甘遂半夏汤中甘遂甘草反药组合加减应用对腹水大鼠CYP450酶的影响

Effect of Kansui Radix and Glycyrrhizae Radix et Rhizoma in Gansui Banxiatang on CYP450 Enzyme in Ascites Rats

目的:从细胞色素(CY)P450酶的角度,以甘遂半夏汤复方为载体,探究腹水病理模型下甘遂甘草配伍“反”与“不反”。方法:150只Wistar大鼠随机分为正常组、模型组、甘遂半夏汤全方组(5.68 g·kg^(-1)·d^(-1))、甘遂半夏汤全方去甘遂组(5.57 g·kg^(-1)·d^(-1))、甘遂半夏汤全方去甘草组(4.01 g·kg^(-1)·d^(-1))、甘遂半夏汤全方去甘草甘遂组(3.90 g·kg^(-1)·d^(-1)),其中甘遂1.1 g、法半夏9 g、白芍15 g、炙甘草16.7 g、蜂蜜15 g,每组25只,除正常组外,其余各组大鼠腹腔注射Walker-256细胞复制大鼠癌性腹水模型,正常组、模型组灌胃蒸馏水,其余给药组灌胃相应药液,连续给药7 d;用高效液相色谱法(HPLC)检测探针药物浓度,通过计算代谢率来测定CYP450酶活性;用实时荧光定量聚合酶链式反应(Real-time PCR)测定CYP450酶基因表达;用蛋白免疫印迹法(Western blot)测定CYP450酶蛋白表达。结果:与正常组比较,模型组CYP1A2、CYP2C9活性升高,CYP2E1、CYP2C19、CYP3A4活性降低,CYP2E1、CYP2C19、CYP2C9、CYP3A4 mRNA表达及CYP2E1、CYP3A4蛋白表达均降低(P<0.05)。与模型组比较,甘遂半夏汤全方组CYP2C9活性降低,CYP3A4 mRNA表达升高(P<0.01);去遂组CYP1A2活性降低、CYP2E1、CYP3A4活性升高,CYP1A2、CYP2E1、CYP2C19、CYP3A4 mRNA表达及CYP2E1、CYP3A4蛋白表达升高(P<0.05);去草组CYP2C9活性降低、CYP3A4活性升高,CYP2E1、CYP2C19 mRNA表达升高(P<0.05);双去组CYP2C9活性降低、CYP3A4活性升高、CYP3A4 mRNA表达升高(P<0.01)。与全方组比较,去遂组CYP2E1、CYP2C9、CYP3A4活性升高,CYP1A2、CYP2C19、CYP3A4 mRNA表达及CYP3A4蛋白表达升高(P<0.05);去草组CYP2C9、CYP3A4活性升高、CYP2C19mRNA表达升高,CYP3A4 mRNA表达降低(P<0.05);双去组CYP2C9、CYP3A4活性升高(P<0.01)。结论:4个给药组在CYP450酶的角度均表现出一定的药效,其中全方去甘遂组效果最佳,优于全方组。甘遂甘草反药组合在甘遂半夏汤全方中未表现出“相反”,但是有一定的减效作用,该实验条件下甘遂甘草配伍更接近于七情配伍中“相恶”的配伍关系。

Objective:To explore the relationship between Kansui Radix and Glycyrrhizae Radix et Rhizoma in the Gansui Banxiatang based on cytochrome P450(CYP450)enzyme and ascites rats.Method:A total of 150 Wistar rats were randomized into blank group(distilled water,ig),model group(distilled water,ig),Gansui Banxiatang group(5.68 g·kg^(-1)·d^(-1),ig),Gansui Banxiatang without Kansui Radix group(5.57 g·kg^(-1)·d^(-1),ig),Gansui Banxiatang without Glycyrrhizae Radix et Rhizoma group(4.01 g·kg^(-1)·d^(-1),ig),and Gansui Banxiatang without Kansui Radix And Glycyrrhizae Radix et Rhizoma group(3.90 g·kg^(-1)·d^(-1),ig),with 25 rats in each group.The Gansui Banxiatang was composed of 1.1 g Kansui Radix,9 g Pinelliae Rhizoma Praeparatum,15 g Paeoniae Radix Alba,16.7 g Glycyrrhizae Radix et Rhizoma praeparata cum melle,and 15 g honey.The rats,except for the blank group,were injected(ip)with Walker-256 cells to induce cancerous ascites.The administration lasted 7 days for each group.The concentration of probe drugs was detected by high performance liquid chromatography(HPLC)and based on the metabolic rates of the drugs,the CYP450 enzyme activity was yielded.The CYP450 enzyme gene expression was determined by Real-time PCR,and CYP450enzyme protein expression by Western blot.Result:Compared with the blank group,the model group showed high activity of CYP1A2 and CYP2C9,low activity of CYP2E1,CYP2C19,and CYP3A4,low mRNA expression of CYP2E1,CYP2C19,CYP2C9,and CYP3A4,and low protein expression of CYP2E1 and CYP3A4(P<0.05).Compared with the model group,Gansui Banxiatang group displayed low activity of CYP2C9 and high expression of CYP3A4 mRNA(P<0.01),and Gansui Banxiatang without Kansui Radix group demonstrated low activity of CYP1A2,high activity of CYP2E1 and CYP3A4,high mRNA expression of CYP1A2,CYP2E1,CYP2C19,and CYP3A4,and high protein expression of CYP2E1 and CYP3A4(P<0.05).In comparison with the model group,the Gansui Banxiatang without Glycyrrhizae Radix Et Rhizoma group showed low activity of CYP2C9,high activity of CYP3A4,and high mRNA expression of CYP2E1 and CYP2C19(P<0.05),and Gansui Banxiatang without Kansui Radix and Glycyrrhizae Radix Et Rhizoma group had low activity of CYP2C9,high activity of CYP3A4,and high CYP3A4 mRNA expression(P<0.01).The activity of CYP2E1,CYP2C9,and CYP3A4 was higher,and the mRNA expression of CYP1A2,CYP2C19,and CYP3A4 and CYP3A4 protein expression were higher in the Gansui Banxiatang without Kansui Radix group than in the Gansui Banxiatang group(P<0.05).The activity of CYP2C9 and CYP3A4,and CYP2C19 mRNA were higher and mRNA expression of CYP3A4 was lower in the Gansui Banxiatang without Glycyrrhizae Radix Et Rhizoma group than in the Gansui Banxiatang group(P<0.05).The activity of CYP2C9 and CYP3A4 in the Gansui Banxiatang without Kansui Radix and Glycyrrhizae Radix et Rhizoma group was higher than that in the Gansui Banxiatang group(P<0.01).Conclusion:The four prescriptions all showed certain efficacy in the perspective of CYP450 enzyme,among which Gansui Banxiatang without Kansui Radix had the best effect and was better than Gansui Banxiatang.The Kansui Radix and Glycyrrhizae Radix et Rhizoma did not show antagonism in Gansui Banxiatang,but reduced the effect.Thus,in this study,the medicinal pair may show mutual inhibition.