全氟辛酸对SD大鼠肝脂质代谢紊乱及脂肪酸代谢相关蛋白表达的影响

Perfluorooctanoic acid-induced lipid metabolism disorder in SD rat liver and its effect on the expression of fatty acid metabolism-related proteins

目的:全氟辛酸(perfluorooctanoic acid,PFOA)能引起动物体脂代谢紊乱,影响脂肪酸的分解与合成,过氧化物酶体增殖剂激活受体(peroxisome proliferators-activated receptor,Ppar)在此过程中发挥极其重要的作用。本研究旨在探讨PFOA对Sprague Dawley(SD)大鼠肝脂质代谢紊乱及其相关蛋白表达的影响。方法:将40只雄性SD大鼠随机分成对照组(注射双蒸水)、低剂量组[PFOA剂量为1.25 mg/(kg·d)]、中剂量组[PFOA剂量为5.00 mg/(kg·d)],高剂量组[PFOA剂量为20.00 mg/(kg·d)],每组10只。正常饮食,采用PFOA经口灌胃14 d,每日观察、称重、记录。经口灌胃后,采血、处死大鼠、迅速剥离其肝脏。取部分肝组织固定于4%多聚甲醛后进行糖原过碘酸希夫(periodic acid-schiff,PAS)染色;检测血清以及肝组织中HDLC、LDLC、TG、TC的含量及相关酶的活性;蛋白质印迹法检测环磷腺苷效应元件结合蛋白(cyclic adenosine monophosphate-response element binding protein,Cbp)、类氨基酸合成酵母同源物通用调控蛋白2(general control of amino acid synthesis 5-like 2,Gcn5L2)、Ppar γ、沉默信息调节因子1(silent information regulator 1,Sirt1)和人类视黄醇X受体α2(retinoid X receptor alpha 2,Rxrα2)的表达水平。结果:PFOA暴露14 d后,与对照组相比,中、高剂量组的SD大鼠肝细胞的胞质及胞核内PAS染色阳性颗粒明显减少。与对照组相比,低、中剂量组血清中LDLC、TC含量显著降低(均P<0.05),高剂量组中LDLC、TC含量升高,但差异无统计学意义(均P>0.05),HDLC、TG差异均无统计学意义(均P>0.05);低、中、高剂量组血清中碱性磷酸酶(alkaline phosphatase,AKP)、谷丙转氨酶(alanine aminotransferase,ALT)的活性显著升高(均P<0.05);高剂量组中ALT/谷草转氨酶(aspartate aminotransferase,AST)比值显著升高(P<0.05);LDH、TG差异均无统计学意义(均P<0.05)。与对照组相比,高剂量组肝组织中的HDLC含量明显降低(P<0.05);低、中、高剂量组肝组织中的TC含量明显升高(均P<0.05),LDLC、TG差异均无统计学意义(均P<0.05);中、高剂量组肝组织中的AKP活性明显升高(均P<0.05),LDH、ALT、ALT/AST比值差异均无统计学意义(均P>0.05)。与对照组相比,高剂量组肝组织中Pparγ、Cbp、Rxrα2的蛋白质表达水平均明显下调(均P<0.05);Sirt1的蛋白质表达则明显上调(均P<0.05)。结论:PFOA暴露可导致SD大鼠肝脂质代谢紊乱以及糖原减少,这可能与PFOA激活Sirt1的表达、抑制Pparγ的表达,从而影响脂肪酸正常代谢及促进糖酵解有关。

Objective:Perfluorooctanoic acid(PFOA)can cause lipid metabolism disorders in animal body and affect the lipolysis and synthesis of fatty acids.Peroxisome proliferators-activated receptor(PPAR)plays an extremely important role in this process.This study aims to explore the effects of PFOA on liver lipid metabolism disorders in Sprague Dewley(SD)rats and the expression of PPAR.Methods:A total of 40 male SD rats were randomly divided into 4 groups(n=10 in each group):a control group(ddH_(2)O),a low-dose PFOA group[PFOA 1.25 mg/(kg·d)],a middle-dose PFOA group[PFOA 5.00 mg/(kg·d)],and a high-dose PFOA group[PFOA20.00 mg/(kg·d)].The rats were fed with normal diet,and PFOA exposure were performed by oral gavage for 14 days,and the rats were observed,weighted and recorded every day during the exposure.After the exposure,the blood was collected,and the livers were quickly stripped after the rats were killed.Part of the liver tissues were fixed in 4% paraformaldehyde for periodic acid-schiff(PAS)staining;the contents of HDLC,LDLC,TG,TC in serum and liver tissues,as well as the activities of their related enzymes were assayed;The expression levels of cyclic adenosine monophosphate-response element binding protein(Cbp),general control of amino acid synthesis 5-like 2(Gcn5 L2),peroxidation peroxisome proliferation factor activated receptor γ(PPAR),silent information regulator 1(Sirt1)and human retinoid X receptor alpha 2(Rxrα2))were detected by Western blotting.Results:After 14 days of PFOA exposure,the PAS staining positive particles in the cytoplasm and nucleus of SD rats in the medium and high dose groups were significantly reduced compared with the control group.The serum levels of LDLC and TC in the lowdose and middle-dose groups were significantly reduced compared with the control group(all P<0.05),while the high-dose group showed an increasing tendency,without siginificant difference(P>0.05),there was no significant difference in HDLC and TG(both P>0.05).The activities of alkaline phosphatase(AKP)and alanine aminotransferase(ALT)were increased significantly(both P<0.05)compared with control group;the ratio of ALT/aspartate aminotransferase(AST)in the high-dose group was increased significantly(P<0.05),there was no significant difference in LDH and TG(both P>0.05);the HDLC content in the liver tissues in the high-dose group was significantly reduced,compared with the control group(P<0.05);the TC contents in the liver tissues in the low,medium and high-dose groups were significantly increased(all P<0.05),there was no significant difference in LDLC and TG(both P>0.05);the AKP activity in the livers in the medium and high-dose groups was significantly increased(both P<0.05),there was no siginificant difference in LDH,ALT,and the ratio of ALT/AST(all P>0.05);the protein expression levels of Pparγ,Cbp and Rxrα2 in the liver in the high dose groups were significantly down-regulated compared with the control group(all P<0.05),while the protein expression levels of Sirt1 were significantly up-regulated(all P<0.05).Conclusion:PFOA exposure can cause lipid metabolism disorder and glycogen reduction in SD rat livers,which may be related to the activation of Sirt1 and inhibition of Pparγexpression,leading to affecting the normal metabolism of fatty acids and promoting glycolysis.