在“三明治”培养大鼠原代肝细胞中研究利福平及联用丹参酮ⅡA对普伐他汀经BSEP转运的影响
|
摘要
目的以普伐他汀为BSEP底物,在"三明治"培养大鼠原代肝细胞模型上(sandwich-cultured rat hepatocytes,SCRH),研究利福平及联用丹参酮ⅡA对BSEP转运能力的影响。方法构建SCRH模型,利用MTT筛选给药剂量;建立普伐他汀的HPLC-MS/MS检测方法并进行方法学验证;考察利福平及联用丹参酮ⅡA对胆管内普伐他汀浓度的影响,并计算胆管外排指数(biliary excretion index,BEI)。结果成功构建了SCRH模型,并确定了利福平、丹参酮ⅡA、格列本脲及普伐他汀的合适给药剂量;建立了稳定可靠的普伐他汀HPLC-MS/MS检测方法;与空白组相比,利福平能够降低胆管内普伐他汀的浓度和普伐他汀的BEI,高浓度利福平的降低效果最明显(P <0. 01);与利福平高浓度组相比,联用丹参酮ⅡA后,胆管中普伐他汀的浓度以及普伐他汀的BEI增大,高浓度丹参酮ⅡA的增加效果最明显(P <0. 01)。结论在SCRH中,利福平能够抑制BSEP的转运能力,丹参酮ⅡA可以逆转利福平对BSEP转运能力的抑制作用。
Aim To elucidate the effect of rifampin and tanshinone ⅡA on BSEP transport capacity using pravastatin as the BSEP substrate in sandwich-cultured rat hepatocytes( SCRH). Methods SCRH model was established. The doses of drugs were determined by MTT. A HPLC-MS/MS method was developed and was conducted method validation to detect the concentration of pravastatin. The effect of rifampin and tanshinone ⅡA on the concentration of pravastatin in the bile duct was investigated. And the biliary excretion index( BEI) was calculated. Results The SCRH model was successfully developed. The appropriate doses of rifampin,tanshinone ⅡA,glibenclamide and pravastatin were determined. A stable and reliable HPLC-MS/MS method for the determination of pravastatin was established. Compared with blank control group, ri-fampin reduced the concentration of pravastatin in the bile duct and the BEI of pravastatin. The high concentration of rifampin caused the steepest downward trends( P < 0. 01). Compared with high concentration group of rifampin,the concentration of pravastatin in the bile duct and the BEI of pravastatin gradually increased after the combination of rifampin and tanshinone ⅡA,and the effect of high concentration of tanshinone ⅡA was the most significant( P < 0. 01). Conclusions Rifampin could inhibit the function of BSEP in SCRH.The combination of tanshinone ⅡA and rifampin could reverse the inhibitory effect of BSEP transport capacity caused by rifampin.
Aim To elucidate the effect of rifampin and tanshinone ⅡA on BSEP transport capacity using pravastatin as the BSEP substrate in sandwich-cultured rat hepatocytes( SCRH). Methods SCRH model was established. The doses of drugs were determined by MTT. A HPLC-MS/MS method was developed and was conducted method validation to detect the concentration of pravastatin. The effect of rifampin and tanshinone ⅡA on the concentration of pravastatin in the bile duct was investigated. And the biliary excretion index( BEI) was calculated. Results The SCRH model was successfully developed. The appropriate doses of rifampin,tanshinone ⅡA,glibenclamide and pravastatin were determined. A stable and reliable HPLC-MS/MS method for the determination of pravastatin was established. Compared with blank control group, ri-fampin reduced the concentration of pravastatin in the bile duct and the BEI of pravastatin. The high concentration of rifampin caused the steepest downward trends( P < 0. 01). Compared with high concentration group of rifampin,the concentration of pravastatin in the bile duct and the BEI of pravastatin gradually increased after the combination of rifampin and tanshinone ⅡA,and the effect of high concentration of tanshinone ⅡA was the most significant( P < 0. 01). Conclusions Rifampin could inhibit the function of BSEP in SCRH.The combination of tanshinone ⅡA and rifampin could reverse the inhibitory effect of BSEP transport capacity caused by rifampin.
引文
[1]赵晶,陈曼玉,李磊.胆酸盐外排泵研究的最新进展[J].中国药理学通报,2017,33(1):18-22.[1]Zhao J,Chen M Y,Li L.The research progress of bile acid efflux pump(BSEP)[J].Chin Pharmacol Bull,2017,33(1):18-22.
[2]徐永吉,李文楷,刘杰,陆远富.利福平对小鼠的肝毒性及胆酸代谢基因的影响[J].中国药理学通报,2016,32(6):841-5.[2]Xu Y J,Li W K,Liu J,Lu Y F.Effects of rifampicin on hepatotoxicity and genes related to bile acid metabolism in mice[J].Chin Pharmacol Bull,2016,32(6):841-5.
[3]郭晓红,刘立新,张东梅,张骞骞.丹参酮ⅡA对损伤肝细胞的保护作用及活化肝星状细胞的抑制作用[J].中国药物与临床,2009,9(1):13-7.[3]Guo X H,Liu L X,Zhang D M,Zhang Q Q.TanshinoneⅡA:protection against damage in cultured hepatocytes and inhibition of activated hepatic stellate cells[J].Chin Remedies Clin,2009,9(1):13-7.
[4]Wang W,Guan C,Sun X,et al.Tanshinone IIA protects against acetaminophen-induced hepatotoxicity via activating the Nrf2 pathway[J].Phytomedicine,2016,23(6):589-96.
[5]Carolina I,Vigo Michael J,Goedken Jose E,et al.Role of nuclear factor-erythroid 2-related factor 2(Nrf2)in the transcriptional regulation of brain ABC transporters during acute acetaminophen(APAP)intoxication in mice[J].Biochem Pharmacol,2015,94(3):203-11.
[6]梁瑞峰,宋献美,葛文静,等.小檗碱、巴马汀和药根碱在“三明治”培养大鼠原代肝细胞中的胆汁外排特征[J].中国药理学通报,2018,34(2):250-6.[6]Liang R F,Song X M,Ge W J,et al.Biliary excretion characteristics of berberine,palmatine and jateorhizine in sandwich-cultured rat hepatocytes[J].Chin Pharmacol Bull,2018,34(2):250-6.
[7]Cheng Y,Woolf T F,Gan J,et al.In vitro model systems to investigate bile salt export pump(BSEP)activity and drug interactions:a review[J].Chem Biol Interact,2016,255:23-30.
[8]Liu X,Le Cluyse E L,Brouwer K R,et al.Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration[J].Am J Physiol,1999,277(1):G12-21.
[9]Seglen P O.Preparation of isolated rat liver cells[J].Methods Cell Biol,1976,13:29-83.
[10]Polagani S R,Pilli N R,Gandu V.High performance liquid chromatography mass spectrometric method for the simultaneous quantification of pravastatin and aspirin in human plasma:Pharmacokinetic application[J].J Pharm Anal,2012,2(3):206-13.
[11]Bauer S,Mwinyi J,Stoeckle A,et al.Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection[J].JChromatogr B Analyt Technol Biomed Life Sci,2005,818(2):257-62.
[12]Chalasani N P,Hayashi P H,Bonkovsky H L,et al.ACG clinical guideline:the diagnosis and management of idiosyncratic drug-induced liver injury[J].Am J Gastroenterol,2014,109(7):950-67.
[13]Zheng H X,Huang Y,Frassetto L A,et al.Elucidating rifampin's inducing and inhibiting effects on glyburide pharmacokinetics and blood glucose in healthy volunteers:unmasking the differential effects of enzyme induction and transporter inhibition for a drug and its primary metabolite[J].Clin Pharmacol Ther,2009,85(1):78-85.
[14]Lengyel G,Veres Z,Tugyi R,et al.Modulation of sinusoidal and canalicular elimination of bilirubin-glucuronides by rifampicin and other cholestatic drugs in a sandwich culture of rat hepatocytes[J].Hepatol Res,2008,38(3):300-9.
[2]徐永吉,李文楷,刘杰,陆远富.利福平对小鼠的肝毒性及胆酸代谢基因的影响[J].中国药理学通报,2016,32(6):841-5.[2]Xu Y J,Li W K,Liu J,Lu Y F.Effects of rifampicin on hepatotoxicity and genes related to bile acid metabolism in mice[J].Chin Pharmacol Bull,2016,32(6):841-5.
[3]郭晓红,刘立新,张东梅,张骞骞.丹参酮ⅡA对损伤肝细胞的保护作用及活化肝星状细胞的抑制作用[J].中国药物与临床,2009,9(1):13-7.[3]Guo X H,Liu L X,Zhang D M,Zhang Q Q.TanshinoneⅡA:protection against damage in cultured hepatocytes and inhibition of activated hepatic stellate cells[J].Chin Remedies Clin,2009,9(1):13-7.
[4]Wang W,Guan C,Sun X,et al.Tanshinone IIA protects against acetaminophen-induced hepatotoxicity via activating the Nrf2 pathway[J].Phytomedicine,2016,23(6):589-96.
[5]Carolina I,Vigo Michael J,Goedken Jose E,et al.Role of nuclear factor-erythroid 2-related factor 2(Nrf2)in the transcriptional regulation of brain ABC transporters during acute acetaminophen(APAP)intoxication in mice[J].Biochem Pharmacol,2015,94(3):203-11.
[6]梁瑞峰,宋献美,葛文静,等.小檗碱、巴马汀和药根碱在“三明治”培养大鼠原代肝细胞中的胆汁外排特征[J].中国药理学通报,2018,34(2):250-6.[6]Liang R F,Song X M,Ge W J,et al.Biliary excretion characteristics of berberine,palmatine and jateorhizine in sandwich-cultured rat hepatocytes[J].Chin Pharmacol Bull,2018,34(2):250-6.
[7]Cheng Y,Woolf T F,Gan J,et al.In vitro model systems to investigate bile salt export pump(BSEP)activity and drug interactions:a review[J].Chem Biol Interact,2016,255:23-30.
[8]Liu X,Le Cluyse E L,Brouwer K R,et al.Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration[J].Am J Physiol,1999,277(1):G12-21.
[9]Seglen P O.Preparation of isolated rat liver cells[J].Methods Cell Biol,1976,13:29-83.
[10]Polagani S R,Pilli N R,Gandu V.High performance liquid chromatography mass spectrometric method for the simultaneous quantification of pravastatin and aspirin in human plasma:Pharmacokinetic application[J].J Pharm Anal,2012,2(3):206-13.
[11]Bauer S,Mwinyi J,Stoeckle A,et al.Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection[J].JChromatogr B Analyt Technol Biomed Life Sci,2005,818(2):257-62.
[12]Chalasani N P,Hayashi P H,Bonkovsky H L,et al.ACG clinical guideline:the diagnosis and management of idiosyncratic drug-induced liver injury[J].Am J Gastroenterol,2014,109(7):950-67.
[13]Zheng H X,Huang Y,Frassetto L A,et al.Elucidating rifampin's inducing and inhibiting effects on glyburide pharmacokinetics and blood glucose in healthy volunteers:unmasking the differential effects of enzyme induction and transporter inhibition for a drug and its primary metabolite[J].Clin Pharmacol Ther,2009,85(1):78-85.
[14]Lengyel G,Veres Z,Tugyi R,et al.Modulation of sinusoidal and canalicular elimination of bilirubin-glucuronides by rifampicin and other cholestatic drugs in a sandwich culture of rat hepatocytes[J].Hepatol Res,2008,38(3):300-9.