论文部分内容阅读
目的观察补肾祛斑颗粒抑制细胞内黑色素合成的分子机制。方法 20只SPF级雌性SD大鼠完全随机分为生理盐水组(对照组)、补肾祛斑颗粒高、中、低剂量组,每组5只。高、中、低剂量组分别灌胃补肾祛斑颗粒4.8、2.4、1.2 g/kg(分别相当于临床剂量24、12、6倍),2次/日,共3日。提取含药血清。RT-PCR检测G蛋白偶联黑皮质素1受体(melanocortin 1 receptor,MC1R)、小眼畸形相关转录因子(microphthalmia-associated transcription factor,MITF)、酪氨酸酶(tyrosinase,TYP)、酪氨酸酶相关蛋白1(tyrosinase-related protein 1,TYRP1)及酪氨酸酶相关蛋白2(tyrosinase-related protein 2,TYRP2)mRNA水平的表达,Wertern-blot检测磷酸化细胞外调节蛋白激酶1/2(phosphorylated-extracellular regulated MAP kinase1/2,p-ERK)、TYP、TYRP1及TYRP2蛋白水平的表达,Na OH溶解法测定细胞内黑色素含量,多巴色素法测定细胞TYP活性,MTT检测细胞活性。结果与对照组比较,各给药组MC1R、MITF、TYP、TYRP1及TYRP2 mRNA水平表达降低(P<0.05),TYP、TYRP1、及TYRP2蛋白水平表达降低(P<0.05),细胞内黑色素含量及TYP活性降低(P<0.05),p-ERK及细胞增殖活力升高(P<0.05);PD98059抑制ERK后,各给药组MC1R、MITFmRNA水平表达差异无统计学意义(P>0.05)。与对照组比较,高剂量组TYP、TYRP1及TYRP2 mRNA表达降低(P<0.05),p-ERK、TYP、TYRP1及TYRP2蛋白表达差异无统计学意义(P>0.05),细胞内黑色素含量、TYP活性及细胞增殖活力差异无统计学意义(P>0.05)。结论补肾祛斑颗粒可能通过上调p-ERK来抑制细胞内TYP及其相关蛋白表达,抑制细胞内黑色素合成。
Objective To observe the molecular mechanism of Bushen Quban Granule in inhibiting intracellular melanin synthesis. Methods Twenty female Sprague-Dawley (SD) rats were randomly divided into normal saline group (control group), Bushen Quban granule group (high, medium and low dose group), with 5 rats in each group. The high, middle and low dose groups were administered with Bushen freckle particles 4.8, 2.4 and 1.2 g / kg respectively (corresponding to 24, 12 and 6 times of the clinical dose respectively) and 2 times / day for 3 days. Extraction of drug-containing serum. RT-PCR was used to detect G protein-coupled melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), tyrosinase (TYP) Tyrosinase-related protein 1 (TYRP1) and tyrosinase-related protein 2 (TYRP2) mRNA expression levels, Wertern-blot detection of phosphorylated extracellular regulated protein kinase 1/2 The expression of phosphorylated-extracellular regulated MAP kinase1 / 2, p-ERK, TYP, TYRP1 and TYRP2 protein were measured. The content of intracellular melanin was measured by Na-OH method. The activity of TYP was detected by the method of cholinergic assay. Results Compared with the control group, the expression of MC1R, MITF, TYP, TYRP1 and TYRP2 mRNA decreased (P <0.05), the expression of TYP, TYRP1 and TYRP2 decreased (P <0.05) (P <0.05). The expression of MC1R and MITF mRNA in each administration group was not significantly different after PD98059 was inhibited by ERK (P> 0.05). Compared with the control group, the expression of TYP, TYRP1 and TYRP2 mRNA in high-dose group was significantly lower than that in the control group (P <0.05), while there was no significant difference in the expression of p-ERK, TYP, TYRP1 and TYRP2 There was no significant difference in the activity and cell proliferation activity (P> 0.05). Conclusion Bushen freckle particles may inhibit the expression of TYP and its related proteins and inhibit intracellular melanin synthesis by up-regulating p-ERK.