6种寒性中药对大鼠肝脏能量代谢的影响

目的:探讨6种寒性中药对大鼠肝脏能量代谢影响的共同规律.方法:6种寒性中药(苦参、栀子、黄柏、黄芩、黄连和龙胆)的水提取物分别6.0,7.0,8.4,6.0,7.0,4.0 g·kg~(-1)灌胃大鼠30 d,测定大鼠肝脏Na~+-K~+-ATP酶、Ca~(2+)-ATP酶,琥珀酸脱氢酶(SDH)的活性,肝糖原含量和肝脏解偶联蛋白2(UCP2)的mRNA表达水平.结果:6种寒性中药能显著降低Na~+-K~+-ATP酶、Ca~(2+)-ATP酶的活性;6种寒性中药均有使SDH活性降低的趋势,其中黄柏、黄芩、黄连和龙胆的降低有统计学意义;6种寒性中药有升高大鼠肝糖原含量的趋势,但是未达到显著水平;6种寒性中药有降低肝脏UCP2 mRNA水平的趋势,其中以苦参、黄柏、黄芩的降低有显著意义.结论:6种寒性中药可能通过降低肝脏线粒体SDH的活性从而减少ATP的生成,降低肝脏Na~+-K~+-ATP酶、Ca~(2+)-ATP酶的活性从而减少ATP的消耗,减少肝脏UCP2 mRNA的表达从而减少产热,起到调节肝脏能量代谢的作用.     

热性中药对大鼠肝脏能量代谢相关因子的影响

目的:探讨6种热性中药对大鼠肝脏能量代谢相关因子的影响.方法:6种热性中药(附子、干姜、高良姜、花椒、肉桂和吴茱萸)水提取物分别10.5,8.4,6.0,4.0,3.5,4.2 g·kg-1灌胃大鼠30 d,测定肝脏Na+-K+-ATP酶、Ca2+-ATP酶、琥珀酸脱氢酶(SDH)的活性、肝糖原含量和解偶联蛋白2(UCP2)的mRNA表达水平.结果:6种热性中药均能升高肝组织Na+-K+-ATP酶活性,其中高良姜、花椒、吴茱萸达到了显著水平(P＜0.05);6种热性中药有升高肝脏Ca2+-ATP酶活性的趋势,其中高良姜达到了显著水平(P＜0.05);6种热性中药均能升高大鼠肝脏SDH活性,除附子组外,其他5种热性中药均达到了极显著水平(P＜0.01);6种热性中药均能显著或极显著降低大鼠肝糖原的含量;6种热性中药对大鼠肝脏UCP2 mRNA表达量的影响不明显.结论:热性中药可能通过促进肝糖原的分解、增加SDH酶活性而产生更多ATP,通过增加Na+-K+-ATP酶和Ca2+-ATP酶活性而增加ATP的消耗,从而起到调节肝脏能量代谢的作用.     

吴茱萸有效部位对大鼠肝脏5种CYP450亚酶mRNA的影响

目的:制备吴茱萸挥发油、总黄酮及总生物碱等有效部位,对大鼠诱导后取肝脏,采用实时荧光定量PCR法考察大鼠肝脏5种CYP450亚酶mRNA的影响。方法:吴茱萸药材以水-乙醇提取,经挥发油提取器制备吴茱萸挥发油,药液浓缩、干燥后制备吴茱萸提取物,提取物经聚酰胺色谱柱分离得总黄酮,经酸提碱沉法制备总生物碱。将雄性SD大鼠分为5组,以生理盐水组为空白对照,连续灌胃诱导7 d后解剖取肝脏,采用实时荧光定量PCR法评价空白组、挥发油组、黄酮组、生物碱组以及提取物组肝脏CYP450亚酶的mRNA表达。结果:吴茱萸挥发油类成分对大鼠肝脏的CYP3A1、CYP2B1的mRNA表达均具有抑制作用,黄酮类成分对CYP2B1具有非常显著抑制作用,生物碱类成分对CYP2E1、CYP3A1具有抑制作用,吴茱萸总提取物对CYP2B1具有抑制作用。结论:从基因水平考查了吴茱萸对大鼠肝脏5种CYP450亚酶的影响,提示在吴茱萸的临床使用中应注意代谢性相互作用和药物不良反应。     

6味热性中药对大鼠骨骼肌能量代谢相关因子的影响

目的:探讨6味热性中药对于大鼠骨骼肌能量代谢相关因子的影响。方法:6味热性中药(制附子、干姜、高良姜、花椒、肉桂和吴茱萸)水提取物分别10.5、8.4、6.0、4.0、3.5、4.2g/kg灌胃大鼠30d,测定大鼠骨骼肌Na+-K+-ATP酶、Ca2+-ATP酶、琥珀酸脱氢酶(SDH)的活性、肌糖原含量和解偶联蛋白3(UCP3)mRNA表达水平。结果:6味热性中药能显著升高骨骼肌Na+-K+-ATP酶、Ca2+-ATP酶、SDH活性;除肉桂外,均能明显降低肌糖元含量;除高良姜和肉桂外,均能明显降低UCP3mRNA的表达量。结论:热性中药可能通过促进了肌糖原的分解、增加SDH酶的活性、减少UCP3mRNA表达减少骨骼肌产热从而产生更多ATP,通过增加Na+-K+-ATP酶和Ca2+-ATP酶活性而增加ATP的消耗,起到调节骨骼肌能量代谢的作用。     

CYP450酶与中药代谢相互作用及酶活性测定的研究进展

药物对于人体属于外源性化合物,进入人体后将被许多酶所代谢。CYP450酶作为主要的代谢酶,且是重要的药物I相代谢酶,人体约75%的药物代谢是通过CYP450酶进行,而且CYP450酶是引起中药-中药(药对)、中药-西药等联合用药时药物相互作用产生的关键因素。为明确代谢酶与中药代谢的相互作用关系,普遍以酶活性作为评价指标,即以CYP450酶活性的增强和降低来推断中药有效成分及提取物等对酶的诱导或抑制作用。目前,代谢酶活性测定的常用方法是Cocktail探针药物法,其中探针底物的选择是关键。这对研究药物在机体内的吸收、分布、代谢及排泄(ADME)过程有着重大意义。该文就中药、中药有效成分、中药提取物及Cocktail探针底物等与CYP450酶的相互作用进行了综述,以期对将来的相关研究有借鉴和指导意义。     

四种抗癌中药提取物对大鼠肝CYP3A酶活性及mRNA表达的影响

目的研究4种抗癌中药提取物对大鼠肝脏细胞色素P450 3A(CYP3A)酶在酶活性及mRNA水平的调节作用.方法采用紫外分光光度法测定大鼠肝微粒体CYP450含量及CYP3A同工酶--红霉素N-脱甲基酶(ERD)的活性;采用反转录PCR(RT-PCR)技术检测CYP3A1、CYP3A2 mRNA的表达.结果莪术、苍术、白术的提取物均可明显增加CYP450蛋白含量及CYP3A酶活性,而茯苓提取物无明显作用.在mRNA水平上,莪术、苍术、白术的提取物均能明显诱导CYP3A1的基因表达,茯苓提取物无明显作用;对于CYP3A2的基因表达,莪术和苍术的提取物均有明显的诱导作用,而白术和茯苓的提取物则有显著的抑制作用.结论莪术、苍术、白术的提取物对CYP3A酶的调控均发生在酶活性水平和转录水平,而茯苓提取物对CYP3A酶仅存在转录水平的调控.4种抗癌中药提取物对CYP3A酶的诱导或抑制可能会影响其他药物的体内代谢.     

热毒宁注射液对大鼠肝脏CYP450酶的影响

研究热毒宁注射液对大鼠肝脏CYP450酶酶活性及其mRNA表达水平的影响。将SD大鼠腹腔注射7 d后,处死,制备大鼠的肝微粒体,与探针药物共孵育,利用LC/MS法测定各同工酶特异性底物在大鼠肝微粒的代谢产物的生成量,间接反映各同工酶的活性。应用荧光定量PCR技术考察热毒宁注射液对大鼠肝脏CYP450酶mRNA表达水平的影响。结果显示,酶活性方面,热毒宁注射液与空白组比较可抑制CYP2B1,2C12,2C13,2D2的酶活性,诱导CYP3A1的酶活性,对CYP1A2酶活性无明显影响。mRNA表达方面,热毒宁注射液对CYP2B1,3A1,1A2,2C11,2D1,2E1 mRNA的表达具有诱导作用。通过对上述结果分析,可以看出热毒宁注射液在酶活性和mRNA水平均对CYP3A1具有显著的诱导作用,与大环内酯类抗生素合用时可能会出现不良反应;对CYP2B1,2C12,2C13,2D2的酶活性均具有抑制作用,与其他药物联合用药时要充分考虑可能出现的药物相互作用或潜在风险。     

基于肝脏药物代谢酶研究附子配伍干地黄的减毒机制

测定大鼠肝微粒体中CYP1A2和CYP3A4酶活性,并从蛋白和转录水平上阐明附子配伍干地黄对CYP450酶蛋白和基因表达的影响,探索附子配伍干地黄对其减毒的分子机制。采用"Cocktail"探针药物法进行CYP450酶活性测定;采用CO还原差示法测定肝微粒体中CYP450酶含量并采用RT-PCR测定大鼠肝脏CYP1A2和CYP3A4 mRNA表达量。结果显示,与空白组比较,附子干地黄组CYP1A2和CYP3A4酶活性及mRNA表达显著升高(P0.05);附子组CYP450含量显著降低(P0.01);干地黄组、附子干地黄组CYP450含量显著升高(P0.05)。因此,附子配伍干地黄能够诱导CYP1A2和CYP3A4酶活性,增加CYP450酶含量,加快附子毒性成分的代谢,达到附子减毒作用。     

板蓝根颗粒、茵栀黄颗粒和复方黄连素片对药物代谢酶CYP_3A_4活性的影响

目的:探讨黄连上清丸、护肝片、牛黄解毒丸对CYP_3A_4活性的影响。方法:利用体外肝微粒体培育体系,以咪达唑仑作为探针底物,研究板蓝根颗粒、茵栀黄颗粒和复方黄连素片对人肝微粒体细胞色素P_(450 3)A_4(CYP_3A_4)活性的影响。结果:与空白组对比,茵栀黄颗粒对CYP_3A_4活性表现为诱导作用,板蓝根颗粒和复方黄连素片对CYP_3A_4活性表现为抑制作用。结论:在与临床上经CYP_3A_4代谢的药物联合应用时,应警惕板蓝根颗粒、茵栀黄颗粒和复方黄连素片潜在药物之间的相互作用。     

临床用量的复方黄黛片及大剂量雄黄对大鼠肝脏主要药物代谢酶的影响

探究临床用量的复方黄黛片及大剂量君药雄黄对大鼠肝脏主要药物代谢酶(CYP450)酶活性的影响及在mRNA水平的调控作用。以Wistar大鼠为研究对象,受试药物给予14 d后,采用Cocktail体外孵育法结合HPLC-MS/MS技术对大鼠肝中CYP1A2,CYP2B,CYP3A,CYP2C各亚酶的活性进行测定,并利用实时荧光定量PCR技术对上述亚型的mRNA水平表达进行检测。结果显示,复方黄黛片显著诱导CYP1A2,CYP2B酶活性,抑制CYP3A酶活性,结果与mRNA的表达具有一致性,但其单味药却呈现出弱于其甚至是相反的现象;不同剂量的雄黄组之间对酶活性及mRNA的表达结果亦呈现非常显著的不一致性。这些现象可能与复方黄黛片的配伍增效或减毒有关。CYP450酶的研究结果亦可以提示,复方黄黛片与其他药物合用时有可能产生药物相互作用。     

应用Cocktail探针药物法分析中药对CYP450酶活性影响的研究进展

Cocktail探针药物法作为一种快速、准确、高通量的评价CYP450酶活性的研究方法广泛应用于药物研发早期筛选、药物代谢途径及机制研究、药物相互作用研究、指导临床合理用药、上市药物再评价及协助肝肾病理研究等诸多领域。该文对Cocktail探针药物法的特点做简要说明,着重介绍其在中医药领域内的应用进展,从中药活性成分的代谢机制研究,炮制过程,方剂配伍对CYP450的影响,揭示中成药CYP450代谢特点规律,民族药学应用情况,中药保肝作用及中医证候模型研究等,并总结归纳现有研究成果,比较其在中西医药领域内应用的异同点,为该领域的深入研究工作提供参考。     

基于iTRAQ技术结合2D-LC-MS/MS研究黄连对CYP450同工酶表达的影响

黄连是临床常用药材,在中医药中被广泛应用,具有清热燥湿、泻火解毒之功,中药成分比较复杂,细胞色素P450(cytochrome P450,CYP450)同工酶是药物的主要代谢酶,研究黄连对CYP450酶的影响具有很重要的临床意义。实验选用大鼠肝脏组织,差速离心法分离肝微粒体,BCA(bicinchoninic acid)蛋白定量,应用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification,iTRAQ)技术结合2D-LC-MS/MS筛选差异蛋白。黄连一共鉴定出30个CYP450同工酶,7个表达上调,8个表达下调,15个没有发生变化。iTRAQ技术结合2D-LC-MS/MS可以全面研究CYP450酶,但黄连有必要进一步在体外水平对其进行评价,预防潜在的临床药物相互作用。     

托法常用辛、温、热药中活性成分的抗炎研究进展

托法是中医外科经典的治法。现代药理研究发现托法中常用的辛、温、热药,如黄芪、肉桂、姜黄,具有抗炎效果。黄芪既可能够抑制病毒,治疗感染性炎症;亦可抑制白介素-1(IL-1)等,治疗非感染性炎症。肉桂、姜黄二药既可通过抑制或杀灭细菌、病毒、乃至真菌,治疗感染性炎症;亦可通过抑制核因子-κB(NF-κB)、丝裂原活化蛋白激酶(MAPK)等信号通路降低IL-1、肿瘤坏死因子α(TNF-α)、前列腺素E2(PGE2)等炎症因子水平,治疗非感染性炎症。现综述三种托法代表药(黄芪、肉桂、姜黄)抗炎研究的最新成果,分析存在的问题并提出相应的解决对策,为进一步研究与应用提供理论依据。     

平性活血化瘀药对寒、热2种血瘀证双向适用的实验研究Ⅱ

目的:通过观察10味平性活血化瘀中药对瘀热互结证和寒凝血瘀证大鼠血液流变学指标的作用特点,进一步探讨平性药“双向适用,条件显性”的药性特征.方法:分别采用角叉菜胶和干酵母联合造模的方法建市瘀热互结证大鼠模型和采用伞身冷冻法建立寒凝血瘀证大鼠模型,并用10味平性药、5味热性药、5味寒性药进行干预,观察各组大鼠全血黏度、血浆黏度、红细胞压积等指标的变化情况,对比分析平性药的作用特点.结果:方差分析结果表明:10味平性药中,蒲黄、肿节风、苏木等6味药物均能明显改善寒、热2种血瘀证大鼠血液流变学指标;5味寒性药中丹参、益母草、大黄均能明显改善瘀热互结证大鼠血液流变学指标(P＜0.01或P＜0.05),仅丹参能够显著改善寒凝血瘀证大鼠血液流变学指标(P＜0.05);5味热性药中红花、三七能够显著改善瘀热瓦结证大鼠血液流变学指标,5味药物均能显著改善寒凝血瘀证大鼠血液流变学指标(P＜0.01).血液平均高切黏度分析表明:在瘀热互结证中平性药与寒性药作用特点类似,在降低伞血黏度方面优于热性药;在寒凝血瘀证中平性药与热性药类似,在降低全血黏度方面优于寒性药.结论:在改善瘀热互结证大鼠血液流变学方面,平性药表现出类似寒性药的作用特征,在改善寒凝血瘀证大鼠血液流变学方面,平性药显示出类似热性药的作用特征,在一定程度上表明平性药在寒证和热证不同的条件下,显示出了双向适用的药性特征.     

Effects of unprocessed versus vinegar-processed Schisandra chinensis on the activity and mRNA expression of CYP1A2, CYP2E1 and CYP3A4 enzymes in rats

Ethnopharmacological relevance

Schisandra chinensis (SC) is a well-known traditional Chinese herbal medicine that has been used in clinical practices for thousands of years. However, the differences between the effects of unprocessed and vinegar-processed Schisandra chinensis (VSC) on cytochrome P450 (CYP450) activities are poorly understood.

Aim of the study

To evaluate the differences between processed and unprocessed SC on the metabolism of CYP1A2, CYP2E1 and CYP3A4 substrates in rats using a cocktail method based on a developed and validated HPLC method. We also investigate the influence of processing on the levels of CYP mRNA.

Materials and methods

Three probe substrates (theophylline, dapsone and chlorzoxazone) were delivered simultaneously into rats treated with single or multiple doses of processed or unprocessed SC extract. The plasma concentrations of the three probes were profiled by HPLC, and their corresponding pharmacokinetic parameters were calculated. Real-time RT-PCR was performed to determine the effects of processed and unprocessed SC on the mRNA expression of CYP1A2, CYP2E1 and CYP3A4 in the liver.

Results

Treatment with single or multiple doses of either extract of SC induced CYP3A4 enzyme activity and inhibited CYP1A2 enzyme activity in rats. Furthermore, the inhibitory effect of SC was more potent after vinegar processing than without vinegar processing. CYP2E1 enzyme activity was induced after treatment with a single dose but was inhibited after multiple doses. The mRNA expression results were in accordance with the pharmacokinetic results.

Conclusions

These results provide useful scientific data for the safe clinical application of either extract of SC in combination with other drugs, which should lack the side effects induced by other herb–drug interactions.     

三七总皂苷对大鼠肝脏药物代谢酶酶活性、mRNA及蛋白表达的影响

研究三七总皂苷对大鼠肝脏药物代谢酶酶活性、mRNA及蛋白表达的影响。将Wistar雄性大鼠随机分为9组,给予受试药后,提取肝脏微粒体,肝脏总RNA和总蛋白,分别采用底物探针法、荧光定量PCR技术和Western blot,检测三七总皂苷对肝脏药物代谢酶相关亚型酶活性、mRNA和蛋白质表达3个水平的调节作用。实验的结果是三七总皂苷能显著诱导CYP1A2和CYP2E1酶活性和mRNA表达,同时对CYP2E1的蛋白表达水平也有显著诱导作用;三七总皂苷显著诱导CYP3A mRNA表达,但对CYP3A酶活性水平无明显的影响;三七总皂苷对CYP1A1和CYP2B mRNA表达和酶活性均无明显影响。三七总皂苷对不同的P450亚型的调节作用具有选择性,主要的调节亚型是CYP1A2和CYP2E1,临床应用时,尤其是与CYP1A2和CYP2E1代谢有关药物合用时,应充分考虑到可能产生的药物相互作用以避免潜在的不良反应和毒副作用,同时对CYP2E1的诱导作用是否与人参皂苷清除自由基作用有关值得进一步研究。     

Effects of hydroxysafflor yellow A on the activity and mRNA expression of four CYP isozymes in rats

Ethnopharmacological relevance

In traditional therapy with Chinese medicine, hydroxysafflor yellow A (HSYA), a main active component isolated from the dried flower of Carthamus tinctorius L., is the principal efficiency ingredient of Safflor Yellow Injection. Now HSYA has been demonstrated to have good pharmacological activities of antioxidation, myocardial and cerebral protective and neuroprotective effects. The purpose of this study was to find out whether HSYA influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D4 and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied.

Materials and methods

A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), dextromethorphan (20 mg/kg) and midazolam (10 mg/kg), was given as oral administration to rats treated with short or long period of intravenous HSYA via the caudal vein. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC–MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time RT-PCR was performed to determine the effect of HSYA on the mRNA expression of CYP1A2, CYP2C11, CYP2D4 and CYP3A1 in rat liver.

Results

HSYA had significant inhibition effects on CYP1A2 and CYP2C11 in rats as oriented from the pharmacokinetic profiles of the probe drugs. Furthermore, HSYA had no effects on rat CYP2D4. However, CYP3A1 enzyme activity was induced by HSYA. The mRNA expression results were in accordance with the pharmacokinetic results.

Conclusions

HSYA can either inhibit or induce activities of CYP1A2, CYP2C11 and CYP3A1. Therefore, co-administration of some CYP substrates with HSYA may need dose adjustment to avoid an undesirable herb–drug interaction.     

Evaluation of protective effects of Chi-Zhi-Huang decoction on Phase I drug metabolism of liver injured rats by cocktail probe drugs

AIM OF THE STUDY: Chi-Zhi-Huang decoction (PGR) is one of the traditional Chinese medicine (TCM) preparations with unique effect on withdrawing jaundice and has been used to treat icteric patients in China for many years. In this research, we aim at to evaluate the potential activity of PGR in restoring hepatic drug metabolism in a damaged liver. MATERIALS AND METHODS: A cocktail approach with caffeine (10mg/kg), dapsone (10mg/kg) and chlorzoxazone (20mg/kg) respectively as probe drug of cytochrome P450 (CYP) isoform of CYP 1A2, 3A4 and 2E1 was used to evaluate its possible effects on Phase I oxidative metabolism. Pretreated with three dosages of PGR water extract (0.75, 1.5 and 3g/kg, po) for 5 days, male Wistar rats (220-240 g) were intoxicated by phenylisothiocyanate (PITC, 100mg/kg, po) 24h before probes intravenous injection. The pharmacokinetics of the probes in the blood was determined simultaneously by HPLC, and their non-compartmental parameters were used to evaluate the metabolic difference among the groups. Moreover, the levels of liver enzymes (ALT, AST, ALP) and bilirubins were also measured for insight of liver function. RESULTS: The findings in this study suggest that PGR induces CYP 3A4, does not have much effect on CYP 2E1, and inhibits CYP 1A2 at high dosage. CONCLUSION: The current pharmacokinetic approach allowed the protective effects of PGR on oxidative drug metabolism in damaged liver to be systemically examined and will certainly help in the explanation of synergistic effect of the composites formula.