京尼平苷酸对ANIT诱导胆汁淤积大鼠的酮洛芬肝代谢和胆汁排泄的影响及机制探究
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摘要
目的探讨京尼平苷酸(GPA)对α-萘异硫氰酸酯(ANIT)诱导胆汁淤积模型大鼠的酮洛芬(Ketoprofen,KP)及结合型代谢产物酮洛芬葡萄糖醛酸复合物(KPG)的肝代谢和胆汁排泄的影响及其可能机制。方法将80只SD大鼠随机分为5组,依次为灌胃100、50、25 mg·kg~(-1)·d~(-1)的GPA高、中、低剂量组,ANIT模型组以及空白对照组,给药10 d;在第8天给药后,除空白对照组外,所有大鼠一次性灌胃65 mg·kg~(-1)ANIT诱导肝内胆汁淤积模型;末次给药后,各组静脉注射KP 20 mg·kg~(-1),以HPLC-UV法测定大鼠血浆、胆汁中KP及其酮洛芬葡萄糖醛酸结合物(S-KPG和R-KPG)含量。检测各组大鼠血清中谷丙转氨酶(GPT)、谷草转氨酶(GOT)、总胆汁酸(TBA)、总胆红素(TB)。反转录-聚合酶链反应(RT-PCR)检测大鼠肝组织中组成型雄甾烷受体(CAR)、孕烷X受体(PXR)以及尿苷二磷酸葡萄糖醛酸转移酶1A1(Ugt1a1)mRNA的转录。蛋白质印迹法(WB)检测CAR、PXR、Ugt1a1的蛋白表达情况。结果与空白对照组比较,ANIT模型组大鼠KPG的累计胆汁排泄量显著降低(P <0.01);与ANIT模型组比较,100和50 mg·kg~(-1)·d-1GPA干预组大鼠KPG的累计胆汁排泄量显著提高(P <0.01)。与空白对照组比较,ANIT模型组大鼠血浆中KP曲线下面积AUC0-∞显著增加(P <0.01)、消除半衰期T1/2显著延长(P <0.01);与ANIT模型组比较,100,50 mg·kg~(-1)·d~(-1)GPA干预组大鼠血浆中KP的曲线下面积AUC0-∞显著降低(P <0.01)、消除半衰期T1/2显著缩短(P <0.01)。与ANIT模型组比较,100和50 mg·kg~(-1)·d~(-1)GPA干预组血清中TB、GPT、GOT和TB的含量明显下降(P <0.01),25 mg·kg~(-1)·d~(-1)GPA干预组降低不明显,无统计学意义。GPA能显著增加CAR、PXR、Ugt1a1 mRNA转录和蛋白表达量,与剂量呈正相关。结论 ANIT诱导的胆汁淤积大鼠KPG胆排泄降低,可引起KP的药动学参数的变化。GPA可改善胆汁淤积大鼠对酮洛芬肝代谢和胆汁排泄的影响,有可能是通过调节CAR和PXR对Ugt1a1基因转录和蛋白表达来实现的。
Objective To study the liver metabolism and bile excretion of ketoprofen(KP)and the conjugated metabolite ketoprofen glucuronide(KPG)treated by geniposidic acid(GPA)in rats with alpha naphthyl thiocyanate(ANIT)induced cholestasis and its possible mechanism.Methods Eighty SD rats were randomly divided into 5groups,which were 100,50,25and low doses)GPA groups,ANIT model group and blank control group.Rats were treated for 10 days.After 8 days’administration,all rats except the blank control group were given 65 mg·kg~(-1)ANIT once to induce intrahepatic cholestasis models.After the last administration,rats were injected 20 mg·kg~(-1)KP.HPLC-UV were used to determine the contents of KP and ketoprofen glucuronide(S-KPG and R-KPG)in plasma and bile samples of rats.The serum levels of alanine aminotransferase(GPT),aspartate aminotransferase(GOT),total bile acid(TBA)and total bilirubin(TB)were measured.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the transcription of constitutive androstane receptor(CAR),the pregnane X receptor(PXR)and the UDP-glucurnosyltransferase 1a1(Ugt1a1)mRNA in the rats’liver.Western Blot(WB)was used to detect the protein expression of CAR,PXR and Ugt1a1.Results Compared with the blank control group,the cumulative bile excretion of KPG in the ANIT model group was significantly decreased(P<0.01),AUC0-∞(P<0.01)under the KP curve in the plasma of the ANIT model rats increased significantly and(P<0.01)was significantly prolonged.Compared with the ANIT model group,the cumulative bile excretion of KPG in the 100 and 50intervention groups were significantly increased
significantly andwas significantly shortened(P<0.01).Compared with the ANIT model group,the contents of TBA,GPT,GOT and TB in the serum of 100 and 50intervention groups decreased significantly(P<0.01).GPA significantly increased the transcription and protein expression of CAR,PXR and Ugt1a1 mRNA with positively dose-effect correlation.Conclusion Cholestasis rats reduces KPG bile excretion,and may cause changes in pharmacokinetic parameters of KP.GPA may improve the liver metabolism and bile excretion of KP in rats with cholestasis,which may be achieved by regulating the transcription of Ugt1a1 by CAR and PXR.
Objective To study the liver metabolism and bile excretion of ketoprofen(KP)and the conjugated metabolite ketoprofen glucuronide(KPG)treated by geniposidic acid(GPA)in rats with alpha naphthyl thiocyanate(ANIT)induced cholestasis and its possible mechanism.Methods Eighty SD rats were randomly divided into 5groups,which were 100,50,25and low doses)GPA groups,ANIT model group and blank control group.Rats were treated for 10 days.After 8 days’administration,all rats except the blank control group were given 65 mg·kg~(-1)ANIT once to induce intrahepatic cholestasis models.After the last administration,rats were injected 20 mg·kg~(-1)KP.HPLC-UV were used to determine the contents of KP and ketoprofen glucuronide(S-KPG and R-KPG)in plasma and bile samples of rats.The serum levels of alanine aminotransferase(GPT),aspartate aminotransferase(GOT),total bile acid(TBA)and total bilirubin(TB)were measured.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the transcription of constitutive androstane receptor(CAR),the pregnane X receptor(PXR)and the UDP-glucurnosyltransferase 1a1(Ugt1a1)mRNA in the rats’liver.Western Blot(WB)was used to detect the protein expression of CAR,PXR and Ugt1a1.Results Compared with the blank control group,the cumulative bile excretion of KPG in the ANIT model group was significantly decreased(P<0.01),AUC0-∞(P<0.01)under the KP curve in the plasma of the ANIT model rats increased significantly and(P<0.01)was significantly prolonged.Compared with the ANIT model group,the cumulative bile excretion of KPG in the 100 and 50intervention groups were significantly increased
significantly andwas significantly shortened(P<0.01).Compared with the ANIT model group,the contents of TBA,GPT,GOT and TB in the serum of 100 and 50intervention groups decreased significantly(P<0.01).GPA significantly increased the transcription and protein expression of CAR,PXR and Ugt1a1 mRNA with positively dose-effect correlation.Conclusion Cholestasis rats reduces KPG bile excretion,and may cause changes in pharmacokinetic parameters of KP.GPA may improve the liver metabolism and bile excretion of KP in rats with cholestasis,which may be achieved by regulating the transcription of Ugt1a1 by CAR and PXR.
引文
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[10]陈浩,闵剑斌,黄小桃,等.京尼平苷酸在胆汁淤积型大鼠体内的药动学分析[J].中国实验方剂学杂志,2015,21(17):75-78.
[11]CULLEN J M,FAIOLA B,MELICH D H,et al.Acute alphanaphthylisothiocyanate induced liver toxicity in germ-free and conventional male rats[J].Toxicologic Pathology,2016,44(7):987-997.
[12]GOLBAR M,IZAWA T,BONDOC A,et al.Attenuation of alphanaphthylisothiocyanate(ANIT)-induced biliary fibrosis by depletion of hepatic macrophages in rats[J].Exp Toxicol Pathol,2017,69(4):221-230.
[13]姜玉才,黄爱文,彭永练,等.酮洛芬丹皮酚酯及其类似物的合成及抗炎活性研究[J].中国现代应用药学,2017,34(3):370-374.
[14]李敏,康晓芳,李婷婷,等.酮洛芬巴布膏治疗膝骨关节炎的临床研究[J].中国药物与临床,2016,16(9):1349-1351.
[15]JOO J,KIM Y W,WU Z,et al.Screening of non-steroidal antiinflammatory drugs for inhibitory effects on the activities of six UDP-glucuronosyltransferases(UGT1A1,1A3,1A4,1A6,1A9 and2B7)using LC-MS/MS[J].Biopharm Drug Dispos,2015,36(4):1-7.
[16]KAKIZAKI S,TAKIZAWA D,TOJIMA H,et al.Nuclear receptors CAR and PXR;herapeutic targets for cholestatic liver disease[J].Front Biosci,2011,16:2988-3005.
[2]CHEN H,HUANG X T,MIN J B,et al.Geniposidic acid protected against ANIT induced hepatotoxity and acute intrahepatic cholestasis,due to Fxr-mediated regulation of Bsep and Mrp2[J].Journal of Ethnopharmacology,2016,179:197-207.
[3]MANO Y,USUI T,KAMIMURA H.In vitro inhibitory effects of non-steroidal anti-inflammatory drugs on 4-methylumbelliferone glucuronidation in recombinant human UDP-glucuronosyltransferase1A9-potent inhibition by niflumic acid[J].Biopharm Drug Dispos2006,27:1-6.
[4]SHIMADA H,KOBAYASHI Y,TANAHASHI S,et al.Correlation between glucuronidation and covalent adducts formation with proteins of nonsteroidal anti-inflammatory drugs[J].European Journal of Pharmaceutical Sciences,2018,112:132-138.
[5]张芳芳,朱心强.PXR、CAR和PPAR对UGT基因转录的调节[J].国外医学(卫生学分册),2005,32(2):99-104.
[6]LEE S Y,LEE J Y,KIM Y M,et al.Expression of hepatic cytochrome P450s and UDP-glucuronosyltransferases in PXR and CAR double humanized mice treated with rifampicin[J].Toxicology Letters,2015,235:107-115.
[7]BOTHE MK,MEYER C,MUELLER U,et al.Characterization of a rat model of moderate liver dysfunction based on alphanaphthylisothiocyanate-induced cholestasis[J].J Toxicol Sci,2017,42(6):715-721.
[8]刘方乐,林朝展,赵威,等.消炎利胆片对ANIT致肝内胆汁淤积大鼠模型的干预作用[J].中药材,2016,39(4):898-901.
[9]闵剑斌,陈浩,石思,等.溪黄草水提物对ANIT诱发的实验性大鼠肝内胆汁淤积模型的干预作用[J].中药新药与临床药理,2015,26(5):635-639.
[10]陈浩,闵剑斌,黄小桃,等.京尼平苷酸在胆汁淤积型大鼠体内的药动学分析[J].中国实验方剂学杂志,2015,21(17):75-78.
[11]CULLEN J M,FAIOLA B,MELICH D H,et al.Acute alphanaphthylisothiocyanate induced liver toxicity in germ-free and conventional male rats[J].Toxicologic Pathology,2016,44(7):987-997.
[12]GOLBAR M,IZAWA T,BONDOC A,et al.Attenuation of alphanaphthylisothiocyanate(ANIT)-induced biliary fibrosis by depletion of hepatic macrophages in rats[J].Exp Toxicol Pathol,2017,69(4):221-230.
[13]姜玉才,黄爱文,彭永练,等.酮洛芬丹皮酚酯及其类似物的合成及抗炎活性研究[J].中国现代应用药学,2017,34(3):370-374.
[14]李敏,康晓芳,李婷婷,等.酮洛芬巴布膏治疗膝骨关节炎的临床研究[J].中国药物与临床,2016,16(9):1349-1351.
[15]JOO J,KIM Y W,WU Z,et al.Screening of non-steroidal antiinflammatory drugs for inhibitory effects on the activities of six UDP-glucuronosyltransferases(UGT1A1,1A3,1A4,1A6,1A9 and2B7)using LC-MS/MS[J].Biopharm Drug Dispos,2015,36(4):1-7.
[16]KAKIZAKI S,TAKIZAWA D,TOJIMA H,et al.Nuclear receptors CAR and PXR;herapeutic targets for cholestatic liver disease[J].Front Biosci,2011,16:2988-3005.