-C=4.946,P<0.05).5.Under the HE microscope,the fat cells in the control group were arranged neatly and evenly.The area of adipocytes in the middle and high dose groups gradually increased,and some cell membranes were partially formed.After rupture,the number of cells in the control group was significantly higher than that of NP.The number of cells under the microscope decreased with the increase of NP dose（F=85.873,P<0.001）.6.The detection of serum lipoprotein by ELISA with the increase in NP treatment groups,serum CEBPα,FAS,PPARγ,SREBP1(F_CEBPα=189.104;F_FAS=51.011;F_PPARγ=114.306,F_SREBP1=30.432,P<0.001).The protein content also increased.7.The expression of lipid metabolism-related proteins（CEBPα,FAS,PPARγ,SREBP1）and apoptosis-related proteins in adipose tissue of NP exposed group increased(F_CEBPα=53.403;F_FAS=295.249;F_PPARγ=169.936;F_SREBP1=213.586;F_bax=245.707;F_caspase-3=87.711,P<0.001).8.Increased expression of lipid metabolism-related genes（CEBPα,FAS,PPARγ,SREBP1）and apoptosis-related（bax,caspase-3）genes in adipose tissue of NP exposed group(F_CEBPα=101.086;F_FAS=439.600;F_PPARγ=10.540;F_SREBP1=123.499,F_bax=31.758;F_caspase-3=42.238,P<0.001).Conclusion:1.2μg/kg/day NP exposure for 21 weeks can lead to obesity in rats,mainly manifested by body weight,fat weight/fat coefficient,and increased blood lipids.2.The serum lipoprotein content and lipid metabolism（FAS,CEBPα,PPARγ,SREBP1）protein/RNA expression levels were abnormal after NP exposure in rats.3.NP exposure induced obesity in rats and resulted in increased expression of apoptotic proteins in adipose tissue.PartⅡ NP promotes the differentiation of 3T3-L1 preadipocytesObjective: To explore the critical period and possible mechanism of NP on differentiation of 3T3-L1 preadipocytes.Methods:The cell viability was measured by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（MTT）assay after exposure inhibition of 3T-3L1 cell culture.The half maximal inhibitory concentration（IC50）was used to determine the NP exposure concentration.According to the MDI induction protocol,the cell NP exposure time points were divided into MDI induction（Pre-induction）and MDI induction（Period of induction）MD mature adipocytes（Period after induction）,NP exposure time was 48 h.The cell state and differentiation were observed by inverted phase contrast microscope.The lipid deposition was observed under the inverted phase contrast microscope after staining with oil red O.The protein was extracted by Western blot to detect theexpression levels of lipid metabolism related proteins（FAS,CEBPα,SREBP-1,PPARγ）,and apoptosis-related proteins（bax,caspase-3）.Results: 1.The IC50 of NP to 3T3-L1 preadipocytes was 40 μM,and the NP exposure dose was determined to be 0,40 p M,40 n M,and 40 μM.2.NP exposed 3T3-L1preadipocytes（Part A）were found to be compared with the control group.The intensity of oil red staining in the visible field of NP exposure was deepened,and NP（40 p M,40 n M）oil red staining was mainly concentrated in the cell membrane site;NP 40 μM Red staining appeared in the cells.Increased expression of lipid metabolism-related proteins（CEBPα,FAS,SREBP1,PPARγ）and apoptosis-related proteins（bax,caspase-3）in NP-exposed groups（FCEBPα=517.864;FFAS=124.005;FPPARγ=286.338;FSREBP1=1640.631;Fbax =220.050;Fcaspase-3=1199.542,P<0.001）.3.The 3T3-L1 preadipocytes were exposed to MDI for 48 h and then cultured to12 d（Part B）.After oil red O staining,the number of lipid droplets in the NP exposed cells increased with the increase of size of lipid droplets,and NP（40 μM）,visual field The number of differentiated cells was significantly higher than that of the control group,and the lipid droplets increased with the increase of size of lipid droplets.The lipid metabolism related protein in the NP exposed group was significantly higher than that in the control group.（FCEBPα=539.103;FFAS =715.740;FPPARγ=114.783;FSREBP1=139.600,Fbax =54.329;Fcaspase-3=449.201,P<0.001）.4.Mature fat cells were exposed to NP for 48 h（Period after induction）,and it was found by oil red O staining that compared with the control group,NP exposure caused a slight increase in intracellular red staining,The expression of intracellular lipid metabolism related proteins（CEBPα,FAS,PPARγ,SREBP-1）and apoptosis-related proteins（bax,caspase-3）increased slightly.(FCEBPα=29.727;FFAS =20.609;FPPARγ=330.951;FSREBP1=27.093;（Fcaspase-3=15.490,P<0.001;Fbax =4.686,P=0.008）.The difference between the groups of Part C are less than the difference between the groups of Period of pre-induction or Period of induction.Conclusion : 1.NP can promote the differentiation of 3T3-L1 preadipocytes,causing the increase of cell lipid metabolism and apoptosis-related protein expression;2.The critical period for NP to promote adipose differentiation is MDI-induced pre-differentiation and MDI-induced differentiation,and the degree of cell differentiation increases with increasing NP exposure dose.