补肾祛斑颗粒抑制细胞内黑色素合成机制

Mechanisms Involved in the Inhibition of Melanin Synthesis by Bushen Quban Granule

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 （BQG） for inhibiting the synthesis of intracellular melanin. Methods Twenty SPF grade female SD rats were di- vided into four groups by completely randomized method, i.e., the control group （fed with normal saline）, high, middle, and low dose BQG groups （administered with BQG at 4.8,2.4, 1.2 g/kg by gastrogavage, equivalent to 24, 12, and 6 times clinical doses, respectively, twice per day for 3 days in total）, 5 in each group. Drug containing serum was collected. Expressions of melanocortin 1 receptor （ MC1 R）, mi- crophthalmia-associated transcription factor （MITF）, tyrosinase （TYP）, tyrosinase-related protein 1 （TYRP1）, and tyrosinase-related protein 2 （TYRP2） at the mRNA level were detected by RT-PCR. Ex- pressions of phosphorylated-extracellular regulated MAP kinasel/2 （p-ERK）, TYP, TYRP1 and TYRP2 at the protein level were detected by Western blot. Intracellular melanin contents were determined by NaOH dissolving method. Activities of tyrosinase were determined by Dopa pigment method, and the cell viability was detected by MTT. Results Compared with the control group, expressions of MC1 R, MITF, TYP, TYRP1 and TYRP2 at the mRNA level were down-regulated （P 〈0.05）, and those of TYP, TYRP1 and TYRP2 at the protein level were also down-regulated （P 〈0.05）, intracellular contents of melanin and the activity of tyrosinase decreased （P 〈0.05）, but the level of p-ERK and the proliferation of cells increased in each medicated group （P 〈0.05）. When ERK was inhibited by its inhibitor PD98059, there was no sta- tistical difference in expressions of MC1R or MITF at the mRNA level among all medicated groups （P 〉 0.05）. Compared with the control group, mRNA expressions of TYP, TYRP1 and TYRP2 decreased in the high dose BQG group （P 〈0.05）, but with no significant difference in protein expressions of p-ERK, TYP, TYRP1 and TYRP2 （P 〉0.05）. There was no statistical difference in the content of melanin, the activity of TYP, or the proliferation of cells between the control group and the high dose BQG group （P 〉0.05）. Conclusion BQG could inhibit the synthesis of intracellular melanin through up-regulating p-ERK to inhibit the expression of tyrosinase and its related proteins.