非诺贝特对DDC诱导的原发性硬化性胆管炎模型小鼠的预防作用及机制
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摘要
目的研究非诺贝特对3,5-二乙氧基羰基-1,4-二氢-2,4,6-三甲基吡啶(DDC)诱导的原发性硬化性胆管炎(PSC)的预防作用及机制。方法将健康雌性野生型和过氧化物酶体增殖物激活受体α(Pparα)基因敲除型小鼠随机各分为4组,分别为正常对照组、PSC模型组、非诺贝特对照组和非诺贝特预防组。非诺贝特对照组和预防组连续10 d每天2次ig给予非诺贝特25 mg·kg~(-1),PSC模型组给予玉米油;给药第4～10天,PSC模型组和非诺贝特预防组连续食饲含0.1%DDC的饲料诱导PSC,正常对照组和非诺贝特对照组正常饲养。采用生化法测定小鼠血清谷草转氨酶(GOT)、谷丙转氨酶(GPT)、碱性磷酸酶(ALP)、总胆汁酸(TBA)、总胆红素(TBIL)和直接胆红素(DBIL)水平;HE染色观察肝组织病理改变;RT-qPCR检测肝组织胆固醇7α-羟化酶(Cyp7α1)、固醇12α羟化酶(Cyp8b1)、有机阴离子转运多肽1(Oatp1)、钠离子-牛磺胆酸协同转运多肽(Ntcp)、多药耐药蛋白1α(Mdr1α)、Mdr2、Mrp4、有机溶质转运体β(Ostβ)、白细胞介素1β(IL-1β)、IL-2、IL-6、IL-10、肿瘤坏死因子-α(Tnf-α)、细胞因子信号抑制物(Socs3)、c-Fos、c-Jun、Bcl-2、Bax和Bcl-xl m RNA表达水平。结果与正常对照组相比,野生型和基因敲除型小鼠的PSC模型组GOT,GPT,TBA和ALP均显著升高(P<0.01),肝组织胆管周围发生炎症并有胆淤,表明在2种基因型小鼠中,DDC均成功地诱导了PSC;且RT-q PCR检测结果表明,PSC模型组Cyp8b1和Ntcp mRNA表达显著降低(P<0.05,P<0.01),Mdr1α,Ostβ,IL-1β和Socs3 mRNA表达升高(P<0.05,P<0.01)。与PSC模型组相比,在野生型小鼠中,非诺贝特预防组肝组织Cyp8b1和Ntcp m RNA表达显著升高(P<0.05),Mdr1α,Mdr2,Ostβ,IL-1β,IL-2,IL-6,IL-10,Socs3,c-Fos,c-Jun,Bcl-2和Bax mRNA表达显著降低(P<0.05,P<0.01);而在基因敲除型小鼠中,非诺贝特预防组肝组织未见明显病理损伤,炎症和胆汁酸代谢相关基因mRNA表达水平均未见改善。在2种基因型小鼠中,非诺贝特对照组上述各指标与正常对照组均无明显差异。结论非诺贝特可通过调节胆汁酸代谢和抑制炎症反应预防DDC诱导的小鼠PSC,且该作用依赖于PPARα介导。
OBJECTIVE To explore the preventive effect and mechanism of fenofibrate on 3,5-diethoxycarbonyl-1,4-dihydrocollidine(DDC)-induced primary sclerosing cholangitis(PSC)in mice.METHODS Female wild-type and Ppara-null mice were divided into four groups:normal control group,PSC model group,PSC+fenofibrate group,and fenofibrate control group.The mice in PSC+fenofibrate group and fenofibrate group were dosed 25 mg·kg-1fenofibrate by ig twice per day for 10 d,the mode group was treated with corn oil simultaneously,and the PSC model group and PSC+fenofibrate group were fed 0.1%DDC-supplemented diet to induce PSC from day 4 to day 10.The mice of normal contro group and fenofibrate control group were fed normal commercial diet.Serum levels of total bile acid(TBA),alkaline phosphatase(ALP),total bilirubin(TBIL),direct bilirubin(DBIL),glutamic-pyruvic transaminase(GPT),glutamic-oxaloacetic transaminase(GOT)were measured.Pathological changes in hepatic tissues were examined by hematoxylin and eosin(HE)staining.The m RNA levels of cholesterol 7-alpha hydroxylase(Cyp7a1),sterol 12α-hydroxylase(Cyp8b1),organic anion transporting polypeptide 1(Oatp1),taurocholate co-transporting polypeptide(Ntcp),multidrug resistance 1α(Mdr1α),Mdr2,Mrp4,organic solute transporterβ(Ostβ),interleukin-1β(IL-1β),IL-2,IL-6,IL-10,tumor necrosis factor-α(Tnf-α),suppressor of cytokine signaling(Socs3),c-Jun N-terminal kinase target gene(c-Fos and c-Jun)apoptotic Bcl-2 family members(Bcl-2,Bax and Bcl-xl)were quantified by real-time quantitative polymerase chain reaction(RT-qPCR).RESULTS Compared with the normal control group,the levels of GOT,GPT,ALP and TBA in the model groups of both wild-type and Ppara-null mice were significantly increased(P<0.01).Histopathologically,obvious inflammation and cholestasis indicated that primary sclerosing cholangitis was induced by DDC.RT-qPCR showed that the expressions of Cyp8b1 and Ntcp mRNA were significantly decreased.The expressions of Mdr1αand OstβmRNA were increased as well as the inflammatory genes IL-1βand Socs3(P<0.05,P<0.01).In the liver tissue of the wild-type PSC+fenofibrate group,the expression levels of Cyp8b1 and Ntcp m RNA were significantly up-regulated(P<0.05),and the mRNA expression levels of Mdr1α,Mdr2 and Ostβwere significantly down-regulated(P<0.01).The m RNA expression levels of IL-1β,IL-2,IL-6,IL-10,Socs3,c-Fos and c-Jun were downregulated by 80%,85%,90%,77%,70%,95%and 75%(P<0.01)respectively.Bcl-2 and Bax mRNA expression levels were significantly inhibited as well(P<0.01).The above changes did not occur in the Ppara-null PSC+fenofibrate mice.Liver histology,serum biomarkers and the expression of genes involved in bile acid metabolism in fenofibrate control groups of two mouse lines were not different from those of normal control groups.CONCLUSION Fenofibrate prevents DDC-induced PSC by regulating the metabolism of bile acids and inhibiting inflammation,and this preventive action depends on PPARα.
OBJECTIVE To explore the preventive effect and mechanism of fenofibrate on 3,5-diethoxycarbonyl-1,4-dihydrocollidine(DDC)-induced primary sclerosing cholangitis(PSC)in mice.METHODS Female wild-type and Ppara-null mice were divided into four groups:normal control group,PSC model group,PSC+fenofibrate group,and fenofibrate control group.The mice in PSC+fenofibrate group and fenofibrate group were dosed 25 mg·kg-1fenofibrate by ig twice per day for 10 d,the mode group was treated with corn oil simultaneously,and the PSC model group and PSC+fenofibrate group were fed 0.1%DDC-supplemented diet to induce PSC from day 4 to day 10.The mice of normal contro group and fenofibrate control group were fed normal commercial diet.Serum levels of total bile acid(TBA),alkaline phosphatase(ALP),total bilirubin(TBIL),direct bilirubin(DBIL),glutamic-pyruvic transaminase(GPT),glutamic-oxaloacetic transaminase(GOT)were measured.Pathological changes in hepatic tissues were examined by hematoxylin and eosin(HE)staining.The m RNA levels of cholesterol 7-alpha hydroxylase(Cyp7a1),sterol 12α-hydroxylase(Cyp8b1),organic anion transporting polypeptide 1(Oatp1),taurocholate co-transporting polypeptide(Ntcp),multidrug resistance 1α(Mdr1α),Mdr2,Mrp4,organic solute transporterβ(Ostβ),interleukin-1β(IL-1β),IL-2,IL-6,IL-10,tumor necrosis factor-α(Tnf-α),suppressor of cytokine signaling(Socs3),c-Jun N-terminal kinase target gene(c-Fos and c-Jun)apoptotic Bcl-2 family members(Bcl-2,Bax and Bcl-xl)were quantified by real-time quantitative polymerase chain reaction(RT-qPCR).RESULTS Compared with the normal control group,the levels of GOT,GPT,ALP and TBA in the model groups of both wild-type and Ppara-null mice were significantly increased(P<0.01).Histopathologically,obvious inflammation and cholestasis indicated that primary sclerosing cholangitis was induced by DDC.RT-qPCR showed that the expressions of Cyp8b1 and Ntcp mRNA were significantly decreased.The expressions of Mdr1αand OstβmRNA were increased as well as the inflammatory genes IL-1βand Socs3(P<0.05,P<0.01).In the liver tissue of the wild-type PSC+fenofibrate group,the expression levels of Cyp8b1 and Ntcp m RNA were significantly up-regulated(P<0.05),and the mRNA expression levels of Mdr1α,Mdr2 and Ostβwere significantly down-regulated(P<0.01).The m RNA expression levels of IL-1β,IL-2,IL-6,IL-10,Socs3,c-Fos and c-Jun were downregulated by 80%,85%,90%,77%,70%,95%and 75%(P<0.01)respectively.Bcl-2 and Bax mRNA expression levels were significantly inhibited as well(P<0.01).The above changes did not occur in the Ppara-null PSC+fenofibrate mice.Liver histology,serum biomarkers and the expression of genes involved in bile acid metabolism in fenofibrate control groups of two mouse lines were not different from those of normal control groups.CONCLUSION Fenofibrate prevents DDC-induced PSC by regulating the metabolism of bile acids and inhibiting inflammation,and this preventive action depends on PPARα.
引文
[1]Cholestatic Liver Disease Diagnosis and Treatment Expert Committee.Consensus on the diagnosis and treatment of cholestatic liver diseases 2013[J/CD].Chin J Exp Clin Infect Dis(Electron Ed)(中华实验和临床感染病杂志:电子版),2013,7(1):134-144.
[2]Chinese Society of Hepatology,Chinese Medical Association;Chinese Society of Gastroenterology,Chinese Medical Association;Chinese Society of Infectious diseases,Chinese Medical Association.Consensus on the diagnosis and treatment of cholestasis liver diseases[J].Chin J Hepatol,2015,23(12):924-933.
[3]Marzioni M,Saccomanno S,Agostinelli L,Rychlicki C,De Minicis S,Pierantonelli I,et al.PDX-1/Hes-1 interactions determine cholangiocyte proliferative response to injury in rodents:possible implications for sclerosing cholangitis[J].JHepatol,2013,58(4):750-756.
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[6]Honda A,Ikegami T,Nakamuta M,Miyazaki T,Iwamoto J,Hirayama T,et al.Anticholestatic effects of bezafibrate in patients with primary biliary cirrhosis treated with ursodeoxycholic acid[J].Hepatology,2013,57(5):1931-1941.
[7]Cindoruk M,Kerem M,Karakan T,Salman B,Akin O,Alper M,et al.Peroxisome proliferatorsactivated alpha agonist treatment ameliorates hepatic damage in rats with obstructive jaundice:an experimental study[J/OL].BMC Gastroenterol,2007,7:44(2007-11-28).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2219993/
[8]El-Sisi A,Hegazy S,El-Khateeb E.Effects of three different fibrates on intrahepatic cholestasis experimentally induced in rats[J/OL].PPAR Res,2013,2013:781348(2013-08-12).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753769/
[9]Ghonem NS,Assis DN,Boyer JL.Fibrates and cholestasis[J].Hepatology,2015,62(2):635-643.
[10]Dai M,Yang J,Xie M,Lin J,Luo M,Hua H,et al.Inhibition of JNK signalling mediates PPARα-dependent protection against intrahepatic cholestasis by fenofibrate[J].Br J Pharmacol,2017,174(18):3000-3017.
[11]Tan Z,Liu AM,Luo M,Yin X,Song DJ,Dai MY,et al.Geniposide inhibits alpha-naphthylisothiocyanate-induced intrahepatic cholestasis:the downregulation of STAT3 and NFκB signaling plays an important role[J].Am J Chin Med,2016,44(4):721-736.
[12]Tan Z,Luo M,Yang J,Cheng Y,Huang J,Lu C,et al.Chlorogenic acid inhibits cholestatic liver injury induced byα-naphthylisothiocyanate:involvement of STAT3 and NFκB signalling regulation[J].JPharm Pharmacol,2016,68(9):1203-1213.
[13]Zhang H,Bian ZL,Wang QX,Lian ZX,Ma X.Establishment and evaluation of cholestatic mice model[J].Chin Hepatol,2015,20(3):218-222.
[14]Fickert P,St?ger U,Fuchsbichler A,Moustafa T,Marschall HU,Weiglein AH,et al.A new xenobioticinduced mouse model of sclerosing cholangitis and biliary fibrosis[J].Am J Pathol,2007,171(2):525-536.
[15]Luo M,Tan Z,Dai M,Song D,Lin J,Xie M,et al.Dual action of peroxisome proliferator-activated receptor alpha in perfluorodecanoic acid-induced hepatotoxicity[J].Arch Toxicol,2017,91(2):897-907.
[16]Li F,Patterson AD,Krausz KW,Tanaka N,Gonzalez FJ.Metabolomics reveals an essential role for peroxisome proliferator-activated receptorαin bile acid homeostasis[J].J Lipid Res,2012,53(8):1625-1635.
[17]Zhou X,Cao L,Jiang C,Xie Y,Cheng X,Krausz KW,et al.PPARα-UGT axis activation represses intestinal FXR-FGF15 feedback signal ing and exacerbates experimental colitis[J/OL].Nat Commun,2014,5:4573(2014-09-03).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164778/
[18]Liu A,Krausz KW,Fang ZZ,Brocker C,Qu A,Gonzalez FJ.Gemfibrozil disrupts lysophosphatidylcholine and bile acid homeostasis via PPARαand its relevance to hepatotoxicity[J].Arch Toxicol,2014,88(4):983-996.
[19]Zhang Y,Lickteig AJ,Csanaky IL,Klaassen CD.Activation of PPARαdecreases bile acids in livers of female mice while maintaining bile flow and biliary bile acid excretion[J].Toxicol Appl Pharmacol,2018,338:112-123.
[2]Chinese Society of Hepatology,Chinese Medical Association;Chinese Society of Gastroenterology,Chinese Medical Association;Chinese Society of Infectious diseases,Chinese Medical Association.Consensus on the diagnosis and treatment of cholestasis liver diseases[J].Chin J Hepatol,2015,23(12):924-933.
[3]Marzioni M,Saccomanno S,Agostinelli L,Rychlicki C,De Minicis S,Pierantonelli I,et al.PDX-1/Hes-1 interactions determine cholangiocyte proliferative response to injury in rodents:possible implications for sclerosing cholangitis[J].JHepatol,2013,58(4):750-756.
[4]Liedtke C,Luedde T,Sauerbruch T,Scholten D,Streetz K,Tacke F,et al.Experimental liver fibrosis research:update on animal models,legal issues and translational aspects[J/OL].Fibrogenesis Tissue Repair,2013,6(1):19(2013-10-01).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3850878/
[5]Pollheimer MJ,Fickert P.Animal models in primary biliary cirrhosis and primary sclerosing cholangitis[J].Clin Rev Allergy Immunol,2015,48(2-3):207-217.
[6]Honda A,Ikegami T,Nakamuta M,Miyazaki T,Iwamoto J,Hirayama T,et al.Anticholestatic effects of bezafibrate in patients with primary biliary cirrhosis treated with ursodeoxycholic acid[J].Hepatology,2013,57(5):1931-1941.
[7]Cindoruk M,Kerem M,Karakan T,Salman B,Akin O,Alper M,et al.Peroxisome proliferatorsactivated alpha agonist treatment ameliorates hepatic damage in rats with obstructive jaundice:an experimental study[J/OL].BMC Gastroenterol,2007,7:44(2007-11-28).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2219993/
[8]El-Sisi A,Hegazy S,El-Khateeb E.Effects of three different fibrates on intrahepatic cholestasis experimentally induced in rats[J/OL].PPAR Res,2013,2013:781348(2013-08-12).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3753769/
[9]Ghonem NS,Assis DN,Boyer JL.Fibrates and cholestasis[J].Hepatology,2015,62(2):635-643.
[10]Dai M,Yang J,Xie M,Lin J,Luo M,Hua H,et al.Inhibition of JNK signalling mediates PPARα-dependent protection against intrahepatic cholestasis by fenofibrate[J].Br J Pharmacol,2017,174(18):3000-3017.
[11]Tan Z,Liu AM,Luo M,Yin X,Song DJ,Dai MY,et al.Geniposide inhibits alpha-naphthylisothiocyanate-induced intrahepatic cholestasis:the downregulation of STAT3 and NFκB signaling plays an important role[J].Am J Chin Med,2016,44(4):721-736.
[12]Tan Z,Luo M,Yang J,Cheng Y,Huang J,Lu C,et al.Chlorogenic acid inhibits cholestatic liver injury induced byα-naphthylisothiocyanate:involvement of STAT3 and NFκB signalling regulation[J].JPharm Pharmacol,2016,68(9):1203-1213.
[13]Zhang H,Bian ZL,Wang QX,Lian ZX,Ma X.Establishment and evaluation of cholestatic mice model[J].Chin Hepatol,2015,20(3):218-222.
[14]Fickert P,St?ger U,Fuchsbichler A,Moustafa T,Marschall HU,Weiglein AH,et al.A new xenobioticinduced mouse model of sclerosing cholangitis and biliary fibrosis[J].Am J Pathol,2007,171(2):525-536.
[15]Luo M,Tan Z,Dai M,Song D,Lin J,Xie M,et al.Dual action of peroxisome proliferator-activated receptor alpha in perfluorodecanoic acid-induced hepatotoxicity[J].Arch Toxicol,2017,91(2):897-907.
[16]Li F,Patterson AD,Krausz KW,Tanaka N,Gonzalez FJ.Metabolomics reveals an essential role for peroxisome proliferator-activated receptorαin bile acid homeostasis[J].J Lipid Res,2012,53(8):1625-1635.
[17]Zhou X,Cao L,Jiang C,Xie Y,Cheng X,Krausz KW,et al.PPARα-UGT axis activation represses intestinal FXR-FGF15 feedback signal ing and exacerbates experimental colitis[J/OL].Nat Commun,2014,5:4573(2014-09-03).https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164778/
[18]Liu A,Krausz KW,Fang ZZ,Brocker C,Qu A,Gonzalez FJ.Gemfibrozil disrupts lysophosphatidylcholine and bile acid homeostasis via PPARαand its relevance to hepatotoxicity[J].Arch Toxicol,2014,88(4):983-996.
[19]Zhang Y,Lickteig AJ,Csanaky IL,Klaassen CD.Activation of PPARαdecreases bile acids in livers of female mice while maintaining bile flow and biliary bile acid excretion[J].Toxicol Appl Pharmacol,2018,338:112-123.