何首乌与制何首乌对人尿源性干细胞的作用差异及机制研究

doi:10.19803/j.1672-8629.2021.03.05

摘要/Abstract

摘要： 目的以人尿源性干细胞（human urine-derived stem cells, hUSCs）为研究对象,探究何首乌（Polygoni Multiflori Radix, PMR）与制何首乌（Polygoni Multiflori Radix Praeparata, PMRP）对人成体干细胞的作用差异及机制。方法利用高效液相色谱法对何首乌水提物（PMR）与制何首乌水提物（PMRP）中主要活性成分大黄素（emodin,EM）和二苯乙烯苷（2,3,5,4'-tetrahydroxy-stilbene-2-O-β-D-glycoside, TSG）进行定量分析;MTT法研究PMR及PMRP对hUSCs活力的影响;根据EM和TSG在2种水提物中的含量和比例,比较单成分及2种成分按比例联用对hUSCs活力的影响;流式细胞术检测hUSCs细胞周期和细胞凋亡,Wes-tern blot检测细胞周期相关蛋白的表达。结果 PMR中TSG和EM的含量分别为77.68、0.53 mg/g,PMRP中TSG和EM的含量分别为29.37、0.36 mg/g;0.5、1.0 mg/mL浓度的PMR对细胞有明显毒性,相同剂量下,PMRP促进细胞增殖;0.5~4 μmol/L EM促细胞增殖,320、640 μmol/L TSG抑制细胞活力;按1∶160（约为PMR比例）和1∶80（约为PMRP比例）联用后对细胞活力的抑制作用较单用显著增加;PMR组及1∶160联用组细胞周期出现G0/G1期阻滞,且细胞凋亡率较PMRP组及1∶80联用组高;Western blot结果显示,PMR组中细胞p21水平上调;CDK1、CDK2及Rb磷酸化水平降低,而PMRP组呈相反趋势。结论相同条件下,PMR较PMRP表现明显细胞毒作用,而PMRP表现促细胞活力作用,说明EM与TSG比例的改变可能是何首乌炮制减毒的机制之一。

关键词: 人尿源性干细胞, 何首乌, 制何首乌, 二苯乙烯苷, 大黄素

Abstract: Objective Human urine-derived stem cells (hUSCs) were used to explore the differentiation effect and mechanism of Polygoni Multiflori Radix(PMR) and Polygoni Multiflori Radix Praeparata(PMRP) on human adult stem cells. Methods Quantitative analysis of Emodin (EM) and 2,3,5,4'-tetrahydroxy-stilbene-2-O-β-D-glycoside (TSG) in the aqueous extract of PMR and PMRP was carried out by high Performance Liquid Chromatography; MTT assay was used to study the influence of PMR and PMRP on hUSCs viability; according to the content and proportion of EM and TSG in the two aqueous extracts, the effects of single component and 2 components combined in proportion on hUSCs viability were compared; cell cycle distribution and cell apoptosis were analyzed by flow cytometry, and cell cycle-related proteins were detected by Western blot. Results The contents of TSG and EM in PMR were 77.68 and 0.53 mg/g, and were 29.37 and 0.36 mg/g in PMRP. PMR had obvious toxicity to cells at 0.5 and 1.0 mg/mL, while PMRP had the effect of promoting cell proliferation at the same dose. EM promoted cell proliferation at 0.5~4 μmol/L,and TSG inhibited cell activity at 320 and 640 μmol/L. The inhibitory effect of the combination of EM and TSG on cell viability was significantly increased compared with the corresponding concentration alone. The G0/G1 cell cycle arrest occurred in the PMR group and the 1∶160 combined group, and the cell apoptosis rates were higher than the PMRP group and the 1∶80 combined group. Western blot results showed that p21 level was up-regulated in the PMR group. CDK1, CDK2 and Rb phosphorylation level were down-regulated in the PMR group, while up-regulated in the PMRP group. Conclusion Under the same conditions, PMR showed obvious cytotoxic effect compared with PMRP, while PMRP showed the effect of promoting cell activity, indicating that the change of EM and TSG proportion may be one of the mechanisms of the PMR processed attenuating toxicity.

Key words: human urine-derived stem cells, polygoni multiflori radix, polygoni multiflori radix praeparata, 2,3,5,4'-tetrahydroxy-stilbene-2-O-β-D-glycoside, Emodin

中图分类号:

R954.3R994.11

谯英固, 周明, 胡英还, 王呈谕, 刘晓璇, 沈亮亮, 孙震晓. 何首乌与制何首乌对人尿源性干细胞的作用差异及机制研究[J]. 中国药物警戒, 2021, 18(3): 228-234.

QIAO Yinggu, ZHOU Ming, HU Yinghuan, WANG Chengyu, LIU Xiaoxuan, SHEN Liangliang, SUN Zhenxiao. Study on the Differential Effects and Mechanism of Polygoni Multiflori Radix and Polygoni Multiflori Radix Praeparata on Human Urine-derived Stem Cells[J]. Chinese Journal of Pharmacovigilance, 2021, 18(3): 228-234.

参考文献

[1] Chinese Pharmacopoeia Commission.Pharmacopoeia of the People's Republic of China (Part 1) (中华人民共和国药典(一部))[M]. Beijing: China Medical Science Press, 2020:183-184.
[2] Zhang Y, Mcneill E, Tian H, et al.Urine derived cells are a potential source for urological tissue reconstruction[J]. J Urol, 2008, 180(5) : 2226-2233.
[3] King NM, Perrin J. Ethical issues in stem cell research and therapy[J/OL]. Stem Cell Res Ther, 2014, 5(4). (2014-07-07)[2020-12-20]. https://pubmed.ncbi.nlm.nih.gov/25157428/.
[4] Falzarano MS, Ferlini A. Urinary stem cells as tools to study genetic disease: overview of the literature[J/OL]. J Clin Med, 2019, 8(5). (2019-05-08)[2020-12-20]. https://pubmed.ncbi.nlm.nih.gov/31071994/.
[5] Gong YS, Zhang YN, Huang W, et al.Research development on chemical compositions of polygonum multiflorum related to efficacy and toxicity[J]. Chinese Journal of Pharmacovigilance(中国药物警戒), 2012, 9(8) : 472-475.
[6] Gao SH, Su ZZ, Xiao X F.Progress in study on chemical composition and pharmacological action of polygonum multiflorum thunb[J]. Journal of Shanxi College of Traditional Chinese Medicine(山西中医学院学报), 2012, 13(2) : 74-77.
[7] Yan SL.Studies on chemical constituents of radix polygoni multiflori praeparata[D]. Tianjin: Tianjin University, 2014.
[8] Chen QT, Zhuo LH, Xu W, et al.Content changes of 5 components in polygohum muhiflorum during processing[J]. Chinese Journal of Experimental Traditional Medical Formulae(中国实验方剂学杂志), 2012, 18(5) : 66-71.
[9] Liu ZL.Changes in chemical components of root of polygonum multiflorum thunb and how maillard reaction involves during the heating process[D]. Beijing: China Academy of Chinese Medical Sciences, 2009.
[10] Shi GB.Study on the influence of concoction over active ingredients in polygonum multiflorum[J]. Chinese Journal of Hospital Pharmacy(中国医院药学杂志), 2003(2) : 33-35.
[11] Liu SQ, Wang L, Yue L.Influence of high pressure processing on contents of active ingredients from polygoni multiflori radix[J]. Chinese Journal of Experimental Traditional Medical Formulae(中国实验方剂学杂志), 2013, 19(21) : 37-40.
[12] Zhou M, Shen LL, Qiao YG, et al.Inducing differentiation of human urine-derived stem cells into hepatocyte-like cells by coculturing with human hepatocyte L02 cells[J]. J Cell Biochem, 2020, 121(1) : 566-573.
[13] Li DK, Chen J, Ge ZZ, et al. Hepatotoxicity in rats induced by aqueous extract of polygoni multiflori radix, root of polygonum multiflorum related to the activity inhibition of CYP1A2 or CYP2E1[J/OL]. Evid Based Complement Alternat Med, 2017. (2017-05-24)[2020-12-20]. https://www.hindawi.com/journals/ecam/2017/9456785/.
[14] Wang CY, Liu XX, Li YQ, et al.Toxicity of polygoni multiflori radix, polygoni cuspidati rhizoma et radix and rhei radix et rhizoma in hepaRG cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis(癌变·畸变·突变), 2020, 32(3) : 215-220.
[15] Moser J, Miller I, Carter D, et al.Control of the restriction point by rb and p21[J]. Proc Natl Acad Sci U S A, 2018, 115(35) : 8219-8227.
[16] Xia XH, Yuan YY, Liu M.The assessment of the chronic hepato-toxicity induced by polygoni multiflori radix in rats: A pilot study by using untargeted metabolomics method[J]. J Ethnopharmacol, 2017, 203 : 182-190.
[17] Lei X, Chen J, Ren J, et al. Liver damage associated with polygonum multiflorum thunb.: a systematic review of case reports and case series[J/OL]. Evid Based Complement Alternat Med, 2015. (2015-01-12)[2020-12-20]. https://www.hindawi.com/journals/ecam/2015/459749/.
[18] Zhang RC, Zhang C, SUN ZX, et al.Damage effect of polygonum multiflorum fractions on human normal liver cells L02 and liver cancer cells HepG2[J]. China Journal of Chinese Materia Medica(中国中药杂志), 2012, 37(12) : 1830-1835.
[19] Quan ZY, Sun ZX.Study on hepatotoxicity of aqueous extract of polygoni multiflori radix and its main components based on zebrafish[J]. Chinese Journal of Pharmacovigilance(中国药物警戒), 2018, 15(1) : 1-5.
[20] Martignoni M, Groothuis GM, de Kanter R. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction[J]. Expert Opin Drug Metab Toxicol, 2006, 2(6) : 875-894.
[21] Kawauchi S, Nakamura T, Horibe S, et al.Down-regulation of hepatic CYP3A1 expression in a rat model of indomethacin-induced small intestinal ulcers[J]. Biopharm Drug Dispos, 2016, 37(9) : 522-532.
[22] Ma KF, Zhang XG, Jia HY.CYP1A2 polymorphism in Chinese patients with acute liver injury induced by polygonum multiflorum[J]. Genet Mol Res, 2014, 13(3) : 5637-5643.
[23] Li C, Rao T, Chen X, et al.HLA-B*35:01 allele is a potential biomarker for predicting polygonum multiflorum-induced liver injury in humans[J]. Hepatology, 2019, 70(1) : 346-357.
[24] Bharadwaj S, Liu G, Shi Y, et al.Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology[J]. Stem Cells, 2013, 31(9) : 1840-1856.
[25] Qin D, Long T, Deng J, et al. Urine-derived stem cells for potential use in bladder repair[J/OL]. Stem Cell Res Ther, 2014. (2014-05-28)[2020-12-20]. https://stemcellres.biomedcentral.com/articles/10. 1186/scrt458.
[26] Slaats GG, Braun F, Hoehne M, et al. Urine-derived cells: a promising diagnostic tool in fabry disease patients[J/OL]. Sci Rep, 2018. (2018-07-23)[2020-12-20]. https://www.nature. com/articles/s41598-018-29240-w.
[27] Zhang QJ, Lin X, Li JJ, et al.Application of urine cells in drug intervention for spinal muscular atrophy[J]. Exp Ther Med, 2017, 14(3) : 1993-1998.
[28] Zhang QJ, He J, Ni W, et al.Noninvasive urine-derived cell lines derived from neurological genetic patients[J]. Neuroreport, 2013, 24(4) : 161-166.