Relationship And Its Mechanism Between Nonylphenol Exposure And The Attack Of Nonalcoholic Fatty Liver Disease In Rats

Non-alcoholic fatty liver disease(NAFLD)is widely prevalent in the world,the incidence rate was increasing year by year,and the pathogenesis is unknown.In order to explore the role of environmental factors(nonylphenol)and unhealthy lifestyles(high-fat-high-sucrose diet)in the development of NAFLD,this subject was divided into two parts to carry out experimental research.In the first part,SD rats fed with normal diet were treated with low-dose NP for 90 days to observe whether NP could cause NAFLD in rats.In the second part,the SD rats were treated with NP for 90 days to observe the attack of NAFLD in rats under high-sucrose-high-fat diet,and investigate whether subchronic low-dose NP exposure could promote the occurrence of NAFLD caused by high-sucrose-high-fat diet.The indicators related to lipid synthesis were further detected,and the mechanism that NP induce NAFLD were explored.Part one The adverse effects of subchronic nonylphenol exposure on hepatic function and lipid metabolism in ratsObjective: To investigate whether sub-chronic nonylphenol exposure induce NAFLD or not.Methods:Forty SD rats were randomly divided into four groups(n = 10): the control group(corn oil),NP low-dose group(0.02 μg/kg),NP middle-dose group(0.2 μg/kg),NP high-dose group(2 μg/kg).Intragastric administration NP for 90 days continuously,four rats in each group were randomly selected for the detection of liver ultrasonography in the10 th week.During the period of intragastric administration,the body weight was weighted1 time in each week,and the dietary intake and the amount of drinking water were recorded.Rats were anesthetized by 10% of the chlorinated aldehyde before the animals were sacrificed,blood was collected from abdominal aorta,and the serum was isolated to measure hepatic function and serum lipid.Liver mass was measured and liver index was calculated,liver specimens were taken after the rats were sacrificed immediately.One liver specimen was fixed to 10% formaldehyde solution for HE staining,and the remaining 2tissues were cryopreserved-80 degrees for real-time quantitative PCR detection of lipid metabolism related genes Fas,Srebp1,Ucp2 expression,Western blot detection of c/EBPα,PPARγ expression.Results:There was no significant difference in the body weight,dietary intake and the amount of drinking water(P> 0.05)among different treatment groups.B-ultrasonography results showed that no significant changes of fatty liver were observed in each group.HE staining showed that a small amount of liver cell steatosis was observed in the high dose NP group.Liver function and blood biochemistry results showed that there were no alterations in the serum ALT,AST,TC,LDL-C levels(P> 0.05).RT-PCR showed that the expressions of Fas,Ucp2 and Srebp1 m RNA in the middle and high dose NP exposure group were up-regulated(P<0.05).The results of western blot showed that the lipid synthesis relative expression of c/EBPα and PPARγ in middle and high dose groups were significantly up-regulated(P<0.05).Conclusions:NP sub-chronic exposure do not cause non-alcoholic fatty liver formation in rats,but could affect the expression of Fas,Ucp2 and Srebp1 genes,c/EBPα and PPARγprotein relate to lipid metabolism.Part two The joint effects of nonylphenol and high-fat-high-sucrose diet on non-alcoholic fatty liver disease in ratsObjective :To investigate whether subchronic NP exposure and high-fat-high-sucrose diet increase the incidence of NAFLD in rats or not.Methods:fourty SD rats(200 ± 20)g were purchased from the Daping Experimental Animal Center of the Third Military Medical University(Grant No.(SCXK)(2012-0005)).Following a 1-week normal period,the rats was randomly divided into 4 groups: diet high-fat-high-sucrose diet(HFD)control group,HFD+low dose NP group(0.02 μg/kg),HFD+middle dose NP group(0.2 μg/kg),HFD+high dose NP group(2 μg/kg).The body weight was weighted 1 time in each week,and the dietary intake and the amount of drinking water were recorded.At the 10 th week,4 rats in each group were randomly selected for liver color ultrasonography.The presence of strong echo area was an indication of success in establishing NAFLD model.The animals were sacrificed at the12 th week after anesthetized by 10% chloral hydrate,the blood from abdominal aorta was used to detect the liver function and serum lipid.The liver mass was weighed and the liver index was calculated.Each rat was given 3 aliquots of 1 mm at the inner edge of the largest leaf margin of the liver,one specimen was fixed to 10% formaldehyde for HE staining,and the remaining 2 specimens were frozen with-80 degrees for real-time fluorescent quantitative PCR was used to detect the expressions of Fas,Srebp1 and Ucp2,and the expressions of c/EBPα and PPARγ was detected by Western blot.Results:All rats occurred fatty liver in control and NP exposure groups of high-fat-high glucose diet,the weight gain was increased at the 12 th week in the high dose group(P<0.05).The drinking water in the low and middle dose groups,food intake in the middle and high dose group was significantly higher than that control group(P<0.05).B-ultrasonography showed that strong echo areas were found in each NP treatment group,suggesting the presence of fatty liver,and the middle and high dose groups were stronger than the control group,high dose group was stronger than low dose group.HE staining showed that bullous steatosis liver cells increased with the increase in NP exposure dose.The serum levels of ALT,AST,TC and LDL-C were higher in the high dose group compared to the control,low,middle dose group.The serum levels of TG,TC and LDL-C were higher in the middle dose group in comparison with the control and low dose group.(P<0.05).RT-PCR results showed that the expressions of Fas and Ucp2 m RNA was up-regulated and the expression of Srebp1 was up-regulated(P<0.05).Western blot results showed that the expression of c/EBPα and PPARγ protein in the middle and high dose groups were higher than those in the control and low dose group(P<0.05).Conclusions:NP can aggravate the effect of high-fat-high-sucrose diet on nonalcoholic fatty liver in rats.The mechanism may be relate to up-regulation of Srebp1,Fas,Ucp2 m RNA expression,enhance c/EBPα,PPARγ expression and increase TG synthesis.