拆方益肝康与药物血清对肝星状细胞PDGF及受体表达的影响
姚希贤, 房红梅, 王军民, 河北医科大学第二医院消化内科
河北省石家庄市 050000
姚希贤, 男, 汉族. 河北省石家庄市人, 1955年河北医学院本科毕业. 河北省消化病研究所所长, 省重点实验室主任, 博士生导师, 中西医结合高徒导师, 主任医师, 教授, 省管优秀专家, 享受国务院特贴. 主要从事慢性肝病研究, 发表论文170篇, 主编书6部.
河北省中医管理局资助项目, No. 200335
通讯作者: 姚希贤,050000, 河北省石家庄市, 河北医科大学第二医院消化内科. yaoxixian@163.com
电话: 0311-7814356
收稿日期: 2005-04-07 接受日期: 2005-04-09
Effects of Yigankang and its separated recipe on expression ofplatelet-derived growth factor and its receptor in hepatic stellate cells
Xi-Xian Yao, Hong-Mei Fang, Jun-Min Wang
Xi-Xian Yao, Hong-Mei Fang, Jun-Min Wang, Department ofGastroenterology, the Second Hospital of Hebei Medical University,Shijiazhuang 050000, Hebei Province, China
Supported by Hebei Administration for Traditional Chinese Medicine,No. 200335
Correspondence to: Dr. Xi-Xian Yao, Department of Gastroenterology,the Second Hospital of Hebei Medical University, Shijiazhuang 050000,Hebei Province, China. yaoxixian@163.com
Received: 2005-04-07 Accepted: 2005-04-09
Abstract
AIM: To investigate the effects of Yigankang and smallcompound of radix salviae militiorrhizae on protein and gene expression ofplatelet-derived growth factor-BB (PDGF-BB) and platelet-derived growthfactor receptor-b(PDGFR-b) in hepatic stellate cells (HSCs).
METHODS: The purified extracts and pharmaceutical serum from normaland liver fibrosis rats of Yigankang and small compound of radixsalviae militiorrhizae were prepared respectively. The expression of PDGF-BBmRNA and protein were detected by reverse transcription polymerase chainreaction (RT-PCR) and Western blot, and the expression of PDGFR-bb mRNA andprotein were measured by RT-PCR and flow cytometry.
RESULTS: The purified extracts of Yigankang and smallcompound of radix salviae militiorrhizae decreased PDGF-BB proteinexpression by 78.79% and 56.77% and its mRNA by 67.13% and 43.59% (P<0.01);lowered PDGFR-bb protein expression by 11.61% and 6.79% and its mRNA by54.85% and 30.51% respectively (P<0.01). Yigankang andsmall compound of radix salviae militiorrhizae pharmaceutical serum fromnormal rats decreased PDGF-BB protein expression by 68.05% and 49.95% andits mRNA by 55.87% and 38.66% (P<0.01); lowered PDGFR-b proteinexpression by 10.98% and 6.48% and its mRNA by 39.67% and 21.38%respectively (P<0.01). Yigankang and small formula ofradix salviae militiorrhizae pharmaceutical serum from liver fibrosis ratsreduced PDGF-BB protein expression by 81.93% and 67.31% and its mRNA by72.68% and 55.49% (P<0.01); decreased PDGFR-bb protein expressionby 17.43% and 11.15% and its mRNA by 60.46% and 46.47% respectively (P<0.01).With respect to the inhibitory effects mentioned above, Yigankanghad more evidently action than its separated recipe did, and thepharmaceutical serum from liver fibrosis rats was superior to that fromnormal rats.
CONCLUSION: The inhibitory effect of Yigankang and itsseparated recipe on PDGF-BB and PDGFR-bb expression may be one of the mainmechanisms of their antifibrotic action, and the whole recipe was superiorto the separated recipe.
Key Words: Yigankang; Small compound of radix salviaemilitiorrhizae; Hepatic fibrosis; Hepatic stellate cell; Platelet-derivedgrowth factor-BB
Yao XX, Fang HM, Wang JM. Effects of Yigankang and its separatedrecipe on expression of platelet-derived growth factor and its receptor inhepatic stellate cells. Shijie Huaren Xiaohua Zazhi 2005;13(12):1412-1416
摘要
目的: 探讨益肝康及其拆方丹参小复方(丹参、黄芪、归尾)对肝星状细胞PDGF与受体表达的影响.
方法: 分别制备益肝康及丹参小复方纯化浸膏、正常大鼠及肝纤维化大鼠药物血清，温育体外培养的HSC.RT-PCR和Westernblot技术检测PDGF-BBmRNA及蛋白表达，RT-PCR和流式细胞技术检测PDGFR-bbmRNA及蛋白表达.
结果: 益肝康及丹参小复方纯化浸膏使PDGF-BB蛋白表达分别下调了78.79%、56.77%(P<0.01)，使其mRNA表达分别下调了67.13%、43.59%(P<0.01)，使PDGFR-b蛋白表达分别下调了11.61%、6.79%(P<0.01)，使其mRNA表达分别下调了54.85%、30.51%(P<0.01);正常大鼠益肝康及丹参小复方药物血清使PDGF-BB蛋白表达分别下调了68.05%、49.95%(P<0.01)，使其mRNA表达分别下调了55.87%、38.66%(P<0.01)，使PDGFR-b蛋白表达分别下调了10.98%、6.48%(P<0.01)，使其mRNA表达分别下调了39.67%、21.38%(P<0.01);肝纤维化大鼠益肝康及丹参小复方药物血清使PDGF-BB蛋白表达分别下调了81.93%、67.31%(P<0.01)，使其mRNA表达分别下调了72.68%、55.49%(P<0.01)，使PDGFR-b蛋白表达分别下调了17.43%、11.15%(P<0.01)，使其mRNA表达分别下调了60.46%、46.47%(P<0.01)，全方作用优于拆方(P<0.01)，肝纤维化大鼠药物血清作用优于正常大鼠药物血清(P<0.01).
结论: 益肝康及丹参小复方可显著抑制PDGF与PDGFR-b基因和蛋白表达，此可能是该方抗肝纤维化的主要机制之一，且全方作用优于拆方.
关键词: 益肝康;丹参小复方;肝纤维化;肝星状细胞;血小板衍生生长因子-BB
姚希贤,房红梅,王军民.拆方益肝康与药物血清对肝星状细胞PDGF及受体表达的影响.世界华人消化杂志 2005;13(12):1412-1416
1 (PDF) 益肝康及丹参小复方对HSC PDGF-BB蛋白表达的影响.
2.2 HSC PDGF-BB mRNA的表达 经益肝康及丹参小复方浸膏(104 g/L)干预后，PDGF-BB基因表达均较对照组显著减少(A3:0.328±0.040，A2:0.563±0.037 vs A1:0.998±0.042，P<0.01);全方对PDGF-BB基因表达的抑制作用强于拆方(P<0.01).两种方法制备的100 mL/L 益肝康及丹参小复方药物血清均可显著抑制PDGF-BB基因表达(B3:0.435±0.038，B2:0.604±0.041 vs B1:0.984±0.047，P<0.01;C3:0.301±0.030，C2:0.489±0.045 vs C1:1.097±0.051，P<0.01)，全方作用优于拆方(P<0.01)，肝纤维化大鼠药物血清组作用优于正常大鼠药物血清组(P<0.01)(图2).
图2 (PDF) 益肝康及丹参小复方对HSC PDGF-BBmRNA表达的影响.
2.3 HSC PDGFR-b蛋白的表达 经益肝康及丹参小复方浸膏(104 g/L)干预后，PDGFR-b蛋白表达均较对照组显著减少(A3:4.95±0.095，A2:5.22±0.143 vs A1:5.60±0.100，P<0.01);全方对PDGFR-b蛋白表达的抑制作用强于拆方(P<0.01).两种方法制备的100 mL/L益肝康及丹参小复方药物血清均可显著抑制PDGFR-b蛋白表达(B3:5.09±0.120，B2:5.30±0.109 vs B1:5.66±0.136，P<0.01;C3:4.81±0.062，C2:5.18±0.117 vs C1:5.82±0.155，P<0.01)，全方作用优于拆方(P<0.01)，肝纤维化大鼠药物血清组作用优于正常大鼠药物血清组(P<0.01).
2.4 HSC PDGFR-bmRNA的表达 经益肝康及丹参小复方浸膏(104 g/L)干预后，PDGFR-b基因表达均较对照组显著减少(A3:0.410±0.027，A2:0.631±0.032 vs A1:0.908±0.035，P<0.01)，全方对PDGFR-b基因表达的抑制作用强于拆方(P<0.01).两种方法制备的100 mL/L益肝康及丹参小复方药物血清均可显著抑制PDGFR-b基因表达(B3:0.555±0.030，B2:0.724±0.035 vs B1:0.920±0.038，P<0.01;C3:0.382±0.029，C2:0.517±0.033 vs C1:0.965±0.040，P<0.01)，全方作用优于拆方(P<0.01)，肝纤维化大鼠药物血清组作用优于正常大鼠药物血清组(P<0.01)(图3).
图3 (PDF) 益肝康及丹参小复方对HSCs PDGFR-bb mRNA表达的影响.
3 讨论
HSC是肝纤维化形成的中心环节与细胞学基础，此在近年来国内外研究业已取得广泛共识[13-15].细胞因子是细胞间信号转导、联系的重要物质基础，HSC的活化过程中始终贯穿着转化生长因子、血管内皮生长因子、白介素-6等细胞网络因子及其受体的变化[16-19].PDGF是目前已知的最强的促HSC分裂增殖因子，与细胞膜上的相应受体结合后发挥其生物学效应.PDGFR由a、b两种亚单位以二硫键连接形成，其与PDGF结合力相差很大，a单位与PDGFA链及B链均有较高亲和力，而b亚单位仅与B链有高亲和力.在肝纤维化时，HSC表面的PDGFR以b受体为主，与PDGF-BB具有较强的亲和力，认为PDGF-BB以及PDGFR-b在肝纤维化过程中的作用尤为突出[20-31].HSC在体外无包被的塑料培养皿中培养，可自发向肌成纤维样细胞转化，具有体内活化的生物学特征[32].HSC株CFSC从CCl4诱发的肝硬化大鼠中分离并通过培养使细胞自发获得永久性，表型为活化的HSC，是较为理想的肝纤维化研究模型[33].本研究分别采用益肝康与拆方的纯化浸膏、正常大鼠及肝纤维化大鼠药物血清进行体外HSCs培养，观察其对肝纤维化形成与发展过程中最重要的细胞因子之一—PDGF与受体蛋白及基因表达的作用情况，结果表明益肝康与丹参小复方的纯化浸膏及药物血清均具有良好的抑制PDGF与PDGFR-b基因和蛋白表达的作用.而且药物配伍后的全方组(A3，B3，C3)对HSC PDGF与PDGFR-b基因及蛋白表达较拆方组(A2，B2，C2)具有更为显著的抑制作用，充分体现了符合中医基础理论君臣佐使配方组合的优势.
中药复方益肝康(前身为益肝冲剂，于1980年投产并应用于临床，益肝康为研制者对该方进一步研究改进而成)系重用丹参，辅以当归、赤芍等活血化瘀，并用黄芪等益气健脾药而成.为明确有效方剂益肝康抗肝纤维化的作用机制，以及为得到专一性更强、疗效更显著的新药，本研究所长期以来围绕肝纤维化的主要病理环节对该复方有关单味药、拆方小复方及全方进行了大量临床与实验研究，结果表明，该药具有消除症状，改善肝功能，减少胶原沉积作用;可抑制HSCs活化、增殖，促进HSCs凋亡，并有抗脂质过氧化，保护受损肝细胞及线粒体等细胞器功能，而且无明显毒副作用[1-7].本研究表明益肝康、丹参小复方的纯化浸膏及药物血清均具有良好的抑制PDGF与PDGFR-b基因和蛋白表达的作用，说明该复方作用机制之一是从mRNA和蛋白水平抑制PDGF及PDGFR-b表达，从而抑制HSC的激活、增殖以及细胞外基质的合成，发挥抗肝纤维化作用.
药物血清中发挥作用的有效物质包括母体药物、药物在机体内的代谢物以及药物诱生的内源性物质，本部分实验采用直接加药法与整体动物给药、分离药物血清作用于体外培养HSC的血清药理学方法进行对比研究，结果表明益肝康、丹参小复方的纯化浸膏及药物血清均具有良好抑制PDGF与PDGFR-b基因和蛋白表达的作用，推测该复方中真正起到抑制PDGF与PDGFR-b表达作用的有效物质可能仍以原药成分为主.本研究结果表明肝纤维化大鼠药物血清对HSC PDGF与PDGFR-b基因及蛋白表达的抑制作用优于正常大鼠药物血清，分析原因，是否与肝纤维化大鼠肝功能下降，对药物的转化、代谢能力降低，从而导致病态机体内药物有效成分浓度提高，作用时间延长，使抗肝纤维化作用增强有关？抑或中医辨证论治的特点使得该方对肝纤维化病态机体更具针对性，疗效更明显.血清药理学的出现为科学阐明中药复方的有效成分及作用机制提供了一种有力的工具，但作为一种新兴的实验方法，在实验技术上还需要进一步探讨、规范和完善.
4 参考文献
1 姚希贤, 唐有为, 姚冬梅, 修贺明. 益肝煎剂对实验性肝纤维化大鼠I, III型胶原蛋白表达的影响.
世界华人消化杂志 2001;9:263-267
2 Yao XX, Tang YW, Yao DM, Xiu HM. Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate
cells. World J Gastroenterol 2002;8:511-514
3 姚希贤, 姚欣, 修贺明, 孙泽明, 宋梅, 冯丽英. “益肝浓缩煎剂”等活血化瘀药抗大鼠肝纤维化作用实验研究.
胃肠病学和肝病学杂志 2001;10:217-222
4 Yao XX, Cui DL, Sun YF, Feng LY, Sun ZM, Song M. Study on the anti-liver fibrosis effect of benefit liver granule
and its mechanism in rats. Chin J Integr Med 2002;8:118-121
5 蒋树林, 姚希贤, 孙泽明, 宋梅, 杨林, 杨川杰, 姚冬梅, 吕涛. 活血化瘀中药对大鼠纤维化肝间质胶原的抑制作用.
中华实用医学 2002;4:10-13
6 唐有为, 姚希贤, 姚洪森, 修贺明. 益肝煎剂对实验性肝纤维化大鼠I, IV型胶原蛋白表达的影响. 中国消化病学杂志
2002;3:16-19
7 崔东来, 姚希贤, 姚金锋, 郭煜. 丹参抗大鼠肝纤维化作用与机制研究. 中华临床医药 2002;3:5-7
8 姚欣, 姚希贤, 姚金锋, 冯丽英, 冯志杰. “益肝浓缩煎剂”对实验性大鼠肝纤维化防治作用的研究.
河北医科大学学报 2002;23:202-204
9 姚欣, 姚希贤,修贺明,高君萍,张玉琢. 活血化瘀中药益肝浓缩煎剂对大鼠肝纤维化的作用. 世界华人消化杂志
2002;10:544-548
10 唐有为, 姚希贤,姚洪森.益肝康对实验性肝纤维化大鼠肝细胞的保护作用及超微结构观察.
中国中西医结合消化杂志 2002;10:76-78
11 蒋树林, 李校天,姚希贤.益肝康对大鼠肝纤维化的防治作用.中国全科医学 2002;5:525-527
12 房红梅, 姚冬梅,姚希贤,王天轶.益肝康的药物血清对大鼠HSCs增殖及PDGF基因表达的影响.
第三军医大学学报 2004;26:611-613
13 Issa R, Williams E, Trim N, Kendall T, Arthur MJ, Reichen J, BenyonRC, Iredale JP. Apoptosis of hepatic stellate
cellsinvolvement in resolution of biliary fibrosis and regulation by solublegrowth factors. Gut 2001;48:548-557
14 Fisher R, Schmitt M, Bode JG, Haussinger D. Expression of theperipheral-type benzodiazepine receptor and
apoptosisinduction in hepatic stellate cells. Gastroenterology 2001;120:1212-1226
15 Nagler A, Pines M, Abadi U, Pappo O, Zeira M, Rabbani E, EngelhardtD, Ohana M, Chowdhury NR, Chowdhury JR, Ilan
Y.Oral tolerization ameliorates liver disorders associated with chronicgraft versus host disease in mice.
Hepatology 2000;31:641-648
16 Paradis V, Dargere D, Bonvoust F, Vidaud M, Segarini P, Bedossa P.Effects and regulation of connective tissue
growthfactor on hepatic stellate cells. Lab Invest 2002;82:767-774
17 Pinzani M, Marra F. Cytokine receptors and signaling in hepaticstellate cells. Semin Liver Dis 2001;21:397-416
18 Li D, Friedman SL. Liver fibrogenesis and the role of hepaticstellate cells: New insights and prospects for
therapy.J Gastroenterol Hepatol 1999;4:618-633
19 Friedman SL. Cytokines and fibrogenesis. Semin Liver Dis 1999;19:129-140
20 Pinzani M. PDGF and signal transduction in hepatic stellate cells. FrontBiosci 2002;7:1720-1726
21 Ikura Y, Morimoto H, Ogami M, Jomura H, Ikeoka N, Sakurai M.Expression of platelet-derived growth factor
andits receptor in livers of patients with chronic liver disease. JCastroentrol 1997;32:496-501
22 Pinani M, Milani S, Herbst H, DeFranco R, Grappone C,Gentilini A, Caligiuri A, Pellegrini G, Ngo DV, Romanelli RG,GentiliniP. Expression of platelet-derived growth factor and its receptors innormal human liver and during active
hepaticfibrosis. Am J Pathol 1996;148:785-800
23 Marra F, Gentilini A, Pinzani M, Choudhury GG, Parola M, Herbst H,Dianzani MU, Laffi G, Abboud HE, Gentilini
P.Phosphatidylinositol 3-kinase is required for platelet-derived growthfactor's actions on hepatic stellate
cells.Gastroenterology 1997;112:1297-1306
24 Isbrucker RA, Oeferson TC. platelet-derived growth factor andpentoxifylline modulation of collagen synthesis
inmyofibroblasts. Toxicology and Applied. Pharmacology 1998;149:120-126
25 Herbst H, Schupan D, Milani S. Fibrogenesis and fibrolysis in theliver. Verh Dtsch Ges Pathol 1995;79:15-27
26 Wong L, Yamasaki G, Johnson RJ, Friedman SL. Induction ofb-platelet-derived growth factor receptor in
rathepatic lipocytes during cellular activation in vivo and in culture. JClin Invest 1994;94:1563-1569
27 Di Sario A, Bendia E, Svegliati-Baroni G, Marzioni M, Ridolfi F,Trozzi L, Ugili L, Saccomanno S, Jezequel AM, Benedetti
A.Rearrangement of the cytoskeletal network induced by platelet-derivedgrowth factor in rat hepatic stellate cells:role
ofdifferent intracellular signalling pathways. J Hepatol 2002;36:179-190
28 Kinnman N, Goria O, Wendum D, Gendron MC, Rey C, Poupon R, HoussetC. Hepatic stellate cell proliferation is an
earlyplatelet-derived growth factor-mediated cellular event in rat cholestaticliver injury. Lab Invest 2001;
81:1709-1716
29 Okuyama H, Shimahara Y, Kawada N, Seki S, Kristensen DB, YoshizatoK, Uyama N, Yamaoka Y. Regulation of cell
growthby redox-mediated extracellular proteolysis of platelet-derived growthfactor receptor beta. J Biol
Chem 2001;276:28274-28280
30 Taylor CC. Platelet-derived growth factor activates porcine thecalcell phosphatidylinositol-3-kinase-Akt/PKB
andras-extracellular signal-regulated kinase-1/2 kinase signaling pathwaysvia the platelet-derived growth
factor-betareceptor. Endocrinology 2000;141:1545-1553
31 Marra F, Arrighi MC, Fazi M, Caligiuri A, Pinzani M, Romanelli RG,Efsen E, Laffi G, Gentilini P.
Extracellularsignal-regulated kinase activation differentially regulatesplatelet-derived growth factor's actions in
hepaticstellate cells, and is induced by in vivo liver injury in the rat.Hepatology 1999;30:951-958
32 Friedman SL. Cellular sources of collagen and regulation ofcollegen production in liver. Sem in Liver Dis
1990;10:20-29
33 Greenwel P, Schwartz M, Rossas M, Peyrol S, Grimaud JA, Rojkind M.Characterization of fat-storing cell lines
derivedfrom normal and CCl4-cirrhoticlivers. Lab Invest 1991;65:644-653
编辑 潘伯荣 审读 张海宁, http://www.100md.com
河北省石家庄市 050000
姚希贤, 男, 汉族. 河北省石家庄市人, 1955年河北医学院本科毕业. 河北省消化病研究所所长, 省重点实验室主任, 博士生导师, 中西医结合高徒导师, 主任医师, 教授, 省管优秀专家, 享受国务院特贴. 主要从事慢性肝病研究, 发表论文170篇, 主编书6部.
河北省中医管理局资助项目, No. 200335
通讯作者: 姚希贤,050000, 河北省石家庄市, 河北医科大学第二医院消化内科. yaoxixian@163.com
电话: 0311-7814356
收稿日期: 2005-04-07 接受日期: 2005-04-09
Effects of Yigankang and its separated recipe on expression ofplatelet-derived growth factor and its receptor in hepatic stellate cells
Xi-Xian Yao, Hong-Mei Fang, Jun-Min Wang
Xi-Xian Yao, Hong-Mei Fang, Jun-Min Wang, Department ofGastroenterology, the Second Hospital of Hebei Medical University,Shijiazhuang 050000, Hebei Province, China
Supported by Hebei Administration for Traditional Chinese Medicine,No. 200335
Correspondence to: Dr. Xi-Xian Yao, Department of Gastroenterology,the Second Hospital of Hebei Medical University, Shijiazhuang 050000,Hebei Province, China. yaoxixian@163.com
Received: 2005-04-07 Accepted: 2005-04-09
Abstract
AIM: To investigate the effects of Yigankang and smallcompound of radix salviae militiorrhizae on protein and gene expression ofplatelet-derived growth factor-BB (PDGF-BB) and platelet-derived growthfactor receptor-b(PDGFR-b) in hepatic stellate cells (HSCs).
METHODS: The purified extracts and pharmaceutical serum from normaland liver fibrosis rats of Yigankang and small compound of radixsalviae militiorrhizae were prepared respectively. The expression of PDGF-BBmRNA and protein were detected by reverse transcription polymerase chainreaction (RT-PCR) and Western blot, and the expression of PDGFR-bb mRNA andprotein were measured by RT-PCR and flow cytometry.
RESULTS: The purified extracts of Yigankang and smallcompound of radix salviae militiorrhizae decreased PDGF-BB proteinexpression by 78.79% and 56.77% and its mRNA by 67.13% and 43.59% (P<0.01);lowered PDGFR-bb protein expression by 11.61% and 6.79% and its mRNA by54.85% and 30.51% respectively (P<0.01). Yigankang andsmall compound of radix salviae militiorrhizae pharmaceutical serum fromnormal rats decreased PDGF-BB protein expression by 68.05% and 49.95% andits mRNA by 55.87% and 38.66% (P<0.01); lowered PDGFR-b proteinexpression by 10.98% and 6.48% and its mRNA by 39.67% and 21.38%respectively (P<0.01). Yigankang and small formula ofradix salviae militiorrhizae pharmaceutical serum from liver fibrosis ratsreduced PDGF-BB protein expression by 81.93% and 67.31% and its mRNA by72.68% and 55.49% (P<0.01); decreased PDGFR-bb protein expressionby 17.43% and 11.15% and its mRNA by 60.46% and 46.47% respectively (P<0.01).With respect to the inhibitory effects mentioned above, Yigankanghad more evidently action than its separated recipe did, and thepharmaceutical serum from liver fibrosis rats was superior to that fromnormal rats.
CONCLUSION: The inhibitory effect of Yigankang and itsseparated recipe on PDGF-BB and PDGFR-bb expression may be one of the mainmechanisms of their antifibrotic action, and the whole recipe was superiorto the separated recipe.
Key Words: Yigankang; Small compound of radix salviaemilitiorrhizae; Hepatic fibrosis; Hepatic stellate cell; Platelet-derivedgrowth factor-BB
Yao XX, Fang HM, Wang JM. Effects of Yigankang and its separatedrecipe on expression of platelet-derived growth factor and its receptor inhepatic stellate cells. Shijie Huaren Xiaohua Zazhi 2005;13(12):1412-1416
摘要
目的: 探讨益肝康及其拆方丹参小复方(丹参、黄芪、归尾)对肝星状细胞PDGF与受体表达的影响.
方法: 分别制备益肝康及丹参小复方纯化浸膏、正常大鼠及肝纤维化大鼠药物血清，温育体外培养的HSC.RT-PCR和Westernblot技术检测PDGF-BBmRNA及蛋白表达，RT-PCR和流式细胞技术检测PDGFR-bbmRNA及蛋白表达.
结果: 益肝康及丹参小复方纯化浸膏使PDGF-BB蛋白表达分别下调了78.79%、56.77%(P<0.01)，使其mRNA表达分别下调了67.13%、43.59%(P<0.01)，使PDGFR-b蛋白表达分别下调了11.61%、6.79%(P<0.01)，使其mRNA表达分别下调了54.85%、30.51%(P<0.01);正常大鼠益肝康及丹参小复方药物血清使PDGF-BB蛋白表达分别下调了68.05%、49.95%(P<0.01)，使其mRNA表达分别下调了55.87%、38.66%(P<0.01)，使PDGFR-b蛋白表达分别下调了10.98%、6.48%(P<0.01)，使其mRNA表达分别下调了39.67%、21.38%(P<0.01);肝纤维化大鼠益肝康及丹参小复方药物血清使PDGF-BB蛋白表达分别下调了81.93%、67.31%(P<0.01)，使其mRNA表达分别下调了72.68%、55.49%(P<0.01)，使PDGFR-b蛋白表达分别下调了17.43%、11.15%(P<0.01)，使其mRNA表达分别下调了60.46%、46.47%(P<0.01)，全方作用优于拆方(P<0.01)，肝纤维化大鼠药物血清作用优于正常大鼠药物血清(P<0.01).
结论: 益肝康及丹参小复方可显著抑制PDGF与PDGFR-b基因和蛋白表达，此可能是该方抗肝纤维化的主要机制之一，且全方作用优于拆方.
关键词: 益肝康;丹参小复方;肝纤维化;肝星状细胞;血小板衍生生长因子-BB
姚希贤,房红梅,王军民.拆方益肝康与药物血清对肝星状细胞PDGF及受体表达的影响.世界华人消化杂志 2005;13(12):1412-1416
1 (PDF) 益肝康及丹参小复方对HSC PDGF-BB蛋白表达的影响.
2.2 HSC PDGF-BB mRNA的表达 经益肝康及丹参小复方浸膏(104 g/L)干预后，PDGF-BB基因表达均较对照组显著减少(A3:0.328±0.040，A2:0.563±0.037 vs A1:0.998±0.042，P<0.01);全方对PDGF-BB基因表达的抑制作用强于拆方(P<0.01).两种方法制备的100 mL/L 益肝康及丹参小复方药物血清均可显著抑制PDGF-BB基因表达(B3:0.435±0.038，B2:0.604±0.041 vs B1:0.984±0.047，P<0.01;C3:0.301±0.030，C2:0.489±0.045 vs C1:1.097±0.051，P<0.01)，全方作用优于拆方(P<0.01)，肝纤维化大鼠药物血清组作用优于正常大鼠药物血清组(P<0.01)(图2).
图2 (PDF) 益肝康及丹参小复方对HSC PDGF-BBmRNA表达的影响.
2.3 HSC PDGFR-b蛋白的表达 经益肝康及丹参小复方浸膏(104 g/L)干预后，PDGFR-b蛋白表达均较对照组显著减少(A3:4.95±0.095，A2:5.22±0.143 vs A1:5.60±0.100，P<0.01);全方对PDGFR-b蛋白表达的抑制作用强于拆方(P<0.01).两种方法制备的100 mL/L益肝康及丹参小复方药物血清均可显著抑制PDGFR-b蛋白表达(B3:5.09±0.120，B2:5.30±0.109 vs B1:5.66±0.136，P<0.01;C3:4.81±0.062，C2:5.18±0.117 vs C1:5.82±0.155，P<0.01)，全方作用优于拆方(P<0.01)，肝纤维化大鼠药物血清组作用优于正常大鼠药物血清组(P<0.01).
2.4 HSC PDGFR-bmRNA的表达 经益肝康及丹参小复方浸膏(104 g/L)干预后，PDGFR-b基因表达均较对照组显著减少(A3:0.410±0.027，A2:0.631±0.032 vs A1:0.908±0.035，P<0.01)，全方对PDGFR-b基因表达的抑制作用强于拆方(P<0.01).两种方法制备的100 mL/L益肝康及丹参小复方药物血清均可显著抑制PDGFR-b基因表达(B3:0.555±0.030，B2:0.724±0.035 vs B1:0.920±0.038，P<0.01;C3:0.382±0.029，C2:0.517±0.033 vs C1:0.965±0.040，P<0.01)，全方作用优于拆方(P<0.01)，肝纤维化大鼠药物血清组作用优于正常大鼠药物血清组(P<0.01)(图3).
图3 (PDF) 益肝康及丹参小复方对HSCs PDGFR-bb mRNA表达的影响.
3 讨论
HSC是肝纤维化形成的中心环节与细胞学基础，此在近年来国内外研究业已取得广泛共识[13-15].细胞因子是细胞间信号转导、联系的重要物质基础，HSC的活化过程中始终贯穿着转化生长因子、血管内皮生长因子、白介素-6等细胞网络因子及其受体的变化[16-19].PDGF是目前已知的最强的促HSC分裂增殖因子，与细胞膜上的相应受体结合后发挥其生物学效应.PDGFR由a、b两种亚单位以二硫键连接形成，其与PDGF结合力相差很大，a单位与PDGFA链及B链均有较高亲和力，而b亚单位仅与B链有高亲和力.在肝纤维化时，HSC表面的PDGFR以b受体为主，与PDGF-BB具有较强的亲和力，认为PDGF-BB以及PDGFR-b在肝纤维化过程中的作用尤为突出[20-31].HSC在体外无包被的塑料培养皿中培养，可自发向肌成纤维样细胞转化，具有体内活化的生物学特征[32].HSC株CFSC从CCl4诱发的肝硬化大鼠中分离并通过培养使细胞自发获得永久性，表型为活化的HSC，是较为理想的肝纤维化研究模型[33].本研究分别采用益肝康与拆方的纯化浸膏、正常大鼠及肝纤维化大鼠药物血清进行体外HSCs培养，观察其对肝纤维化形成与发展过程中最重要的细胞因子之一—PDGF与受体蛋白及基因表达的作用情况，结果表明益肝康与丹参小复方的纯化浸膏及药物血清均具有良好的抑制PDGF与PDGFR-b基因和蛋白表达的作用.而且药物配伍后的全方组(A3，B3，C3)对HSC PDGF与PDGFR-b基因及蛋白表达较拆方组(A2，B2，C2)具有更为显著的抑制作用，充分体现了符合中医基础理论君臣佐使配方组合的优势.
中药复方益肝康(前身为益肝冲剂，于1980年投产并应用于临床，益肝康为研制者对该方进一步研究改进而成)系重用丹参，辅以当归、赤芍等活血化瘀，并用黄芪等益气健脾药而成.为明确有效方剂益肝康抗肝纤维化的作用机制，以及为得到专一性更强、疗效更显著的新药，本研究所长期以来围绕肝纤维化的主要病理环节对该复方有关单味药、拆方小复方及全方进行了大量临床与实验研究，结果表明，该药具有消除症状，改善肝功能，减少胶原沉积作用;可抑制HSCs活化、增殖，促进HSCs凋亡，并有抗脂质过氧化，保护受损肝细胞及线粒体等细胞器功能，而且无明显毒副作用[1-7].本研究表明益肝康、丹参小复方的纯化浸膏及药物血清均具有良好的抑制PDGF与PDGFR-b基因和蛋白表达的作用，说明该复方作用机制之一是从mRNA和蛋白水平抑制PDGF及PDGFR-b表达，从而抑制HSC的激活、增殖以及细胞外基质的合成，发挥抗肝纤维化作用.
药物血清中发挥作用的有效物质包括母体药物、药物在机体内的代谢物以及药物诱生的内源性物质，本部分实验采用直接加药法与整体动物给药、分离药物血清作用于体外培养HSC的血清药理学方法进行对比研究，结果表明益肝康、丹参小复方的纯化浸膏及药物血清均具有良好抑制PDGF与PDGFR-b基因和蛋白表达的作用，推测该复方中真正起到抑制PDGF与PDGFR-b表达作用的有效物质可能仍以原药成分为主.本研究结果表明肝纤维化大鼠药物血清对HSC PDGF与PDGFR-b基因及蛋白表达的抑制作用优于正常大鼠药物血清，分析原因，是否与肝纤维化大鼠肝功能下降，对药物的转化、代谢能力降低，从而导致病态机体内药物有效成分浓度提高，作用时间延长，使抗肝纤维化作用增强有关？抑或中医辨证论治的特点使得该方对肝纤维化病态机体更具针对性，疗效更明显.血清药理学的出现为科学阐明中药复方的有效成分及作用机制提供了一种有力的工具，但作为一种新兴的实验方法，在实验技术上还需要进一步探讨、规范和完善.
4 参考文献
1 姚希贤, 唐有为, 姚冬梅, 修贺明. 益肝煎剂对实验性肝纤维化大鼠I, III型胶原蛋白表达的影响.
世界华人消化杂志 2001;9:263-267
2 Yao XX, Tang YW, Yao DM, Xiu HM. Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate
cells. World J Gastroenterol 2002;8:511-514
3 姚希贤, 姚欣, 修贺明, 孙泽明, 宋梅, 冯丽英. “益肝浓缩煎剂”等活血化瘀药抗大鼠肝纤维化作用实验研究.
胃肠病学和肝病学杂志 2001;10:217-222
4 Yao XX, Cui DL, Sun YF, Feng LY, Sun ZM, Song M. Study on the anti-liver fibrosis effect of benefit liver granule
and its mechanism in rats. Chin J Integr Med 2002;8:118-121
5 蒋树林, 姚希贤, 孙泽明, 宋梅, 杨林, 杨川杰, 姚冬梅, 吕涛. 活血化瘀中药对大鼠纤维化肝间质胶原的抑制作用.
中华实用医学 2002;4:10-13
6 唐有为, 姚希贤, 姚洪森, 修贺明. 益肝煎剂对实验性肝纤维化大鼠I, IV型胶原蛋白表达的影响. 中国消化病学杂志
2002;3:16-19
7 崔东来, 姚希贤, 姚金锋, 郭煜. 丹参抗大鼠肝纤维化作用与机制研究. 中华临床医药 2002;3:5-7
8 姚欣, 姚希贤, 姚金锋, 冯丽英, 冯志杰. “益肝浓缩煎剂”对实验性大鼠肝纤维化防治作用的研究.
河北医科大学学报 2002;23:202-204
9 姚欣, 姚希贤,修贺明,高君萍,张玉琢. 活血化瘀中药益肝浓缩煎剂对大鼠肝纤维化的作用. 世界华人消化杂志
2002;10:544-548
10 唐有为, 姚希贤,姚洪森.益肝康对实验性肝纤维化大鼠肝细胞的保护作用及超微结构观察.
中国中西医结合消化杂志 2002;10:76-78
11 蒋树林, 李校天,姚希贤.益肝康对大鼠肝纤维化的防治作用.中国全科医学 2002;5:525-527
12 房红梅, 姚冬梅,姚希贤,王天轶.益肝康的药物血清对大鼠HSCs增殖及PDGF基因表达的影响.
第三军医大学学报 2004;26:611-613
13 Issa R, Williams E, Trim N, Kendall T, Arthur MJ, Reichen J, BenyonRC, Iredale JP. Apoptosis of hepatic stellate
cellsinvolvement in resolution of biliary fibrosis and regulation by solublegrowth factors. Gut 2001;48:548-557
14 Fisher R, Schmitt M, Bode JG, Haussinger D. Expression of theperipheral-type benzodiazepine receptor and
apoptosisinduction in hepatic stellate cells. Gastroenterology 2001;120:1212-1226
15 Nagler A, Pines M, Abadi U, Pappo O, Zeira M, Rabbani E, EngelhardtD, Ohana M, Chowdhury NR, Chowdhury JR, Ilan
Y.Oral tolerization ameliorates liver disorders associated with chronicgraft versus host disease in mice.
Hepatology 2000;31:641-648
16 Paradis V, Dargere D, Bonvoust F, Vidaud M, Segarini P, Bedossa P.Effects and regulation of connective tissue
growthfactor on hepatic stellate cells. Lab Invest 2002;82:767-774
17 Pinzani M, Marra F. Cytokine receptors and signaling in hepaticstellate cells. Semin Liver Dis 2001;21:397-416
18 Li D, Friedman SL. Liver fibrogenesis and the role of hepaticstellate cells: New insights and prospects for
therapy.J Gastroenterol Hepatol 1999;4:618-633
19 Friedman SL. Cytokines and fibrogenesis. Semin Liver Dis 1999;19:129-140
20 Pinzani M. PDGF and signal transduction in hepatic stellate cells. FrontBiosci 2002;7:1720-1726
21 Ikura Y, Morimoto H, Ogami M, Jomura H, Ikeoka N, Sakurai M.Expression of platelet-derived growth factor
andits receptor in livers of patients with chronic liver disease. JCastroentrol 1997;32:496-501
22 Pinani M, Milani S, Herbst H, DeFranco R, Grappone C,Gentilini A, Caligiuri A, Pellegrini G, Ngo DV, Romanelli RG,GentiliniP. Expression of platelet-derived growth factor and its receptors innormal human liver and during active
hepaticfibrosis. Am J Pathol 1996;148:785-800
23 Marra F, Gentilini A, Pinzani M, Choudhury GG, Parola M, Herbst H,Dianzani MU, Laffi G, Abboud HE, Gentilini
P.Phosphatidylinositol 3-kinase is required for platelet-derived growthfactor's actions on hepatic stellate
cells.Gastroenterology 1997;112:1297-1306
24 Isbrucker RA, Oeferson TC. platelet-derived growth factor andpentoxifylline modulation of collagen synthesis
inmyofibroblasts. Toxicology and Applied. Pharmacology 1998;149:120-126
25 Herbst H, Schupan D, Milani S. Fibrogenesis and fibrolysis in theliver. Verh Dtsch Ges Pathol 1995;79:15-27
26 Wong L, Yamasaki G, Johnson RJ, Friedman SL. Induction ofb-platelet-derived growth factor receptor in
rathepatic lipocytes during cellular activation in vivo and in culture. JClin Invest 1994;94:1563-1569
27 Di Sario A, Bendia E, Svegliati-Baroni G, Marzioni M, Ridolfi F,Trozzi L, Ugili L, Saccomanno S, Jezequel AM, Benedetti
A.Rearrangement of the cytoskeletal network induced by platelet-derivedgrowth factor in rat hepatic stellate cells:role
ofdifferent intracellular signalling pathways. J Hepatol 2002;36:179-190
28 Kinnman N, Goria O, Wendum D, Gendron MC, Rey C, Poupon R, HoussetC. Hepatic stellate cell proliferation is an
earlyplatelet-derived growth factor-mediated cellular event in rat cholestaticliver injury. Lab Invest 2001;
81:1709-1716
29 Okuyama H, Shimahara Y, Kawada N, Seki S, Kristensen DB, YoshizatoK, Uyama N, Yamaoka Y. Regulation of cell
growthby redox-mediated extracellular proteolysis of platelet-derived growthfactor receptor beta. J Biol
Chem 2001;276:28274-28280
30 Taylor CC. Platelet-derived growth factor activates porcine thecalcell phosphatidylinositol-3-kinase-Akt/PKB
andras-extracellular signal-regulated kinase-1/2 kinase signaling pathwaysvia the platelet-derived growth
factor-betareceptor. Endocrinology 2000;141:1545-1553
31 Marra F, Arrighi MC, Fazi M, Caligiuri A, Pinzani M, Romanelli RG,Efsen E, Laffi G, Gentilini P.
Extracellularsignal-regulated kinase activation differentially regulatesplatelet-derived growth factor's actions in
hepaticstellate cells, and is induced by in vivo liver injury in the rat.Hepatology 1999;30:951-958
32 Friedman SL. Cellular sources of collagen and regulation ofcollegen production in liver. Sem in Liver Dis
1990;10:20-29
33 Greenwel P, Schwartz M, Rossas M, Peyrol S, Grimaud JA, Rojkind M.Characterization of fat-storing cell lines
derivedfrom normal and CCl4-cirrhoticlivers. Lab Invest 1991;65:644-653
编辑 潘伯荣 审读 张海宁, http://www.100md.com
