疏肝健脾活血方含药血清对肝纤维化模型大鼠肝窦内皮细胞失窗孔化的影响
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摘要
目的探讨疏肝健脾活血方治疗肝纤维化的可能作用机制。方法 10只SD大鼠以10 ml/kg疏肝健脾方浓缩液(浓度2. 7 g/ml)灌胃,每日1次,连续3天后制备含药血清。中药血清设5%、10%、15%、20%、25%共5个浓度,采用MTT法筛选实验药物浓度。采用四氯化碳(CCl4)腹腔注射建立肝纤维化大鼠模型后分离肝纤维化肝窦内皮细胞(SEC),同时集正常大鼠肝星状细胞(HSC)和SEC。实验分为正常组、模型组、DAPT组、NF组、中药组、中药加DAPT组、中药加NF组,共7组。各组均加入HSC,正常组加入正常大鼠SEC,其余组加入肝纤维化大鼠SEC,常规培养24 h。吸出上清液,DAPT组、中药加DAPT组加入0. 2μmol/L DAPT (Notch通路阻断剂),NF组、中药加NF组加入1 gsu/ml rh NF-κB (Notch通路激动剂)。前4组加入含10%FBS DMEM/12培养基,后3组加入含预筛选浓度中药血清、10%FBS DMEM/12培养基,培养72 h后MTT法检测各组HSC活力,Western Blot法检测SEC中CD31、vWF蛋白表达情况。结果筛选出10%浓度的中药血清对细胞增殖有抑制作用。与正常组比较,造模2、4、8周模型组HSC活力上升,造模4、8周CD31蛋白表达升高(P <0. 05或P <0. 01);与模型组比较,NF组HSC活力上升,DAPT组和中药组HSC活力、CD31、vWF蛋白表达下降(P <0. 05或P <0. 01);与NF组比较,中药加NF组HSC活力、CD31、vWF蛋白表达下降(P <0. 05或P <0. 01);与DAPT组比较,中药加DAPT组HSC活力、CD31、vWF蛋白表达均下降(P <0. 05或P <0. 01)。结论疏肝健脾活血方可通过干预Notch通路,抑制HSC活力及SEC失窗孔化,可能是其治疗肝纤维化的作用机制之一。
Objective To explore the possible mechanism of Shugan Jianpi Huoxue Decoction(疏肝健脾活血方)(STHD) in treating hepatic fibrosis.Methods Ten SD rats were orally administered with 10 ml/kg Shugan Jianpi Huoxue Decoction(concentration 2.7 g/ml) once a day for 3 consecutive days to prepare drug-containing serum.The serum of traditional Chinese medicine is set at 5 concentrations of 5%,10%,15%,20% and 25%.The concentration of the experimental drug was screened by MTT method,and the liver fibrosis sinusoidal endothelial cells(SEC)were isolated by intraperitoneal injection of carbon tetrachloride(CCl4).The normal rat hepatic stellate cells(HSC)and SEC were collected.In this experiment,the rats were divided into 7 groups including normal group,model group,DAPT group,NF group,Chinese medicine group,Chinese medicine plus DAPT group and Chinese medicine plus NF group.HSC was added to each group,and SEC of normal rats was added to the normal group,and SEC of liver fibrosis rats were added to the other groups for 24 hours.The supernatant was aspirated,and the DAPT group,the Chinese medicine plus DAPT group were added with Notch pathway blocker DAPT 0.2 μmol/L,and the NF group and the Chinese medicine plus NF group were added with Notch pathway agonist rhNF-κB 1 gsu/ml.The first4 groups were added with 10% FBS DMEM/12 medium,and the last 3 groups were added with pre-screened concentration Chinese medicine serum,10% FBS DMEM/12 medium.After 72 hours of culture,the MTC activity of each group was detected by MTT method,and the expression of CD31 and vWF proteins in SEC was detected by Western Blot method.Results The screening out 10% concentration of traditional Chinese medicine in serum had an inhibitory effect on cell proliferation.Compared with the normal group,the HSC activity of the model group was increased after 2,4,and 8 weeks,and the CD31 protein expression was increased after 4 and 8 weeks since model establishment(P<0.05 or P<0.01).Compared with the model group,the HSC activity of the NF group was increased.The HSC activity,CD31 and vWF protein expression were decreased in DAPT group and Chinese medicine group(P <0.05 or P<0.01).Compared with the NF group,the HSC activity,CD31 and vWF protein expression decreased in traditional Chinese medicine plus NF group(P<0.05 or P<0.01).Compared with the DAPT group,the expression of HSC,CD31 and v WF protein in Chinese medicine plus DAPT group were decreased(P<0.05 or P<0.01).Conclusion Shugan Jianpi Huoxue Decoction could inhibit HSC activity and the defenestration of SEC by interfering plu Notch pathway,which may be one of the mechanisms of its treatment of liver fibrosis.
Objective To explore the possible mechanism of Shugan Jianpi Huoxue Decoction(疏肝健脾活血方)(STHD) in treating hepatic fibrosis.Methods Ten SD rats were orally administered with 10 ml/kg Shugan Jianpi Huoxue Decoction(concentration 2.7 g/ml) once a day for 3 consecutive days to prepare drug-containing serum.The serum of traditional Chinese medicine is set at 5 concentrations of 5%,10%,15%,20% and 25%.The concentration of the experimental drug was screened by MTT method,and the liver fibrosis sinusoidal endothelial cells(SEC)were isolated by intraperitoneal injection of carbon tetrachloride(CCl4).The normal rat hepatic stellate cells(HSC)and SEC were collected.In this experiment,the rats were divided into 7 groups including normal group,model group,DAPT group,NF group,Chinese medicine group,Chinese medicine plus DAPT group and Chinese medicine plus NF group.HSC was added to each group,and SEC of normal rats was added to the normal group,and SEC of liver fibrosis rats were added to the other groups for 24 hours.The supernatant was aspirated,and the DAPT group,the Chinese medicine plus DAPT group were added with Notch pathway blocker DAPT 0.2 μmol/L,and the NF group and the Chinese medicine plus NF group were added with Notch pathway agonist rhNF-κB 1 gsu/ml.The first4 groups were added with 10% FBS DMEM/12 medium,and the last 3 groups were added with pre-screened concentration Chinese medicine serum,10% FBS DMEM/12 medium.After 72 hours of culture,the MTC activity of each group was detected by MTT method,and the expression of CD31 and vWF proteins in SEC was detected by Western Blot method.Results The screening out 10% concentration of traditional Chinese medicine in serum had an inhibitory effect on cell proliferation.Compared with the normal group,the HSC activity of the model group was increased after 2,4,and 8 weeks,and the CD31 protein expression was increased after 4 and 8 weeks since model establishment(P<0.05 or P<0.01).Compared with the model group,the HSC activity of the NF group was increased.The HSC activity,CD31 and vWF protein expression were decreased in DAPT group and Chinese medicine group(P <0.05 or P<0.01).Compared with the NF group,the HSC activity,CD31 and vWF protein expression decreased in traditional Chinese medicine plus NF group(P<0.05 or P<0.01).Compared with the DAPT group,the expression of HSC,CD31 and v WF protein in Chinese medicine plus DAPT group were decreased(P<0.05 or P<0.01).Conclusion Shugan Jianpi Huoxue Decoction could inhibit HSC activity and the defenestration of SEC by interfering plu Notch pathway,which may be one of the mechanisms of its treatment of liver fibrosis.
引文
[1]王冰琼,孙亚朦,尤红,等.肝纤维化逆转的病理评估[J].临床肝胆病杂志,2017,33(3):435-437.
[2]KIM W,MATSUMOTO K,BESSHO K,et al. Growth inhibition and apoptosis in liver myofibroblasts promoted by hepatocyte growth factor leads to resolution from liver cirrhosis[J]. Am J Pathol,2005,166(4):1017-1028.
[3]张晨曦,卞勉励,陈星燃,等.肝窦内皮细胞在肝脏疾病中的作用[J].中国药理学通报,2017,33(2):149-153.
[4]DELEVE LD. Hepatic microvasculature in liver injury[J]. Semin Liver Dis,2007,27(4):390-400.
[5]陈刚,李宏波,邱少敏. CCl4皮下注射制备大鼠肝纤维化模型的研究[J].现代医学,2010,38(3):225-229.
[6]中华肝脏病学会肝纤维化学组.肝纤维化诊断及疗效评估共识[J].中华肝脏病杂志,2002,10(5):8-9.
[7]吕文良,徐晨光,张莎莎,等.肝窦内皮细胞的分离培养及体外生长规律的观察[J].中华生物医学工程杂志,2015,21(1):20-25.
[8]顾妍秋,原永芳.肝纤维化与炎性细胞因子介导的相关信号通路的研究进展[J].医学综述,2017,23(9):1670-1674.
[9]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.
[10]赵海军,郭顺根,贾文婷,等.肝纤维化肝窦内皮细胞对肝星状细胞的影响[J].解剖学杂志,2006(4):514-516.
[11]GEISLER F,STRAZZABOSCO M. Emerging roles of Notch signaling in liver disease[J]. Hepatology,2015,61(1):382-392.
[12]CHEN Y,ZHENG S,QI D,et al. Inhibition of Notch signaling by aγ-secretase inhibitor attenuates hepatic fibrosis in rats[J]. PLOS One,2012,7(10):e46512.
[13]BANSAL R,VAN BAARLEN J,STORM G,et al. The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis[J]. Sci Rep, 2015, 5:18272. doi:10. 1038/srep18272.
[14]张秋霞,孔心涓,姜英俊,等. Notch通路在大鼠肝纤维化发生发展中作用的初探[J].现代生物医学进展,2012,12(11):2046-2048.
[15]张衣北,陈安民,郭风劲,等.激活的Notch1信号系统抑制前软骨干细胞凋亡及其机制研究[J].中华小儿外科杂志,2006,27(11):592-595.
[16]LI Z,WANG J,ZHAO C,et al. Acute blockage of notch signaling by DAPT induces neuroprotection and neurogenesis in the neonatal rat brain after stroke[J]. Transl Stroke Res,2016,7(2):132-140.
[17]DILL MT,ROTHWEILER S,DJONOV V,et al. Disruption of Notch1 induces vascular remodeling,intussusceptive angiogenesis,and angiosarcomas in livers of mice[J]. Gastroenterology,2012,142(4):967-977.
[18]金超,任凯夕,晏贤春,等.γ分泌酶抑制剂对肝血窦内皮细胞的影响[J].现代生物医学进展,2013,13(16):3019-3023.
[19]张俊杰.中医药在抗肝纤维化过程中对病理性血管新生干预作用的研究进展[J].中国当代医药,2013,20(11):22-24.
[20]杨欣,李清林,陈卓辉,等.疏肝健脾活血方对肝纤维化过程中大鼠TGF-β1/Smad信号通路的影响[J].中华中医药杂志,2017,32(4):1694-1698.
[21]朱瑾,陈平,李晓武,等.原代培养大鼠肝窦内皮细胞凋亡的检测及意义[J].第三军医大学学报,2006,28(6):515-517.
[2]KIM W,MATSUMOTO K,BESSHO K,et al. Growth inhibition and apoptosis in liver myofibroblasts promoted by hepatocyte growth factor leads to resolution from liver cirrhosis[J]. Am J Pathol,2005,166(4):1017-1028.
[3]张晨曦,卞勉励,陈星燃,等.肝窦内皮细胞在肝脏疾病中的作用[J].中国药理学通报,2017,33(2):149-153.
[4]DELEVE LD. Hepatic microvasculature in liver injury[J]. Semin Liver Dis,2007,27(4):390-400.
[5]陈刚,李宏波,邱少敏. CCl4皮下注射制备大鼠肝纤维化模型的研究[J].现代医学,2010,38(3):225-229.
[6]中华肝脏病学会肝纤维化学组.肝纤维化诊断及疗效评估共识[J].中华肝脏病杂志,2002,10(5):8-9.
[7]吕文良,徐晨光,张莎莎,等.肝窦内皮细胞的分离培养及体外生长规律的观察[J].中华生物医学工程杂志,2015,21(1):20-25.
[8]顾妍秋,原永芳.肝纤维化与炎性细胞因子介导的相关信号通路的研究进展[J].医学综述,2017,23(9):1670-1674.
[9]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.
[10]赵海军,郭顺根,贾文婷,等.肝纤维化肝窦内皮细胞对肝星状细胞的影响[J].解剖学杂志,2006(4):514-516.
[11]GEISLER F,STRAZZABOSCO M. Emerging roles of Notch signaling in liver disease[J]. Hepatology,2015,61(1):382-392.
[12]CHEN Y,ZHENG S,QI D,et al. Inhibition of Notch signaling by aγ-secretase inhibitor attenuates hepatic fibrosis in rats[J]. PLOS One,2012,7(10):e46512.
[13]BANSAL R,VAN BAARLEN J,STORM G,et al. The interplay of the Notch signaling in hepatic stellate cells and macrophages determines the fate of liver fibrogenesis[J]. Sci Rep, 2015, 5:18272. doi:10. 1038/srep18272.
[14]张秋霞,孔心涓,姜英俊,等. Notch通路在大鼠肝纤维化发生发展中作用的初探[J].现代生物医学进展,2012,12(11):2046-2048.
[15]张衣北,陈安民,郭风劲,等.激活的Notch1信号系统抑制前软骨干细胞凋亡及其机制研究[J].中华小儿外科杂志,2006,27(11):592-595.
[16]LI Z,WANG J,ZHAO C,et al. Acute blockage of notch signaling by DAPT induces neuroprotection and neurogenesis in the neonatal rat brain after stroke[J]. Transl Stroke Res,2016,7(2):132-140.
[17]DILL MT,ROTHWEILER S,DJONOV V,et al. Disruption of Notch1 induces vascular remodeling,intussusceptive angiogenesis,and angiosarcomas in livers of mice[J]. Gastroenterology,2012,142(4):967-977.
[18]金超,任凯夕,晏贤春,等.γ分泌酶抑制剂对肝血窦内皮细胞的影响[J].现代生物医学进展,2013,13(16):3019-3023.
[19]张俊杰.中医药在抗肝纤维化过程中对病理性血管新生干预作用的研究进展[J].中国当代医药,2013,20(11):22-24.
[20]杨欣,李清林,陈卓辉,等.疏肝健脾活血方对肝纤维化过程中大鼠TGF-β1/Smad信号通路的影响[J].中华中医药杂志,2017,32(4):1694-1698.
[21]朱瑾,陈平,李晓武,等.原代培养大鼠肝窦内皮细胞凋亡的检测及意义[J].第三军医大学学报,2006,28(6):515-517.