疏肝健脾活血方对肝星状细胞/肝窦内皮细胞共培养体系中VEGF/VEGFR表达的影响
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摘要
目的探讨疏肝健脾活血方逆转肝纤维化进程中肝窦毛细血管化的可能作用机制。方法分离正常大鼠肝星状细胞(HSC)、肝窦内皮细胞(LSEC)和肝纤维化模型大鼠LSEC,利用慢病毒转染得到血管内皮生长因子(VEGF)过表达HSC。实验分为6组,1)正常HSC组:正常大鼠HSC空白血清培养; 2)正常组:正常大鼠HSC及正常LSEC空白血清共培养; 3)模型组:正常大鼠HSC及模型大鼠LSEC空白血清共培养; 4)过表达组:VEGF过表达HSC及模型大鼠LSEC空白血清共培养; 5)模型加中药组:正常大鼠HSC及模型大鼠LSEC 10%含药血清共培养; 6)过表达加中药组:VEGF过表达HSC及模型大鼠LSEC10%含药血清共培养。采用Western Blot法检测各组LSEC中血小板内皮细胞黏附因子CD31及血友病因子(vWF)蛋白表达,HSC中胶原Ⅳ、VEGF及血管内皮生长因子受体2 (VEGFR-2)蛋白表达;采用PCR法检测各组HSC中VEGF、血管内皮生长因子受体1 (VEGFR-1)及VEGFR-2 mRNA表达。结果与正常组比较,模型组及过表达组CD31及vWF蛋白表达升高(P <0. 01),而模型加中药组较模型组CD31及v WF蛋白表达降低(P <0. 01);与正常HSC组和正常组比较,模型组及过表达组中胶原Ⅳ、VEGF蛋白表达升高(P <0. 05),而模型加中药组较模型组VEGF、VEGFR-2蛋白表达降低(P <0. 05);与正常HSC组和正常组比较,模型组及过表达组VEGF mRNA表达升高(P <0. 01);模型加中药组VEGF mRNA表达较模型组降低(P <0. 05)。结论疏肝健脾活血方可能通过下调VEGF、VEGFR-2蛋白表达,进而抑制VEGF信号通路,恢复LSEC窗孔结构,从而逆转肝窦毛细血管化。
Objective To explore the possible mechanism of Shugan Jianpi Huoxue Decoction( 疏肝健脾活血方)in treating liver sinus capillarification in liver fibrosis. Methods The hepatic stellate cells( HSC) and liver sinusoidal endothelial cell( LSEC) from normal rats,LSEC from liver fibrosis model rats were separated. The vascular endothelial growth factor( VEGF) overexpressed HSC was obtained by virus transfection. The cells were divided into six groups: a) normal HSC group,cultured HSC by blank serum; b) normal group: co-culture normal HSC and normal LSEC by blank serum; c) model group: co-culture normal HSC and model LSEC by blank serum; d) overexpression group: co-culture VEGF overexpressed HSC and model LSEC by blank serum; e) model with herbs group:co-culture normal HSC and model LSEC by 10% drug containing serum; f) overexpression with herbs group: coculture VEGF overexpressed HSC and model LSEC by 10% drug containing serum. Western Blot method was used to detect the expression of CD31 and von Willebrand factor( vWF) in LSEC and the expression of collagen Ⅳ,VEGF and vascular endothelial growth factor receptor 2( VEGFR-2) in HSC. The expression of VEGF,vascular endothelial growth factor receptor 1( VEGFR-1) and VEGFR-2 mRNA in HSC was detected by PCR. Results Compared with normal group,the expression of CD31 and vWF in model group and overexpression group was increased( P < 0. 01),and the expression of CD31 and v WF in the model with herbs group was decreased when compared with model group( P < 0. 01). Compared with normal HSC group and normal group,the expression of collagen Ⅳ and VEGF in model group and overexpression group was increased( P < 0. 01),and the expression of VEGF and VEGFR-2 in the model with herbs group was decreased when compared with model group( P < 0. 05). Compared with normal HSC group and normal group,the expression of VEGF mRNA in model group and overexpression group was increased( P < 0. 01).The expression of VEGF mRNA in model with herbs group was decreased than that in model group( P < 0. 05).Conclusion Shugan Jianpi Huoxue Decoction reverse the capillary vascularization of hepatic sinus possibly by downregulating the expression of VEGF and VEGFR-2,inhibiting the signal pathway of VEGF and restoring the window structure of LSEC.
Objective To explore the possible mechanism of Shugan Jianpi Huoxue Decoction( 疏肝健脾活血方)in treating liver sinus capillarification in liver fibrosis. Methods The hepatic stellate cells( HSC) and liver sinusoidal endothelial cell( LSEC) from normal rats,LSEC from liver fibrosis model rats were separated. The vascular endothelial growth factor( VEGF) overexpressed HSC was obtained by virus transfection. The cells were divided into six groups: a) normal HSC group,cultured HSC by blank serum; b) normal group: co-culture normal HSC and normal LSEC by blank serum; c) model group: co-culture normal HSC and model LSEC by blank serum; d) overexpression group: co-culture VEGF overexpressed HSC and model LSEC by blank serum; e) model with herbs group:co-culture normal HSC and model LSEC by 10% drug containing serum; f) overexpression with herbs group: coculture VEGF overexpressed HSC and model LSEC by 10% drug containing serum. Western Blot method was used to detect the expression of CD31 and von Willebrand factor( vWF) in LSEC and the expression of collagen Ⅳ,VEGF and vascular endothelial growth factor receptor 2( VEGFR-2) in HSC. The expression of VEGF,vascular endothelial growth factor receptor 1( VEGFR-1) and VEGFR-2 mRNA in HSC was detected by PCR. Results Compared with normal group,the expression of CD31 and vWF in model group and overexpression group was increased( P < 0. 01),and the expression of CD31 and v WF in the model with herbs group was decreased when compared with model group( P < 0. 01). Compared with normal HSC group and normal group,the expression of collagen Ⅳ and VEGF in model group and overexpression group was increased( P < 0. 01),and the expression of VEGF and VEGFR-2 in the model with herbs group was decreased when compared with model group( P < 0. 05). Compared with normal HSC group and normal group,the expression of VEGF mRNA in model group and overexpression group was increased( P < 0. 01).The expression of VEGF mRNA in model with herbs group was decreased than that in model group( P < 0. 05).Conclusion Shugan Jianpi Huoxue Decoction reverse the capillary vascularization of hepatic sinus possibly by downregulating the expression of VEGF and VEGFR-2,inhibiting the signal pathway of VEGF and restoring the window structure of LSEC.
引文
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[16]吕靖,陆雄,陶艳艳,等.肝纤维化小鼠肝组织血管新生特点及其形成机制[J].肝脏,2011,16(1):35-40.
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[3]黄月红,郭杞兰.四氯化碳诱导肝纤维化早期大鼠肝窦内皮细胞及基底膜形态改变的动态研究[J].中国病理生理杂志,2014,30(8):1501-1505,1531.
[4]LEMOS QT,ANDRADE ZA. Angiogenesis and experimental hepatic fibrosis[J]. Mem Inst Oswaldo Cruz,2010,105(5):611-614.
[5]CHAMORRO-JORGANES A,LEE MY,ARALDI E,et al.VEGF-induced expression of miR-17-92 cluster in endothelial cells is mediated by ERK/ELK1 activation and regulates angiogenesis[J]. Circ Res,2016,118(1):38-47.
[6]LIU Y,LUI EL,FRIEDMAN SL,et al. PTK787/ZK22258attenuates stellate cell activation and hepatic fibrosis in vivo by inhibiting VEGF signaling[J]. Lab Invest,2009,89(2):209-221.
[7]COULOUARN C,CORLU A,GLAISE D,et al. Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma[J]. Cancer Res,2012,72(10):2533-2542.
[8]FORD AJ,JAIN G,RAJAGOPALAN P,et al. Designing a fibrotic microenvironment to investigate changes in human liver sinusoidal endothelial cell function[J]. Acta Biomater,2015,24:220-227. doi:10. 1016/j. actbio. 2015.06. 028.
[9]周凝,杨欣,夏雪皎,等.疏肝健脾活血方对大鼠肝星状细胞TGF-β1/Smads信号通路的影响[J].浙江中西医结合杂志,2018,28(4):267-270.
[10]杨开选,程薇波,王文冬,等. 40%四氯化碳大鼠慢性肝损伤模型的研究[J].现代预防医学,2005,32(1):20-21,40.
[11]李洋,蔡双明,张莉莉,等.大鼠原代肝细胞、星状细胞、枯否细胞和肝窦内皮细胞的同步分离与培养[J].南方医科大学学报,2014,34(4):532-537.
[12]VALFREDI BONZO L,NOVO E,CANNITO S,et al. Angiogenesis and liver fibrogenesi[J]. Histol Histopathol,2009,24(10):1323-1341.
[13]郭枸兰,王小众.肝窦内皮细胞在肝纤维化形成中的作用[J].肝脏,2013,18(4):269-273.
[14]XIE G,WANG X,WANG L,et al. Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in Rats[J]. Gastroenterology,2012,142(4):918-927.
[15]THABUT D,SHAH V. lntrahepatic angiogenesis and sinusoidal remodeling in chronic liver disease:New targets for the treatment of portal hypertension?[J]. J Hepatol,2010,53(5):976-980.
[16]吕靖,陆雄,陶艳艳,等.肝纤维化小鼠肝组织血管新生特点及其形成机制[J].肝脏,2011,16(1):35-40.
[17]SCHACHTNER H,CALAMINS SDJ,THOMAS SG.Podosomes in adhesion,migration,mechanosensing and matrix remodeling[J]. Cytoskeleton(Hoboken),2013,70(10):572-589.
[18]原雪,郭青龙. VEGF通路和Notch通路在肿瘤血管生成中的相互作用及靶向药物的研发[J].药学进展,2009,33(5):199-203.