【摘要】：目的：艾滋病(AIDS)患者常用唐草片進(jìn)行輔助甚至替代治療,然而中藥成分復(fù)雜,其對抗病毒藥物的影響及其作用機(jī)制尚不清楚。為了減少潛在的藥物與藥物相互作用,臨床上推薦服用依非韋倫(EFV)1小時(shí)后再服唐草片。然而這種服用方式影響患者的依次性,本研究探討唐草片與EFV同時(shí)服用時(shí),唐草片對EFV藥代動(dòng)力學(xué)的影響及作用機(jī)制；且在此基礎(chǔ)上深入研究唐草片對CYP450酶的影響。方法：本研究首先研究不同給藥方式下唐草片對依非韋倫(EFV)藥動(dòng)學(xué)特性的影響,將18只SD大鼠隨機(jī)分為三組：單獨(dú)服用EFV、EFV和唐草片同時(shí)服用、以及服用EFV 60分鐘后服唐草片。用色譜串聯(lián)質(zhì)譜(LC-MS/MS)法檢測EFV的血藥濃度。然后采用定量PCR技術(shù)研究唐草片對EFV代謝酶CYP450酶CYP1A1、CYP1B1、CYP2B1、CYP2B2、CYP2E1和CYP3A1的調(diào)控。此外,我們采用差速離心法分離肝微粒體,SDS-PAGE分離其蛋白質(zhì),切取CYP450所在的三條電泳帶,用液相色譜串聯(lián)質(zhì)譜進(jìn)行鑒定。對鑒定的蛋白質(zhì)進(jìn)行生物信息學(xué)分析,Expasy blast搜索分析CYP2C11的特征多肽及限定片斷離子的m.z800,選擇CYP2C11中m.z為711.5的多肽進(jìn)行MRM定量分析,該多肽的離子對為：711.5→232.1、711.5→319.2、711.5→466.2和711.5→595.3；內(nèi)標(biāo)(IS)ESAT-6肽的離子對為：735.5→215.3、735.5→389.3、735.5→460.3和735.5→524.3。采用相對峰(分析物／內(nèi)標(biāo))面積進(jìn)行相對定量分析。結(jié)果：我們研究了唐草片對EFV藥代動(dòng)力學(xué)的影響及作用機(jī)制。具體從以下二方面展開：1) EFV以及唐草片單次給藥大鼠模型的藥代動(dòng)力學(xué)研究顯示：EFV單獨(dú)服用組、EFV和唐草片同時(shí)服用以及服用EFV 60分鐘后服唐草片組中EFV的曲線下面積(AUC0-∞)分別為17.97、10.06和14.32 mg.1.h。統(tǒng)計(jì)學(xué)分析顯示：與EFV單獨(dú)服用相比,EFV和唐草片同時(shí)服用時(shí)的曲線下面積(AUC0-∞)、半衰期(T1.2)以及保留時(shí)間(MRT)顯著下調(diào)(p0.05)。當(dāng)EFV和唐草片同時(shí)服用時(shí),唐草片顯著減低EFV的生物利用度和血漿半衰期,而在服用EFV60分鐘后服用唐草片,兩者的AUC0-∞接近,沒有統(tǒng)計(jì)學(xué)差異。2) 對EFV代謝相關(guān)代謝酶的定量PCR分析結(jié)果顯示：唐草片不影響EFV的6個(gè)CYP450代謝酶的mRNA水平。進(jìn)一步的蛋白質(zhì)組研究在肝微粒體中鑒定16個(gè)CYP450同工酶。為了分析唐草片對CYP450酶的影響,采用基于質(zhì)譜的多反應(yīng)監(jiān)測技術(shù)(MRM)進(jìn)行定量分析。發(fā)現(xiàn)：與EFV單獨(dú)給藥組相比,在EFV與唐草片合并用藥組中CYP2C11上調(diào)了1.6倍。結(jié)論：唐草片與EFV同時(shí)服用時(shí),降低EFV的生物利用度；體外酸性環(huán)境中唐草片提高EFV的溶解；唐草片不影響EFV直接相關(guān)代謝酶的表達(dá),唐草片提高CYP2C11的蛋白質(zhì)表達(dá)。但唐草片對EFV調(diào)控的具體機(jī)制有待深入研究。
[Abstract]:Objective: in (AIDS) patients, Tangcao tablets are commonly used to assist or even substitute therapy. However, the effect of Chinese traditional medicine on the antiviral drugs and its mechanism are not clear. To reduce potential drug-drug interactions, it is recommended to take efeveron (EFV) 1 hour later before taking Tangcao tablets. However, the effect of Tangcao tablet on the pharmacokinetics of EFV and the mechanism of its action were investigated, and the effect of Tangcao tablet on CYP450 enzyme was also studied. Methods: the effect of Tangcao tablets on the pharmacokinetic characteristics of (EFV) was studied. Eighteen Sprague-Dawley rats were randomly divided into three groups: taking both EFV and Tangcao tablets alone and taking Tangcao tablets 60 minutes after taking EFV. The plasma concentration of EFV was determined by tandem mass spectrometry (LC-MS/MS). Then the quantitative PCR technique was used to study the regulation of EFV metabolizing enzyme CYP450 enzyme CYP1A1C CYP2B1C CYP2B1CY CYP2B2CY CYP2E1 and CYP3A1 in Tangcao tablets. In addition, SDS-PAGE was used to isolate the protein from liver microsomes by differential centrifugation. Three electrophoresis bands of CYP450 were cut and identified by liquid chromatography tandem mass spectrometry (LC-MS). Bioinformatics analysis was carried out on the identified protein. The characteristic polypeptide of CYP2C11 and the specific fragment ion of CYP2C11 were analyzed by blast search. The polypeptides with m 路z = 711.5 in CYP2C11 were selected for MRM quantitative analysis. The ion pairs of this peptide were: 711.5 / 232.1711.5 / 319.2711.5 / 466.2 and 711.5 / 595.3respectively. The ion pairs of the internal standard (IS) ESAT-6 peptide are: 1: 735.5, 215.3735.5, 389.3735.5, 460.3, and 735.5, 524.3. Relative peak (analyte / internal standard) area was used for relative quantitative analysis. Results: the effect of Tangcao tablet on the pharmacokinetics of EFV and its mechanism were studied. The pharmacokinetic study of EFV and Tangcao tablets in rat model showed that the EFV in the group treated with EFV alone and Tangcao tablets were taken at the same time and after taking EFV for 60 minutes. The area under line (AUC0- 鈭，

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