(1. School of Pharmacy, Jiangsu University, Zhenjiang Jiangsu 212013; 2. Clinical School of Traditional Chinese Medicine, Hubei University of TCM, Wuhan Hubei 430065, China)
Abstract：Objective: To study the effect of drug-containing serum of Si-Ni-San on differentiation of rat′s hepatic stem cells and its mechanism, and explore the scientific basis for treatment of liver disease. Methods: HPLC-MS-MS method was used to detect drugcontaining serum of Si-Ni-San; WB-F344 cells were treated with drugcontaining serum, the activity of cells was detected by CCK-8 assay, the glucose consumption was assessed by glucometer, generation of lactic acid was determined by LA kit, ATP content was measured by luciferin luciferase method,and the expressions of epidermal growth factor receptor(EGFR), fibroblast growth factor receptor(FGFR),transforming growth factor beta receptor type I(TGF-βRⅠ),hepatocyte growth factor receptor(HGFR) were observed by western blotting. Results: In drug-containing serum of Si-Ni-San, some ingredients in prototype like paeoniflorin and aurantiamarin, others in metabolite type such as naringenin- glucuronic acid and glycyrrhetinic acid. Different concentrations of drug-containing serum showed no toxic effects on WB-F344 cells after been incubated with drug-containing serum, 10% concentration of drug-containing serum could significantly promote proliferation of WB-F344 cells. The levels of glucose consumption and lactic acid decreased with the time of differentiation, but ATP took on a up-regulating trend. the expressions of EGFR, FGFR, TGFβRI and HGFR also increased as drug-containing serum incubation. Conclusion: The Si-Ni-San containing serum could promote hepatic stem cells′ proliferation and induce their differentiation, and maybe the regulatory mechanism is related to cytokine receptors, such as EGFR,HGFR.
\[1\]王付,尚立芝,苗小玲,等.四逆散加味治疗肝纤维化的临床疗效观察\[J\].中医药通报,2012,11(1):42-45.［2］陆惠光.四逆散治疗慢性乙型肝炎的临床效果体会\[J\].中国保健营养,2012,22(9):3128-3129.［3］伊书红,武水斗.以四逆散为主治疗肝硬化腹水疗效分析\[J\].慢性病学杂志,2010,12(4): 325-326.［4］程红杰,牛少强.四逆散加味治疗非酒精性脂肪肝40例疗效观察\[J\].北京中医药, 2012, 31(10):761-763.［5］蒋洁云,徐强.四逆散及其各组成中药对实验性肝损伤的影响\[J\].中国天然药物, 2004, 2(1): 45-49.［6］龚莉,周娟妮,罗瑞钦.四逆散对肝损害性小鼠保护作用的实验研究\[J\].江西中医学院学报, 2008, 20(6): 60.［7］王巍.复方四逆散防治脂肪肝的实验研究\[D\].沈阳：辽宁中医学院,2003.［8］郭青文,何银,何蓉蓉.四逆散对应激性肝郁小鼠的保护作用机制研究\[J\].中国药业,2013, 22(24):19-22.［9］Russo FP,Parola M.Stem cells in liver failure\[J\].Best Pract Res Clin Gastroenterol,2012,26(1):35-45.［10］Christ B, Pelz S.Implication of hepatic stem cells in functional liver repopulation\[J\].Cytometry A,2013,83(1):90-102.［11］Forbes S,Vig P,Poulsom R,et al.Hepatic stem cells\[J\]. J Pathol,2002,197(4):510-518.［12］Oh SH,Hatch HM,Petersen BE.Hepatic oval ‘stem’ cell in liver regeneration\[J\]. Semin Cell Dev Biol,2002,13(6): 405-409.［13］Petersen BE,Zajac VF,Michalopoulos GK.Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats\[J\]. Hepatology, 1998, 27(4): 1030-1038.［14］胡俊,黄秀榕,祁明信,等.中药诱导干细胞定向分化的研究进展\[J\].中草药,2006,37(1):147-149.［15］Vacanti NM, Metallo CM.Exploring metabolic pathways that contribute to the stem cell phenotype\[J\].Biochim Biophys Acta,2013,1830(2): 2361-2369.［16］Chen CT, Hsu SH, Wei YH.Mitochondrial bioenergetic function and metabolic plasticity in stem cell differentiation and cellular reprogramming\[J\]. Biochim Biophys Acta, 2012,1820(5): 571-576.［17］Rehman J.Empowering self-renewal and differentiation: the role of mitochondria in stem cells\[J\]. J Mol Med (Berl), 2010,88(10): 981-986.［18］Chen CT, Hsu SH, Wei YH. Upregulation of mitochondrial function and antioxidant defense in the differentiation of stem cells\[J\]. Biochim Biophys Acta, 2010,1800(3): 257-263.［19］Trosko JE, Kang KS. Evolution of energy metabolism, stem cells and cancer stem cells: how the Warburg and Barker hypothesis might be linked\[J\].Int J Stem Cells,2012,5(1):39-56.［20］Baumann K.Stem cells: A metabolic switch\[J\]. Nat Rev Mol Cell Biol, 2013,14(2): 64-65.［21］Ochocki JD, Simon MC.Nutrientsensing pathways and metabolic regulation in stem cells\[J\]. J Cell Biol, 2013,203(1): 23-33.［22］小池弘美.肝细胞生长因子\[J\].日本医学介绍,2002,23(5):226-227.［23］Ishikawa T, Factor VM,Marquardt JU,et al.Hepatocyte growth factor/c-met signaling is required for stem-cell-mediated liver regeneration in mice\[J\].Hepatology,2012,55(4): 1215-1226.［24］Sanchez A, Fabregat I.Growth factor-and cytokine-driven pathways governing liver stemness and differentiation\[J\]. World J Gastroenterol, 2010,16(41): 5148-5161.［25］Okano J, Shiota G,Matsumoto K,et al.Hepatocyte growth factor exerts a proliferative effect on oval cells through the PI3K/AKT signaling pathway\[J\]. Biochem Biophys Res Commun,2003,309(2): 298-304.［26］MartinezPalacian A, Del Castillo G,Herrera B,et al.EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells\[J\]. Cell Signal, 2012,24(2): 505-513.［27］Michalopoulos GK,Bowen WC,Mul K,et al.HGF,EGF and dexamethasone induced gene expression patterns during formation of tissue in hepatic organoid cultures\[J\]. Gene Expr, 2003,11(2): 55-75.［28］Greber B, Wu GM,Bernemann C,et al.Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells\[J\]. Cell Stem Cell, 2010,6(3): 215-226.［29］Xu X, Browning VL, Odorico JS.Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells\[J\]. Mech Dev, 2011,128(7): 412-427.［30］Minagawa K,Koyama T, Miyoshi H.Stimulating effects of fibroblast growth factors on hepatic function of fetal liver cells synergistically with oncostatin M in threedimensional culture\[J\]. J Biosci Bioeng, 2009,107(3): 307-311.［31］Huang T, David L,Mendoza V,et al.TGF-β signalling is mediated by two autonomously functioning TβRⅠ: TβRII pairs\[J\].EMBO J,2011,30(7): 1263-1276.［32］Santibaez JF, Quintanilla M, Bernabeu C.TGF-β/TGF-β receptor system and its role in physiological and pathological conditions\[J\].Clin Sci(Lond),2011,121(6): 233-251.［33］Li WQ,Li YM,Guo J,et al.Hepatocytic precursor (stem-like) WB-F344 cells reduce tumorigenicity of hepatoma CBRH7919 cells via TGFbeta/Smad pathway\[J\]. Oncol Rep, 2010,23(6): 1601-1607.
2014, Vol. 24 
