下瘀血汤抑制胰腺巨噬细胞浸润改善肝纤维化的机制研究
|
摘要
目的:评价下瘀血汤对四氯化碳(CCl_4)诱导的肝纤维化模型小鼠肝脏和胰腺病变的改善作用,并探讨其相关机制。方法:24只雄性C57BL/6小鼠随机分为正常组、模型组和下瘀血汤组,每组8只。模型组和下瘀血汤组小鼠腹腔注射10%CCl_4诱导肝纤维化模型,正常组小鼠给予等体积橄榄油,每周3次,连续6周。造模第4周起,下瘀血汤组小鼠灌胃给予0.467 8 g/kg下瘀血汤药液,正常组和模型组小鼠给予等体积蒸馏水,每日1次,连续3周。药物干预期间,每周称量各组小鼠体质量。末次给药后,腹主动脉采血,分离肝、脾、胰腺组织并称重,计算肝脏指数和脾脏指数;试剂盒检测血清ALT、AST水平,HE染色和天狼星红染色后光镜下观察肝脏和胰腺组织病理形态学变化;免疫荧光检测肝组织α-SMA蛋白表达,免疫组化检测胰腺组织CD68、MCP-1及α-SMA蛋白表达。结果:①与正常组相比,模型组小鼠体质量明显降低(P<0.01),肝脏指数、脾脏指数显著增大(P<0.01),血清ALT、AST水平显著升高(P<0.05,P<0.01);与模型组相比,下瘀血汤组小鼠体质量显著升高,肝脏指数、脾脏指数及血清ALT、AST水平显著降低(P<0.01)。②病理学观察显示,模型组小鼠肝组织可见明显肝细胞肿胀、小叶中心性出血及灶状坏死,并伴有炎症细胞浸润,纤维组织大量增生,增生的胶原纤维分割肝小叶形成较粗大的间隔;胰腺小叶间隙增大,腺泡肿胀,腺管区有较多炎症细胞浸润。下瘀血汤组小鼠肝组织胶原纤维间隔较窄、排列疏松,肝细胞变性、坏死及肝和胰腺组织炎症细胞浸润明显减轻。③与正常组相比,模型组肝组织α-SMA表达明显增多,胰腺组织CD68、MCP-1及α-SMA阳性表达显著升高(P<0.01);下瘀血汤干预3周后,肝组织α-SMA表达明显减少,胰腺组织CD68、MCP-1及α-SMA阳性表达显著降低(P<0.01)。结论:肝纤维化可引起胰腺巨噬细胞浸润和星状细胞激活;下瘀血汤可显著减轻肝纤维化过程中胰腺巨噬细胞浸润,抑制胰腺星状细胞活化。
Objective: To study the effects of Xiayuxue Decoction on the liver and pancreas lesions in mice with liver fibrosis induced by carbon tetrachloride(CCl_4),and explore its related mechanisms. Methods: 24 male C57 BL/6 mice were divided into the normal group,modelgroup and Xiayuxue Decoction group,8 mice in each group. The mice in the model group and Xiayuxue Decoction group were treated with 10% CCl_4 by intraperitoneal injection to induce liver fibrosis model,and the mice in the normal group were treated with olive oil at equal volume,3 times a week for 6 weeks. From the 4 th week of modeling,the Xiayuxue Decoction group was treated with Xiayuxue Decoction at dose of 0.467 8 g/kg,and the normal group and model group were treated with distilled water at equal volume,once every day for 3 weeks. During the drug intervention,the body mass of mice in each group was weighed weekly. After the last administration,the blood was collected from the abdominal aorta,and the liver,spleen and pancreas tissues were separated and weighed,then the indexes of liver and spleen were calculated. The serum levels of ALT and AST were detected by kits. The pathological changes of liver and pancreas were observed under light microscope after HE staining and Sirius red staining. The expression of α-SMA in liver tissue was detected by immunofluorescence,and the protein expressions of CD68,MCP-1 and α-SMA in pancreatic tissue were detected by immunohistochemistry.Results:(1)Compared with the normal group,the body weight of mice in the model group was significantly decreased( P<0.01),the liver index and the spleen index were significantly increased( P<0.01),and the serum levels of ALT and AST were remarkably increased( P<0.05,P<0.01). Compared with the model group,the body weight of mice in Xiayuxue Decoction group was obviously increased,and the liver index,the spleen index and the serum levels of ALT and AST were significantly decreased( P<0.01).(2)Pathological observation showed that hepatocyte swelling,central lobe hemorrhage and focal necrosis were observed in the model group,accompanied by inflammatory cell infiltration and fibrous tissue hyperplasia,and the hyperplastic collagen fibers segmented the hepatic lobule to form a larger interval;while the space in pancreatic lobular was enlarged,the acinus was swelling,and more inflammatory cells were infiltrated the glandular duct area. In the Xiayuxue Decoction group,the collagen fibers in the liver tissue arranged loosely with narrow spaces,and the hepatocyte degeneration and necrosis,the inflammatory cells infiltration in the liver and pancreas were significantly alleviated.(3)Compared with the normal group,the expression of α-SMA in the liver tissue and the positive expressions of CD68,MCP-1 and α-SMA in the pancreatic tissue were significantly increased( P<0.01). After intervention with Xiayuxue Decoction for 3 weeks,the expression of α-SMA in the liver tissue and the positive expressions of CD68,MCP-1 and α-SMA in the pancreatic tissue were significantly decreased( P<0.01). Conclusion:Hepatic fibrosis can cause pancreatic macrophage infiltration and pancreatic stellate cells activation. Xiayuxue Decoction can significantly alleviate the pancreatic macrophage infiltration and inhibit the pancreatic stellate cells activation during liver fibrosis.
Objective: To study the effects of Xiayuxue Decoction on the liver and pancreas lesions in mice with liver fibrosis induced by carbon tetrachloride(CCl_4),and explore its related mechanisms. Methods: 24 male C57 BL/6 mice were divided into the normal group,modelgroup and Xiayuxue Decoction group,8 mice in each group. The mice in the model group and Xiayuxue Decoction group were treated with 10% CCl_4 by intraperitoneal injection to induce liver fibrosis model,and the mice in the normal group were treated with olive oil at equal volume,3 times a week for 6 weeks. From the 4 th week of modeling,the Xiayuxue Decoction group was treated with Xiayuxue Decoction at dose of 0.467 8 g/kg,and the normal group and model group were treated with distilled water at equal volume,once every day for 3 weeks. During the drug intervention,the body mass of mice in each group was weighed weekly. After the last administration,the blood was collected from the abdominal aorta,and the liver,spleen and pancreas tissues were separated and weighed,then the indexes of liver and spleen were calculated. The serum levels of ALT and AST were detected by kits. The pathological changes of liver and pancreas were observed under light microscope after HE staining and Sirius red staining. The expression of α-SMA in liver tissue was detected by immunofluorescence,and the protein expressions of CD68,MCP-1 and α-SMA in pancreatic tissue were detected by immunohistochemistry.Results:(1)Compared with the normal group,the body weight of mice in the model group was significantly decreased( P<0.01),the liver index and the spleen index were significantly increased( P<0.01),and the serum levels of ALT and AST were remarkably increased( P<0.05,P<0.01). Compared with the model group,the body weight of mice in Xiayuxue Decoction group was obviously increased,and the liver index,the spleen index and the serum levels of ALT and AST were significantly decreased( P<0.01).(2)Pathological observation showed that hepatocyte swelling,central lobe hemorrhage and focal necrosis were observed in the model group,accompanied by inflammatory cell infiltration and fibrous tissue hyperplasia,and the hyperplastic collagen fibers segmented the hepatic lobule to form a larger interval;while the space in pancreatic lobular was enlarged,the acinus was swelling,and more inflammatory cells were infiltrated the glandular duct area. In the Xiayuxue Decoction group,the collagen fibers in the liver tissue arranged loosely with narrow spaces,and the hepatocyte degeneration and necrosis,the inflammatory cells infiltration in the liver and pancreas were significantly alleviated.(3)Compared with the normal group,the expression of α-SMA in the liver tissue and the positive expressions of CD68,MCP-1 and α-SMA in the pancreatic tissue were significantly increased( P<0.01). After intervention with Xiayuxue Decoction for 3 weeks,the expression of α-SMA in the liver tissue and the positive expressions of CD68,MCP-1 and α-SMA in the pancreatic tissue were significantly decreased( P<0.01). Conclusion:Hepatic fibrosis can cause pancreatic macrophage infiltration and pancreatic stellate cells activation. Xiayuxue Decoction can significantly alleviate the pancreatic macrophage infiltration and inhibit the pancreatic stellate cells activation during liver fibrosis.
引文
[1] CHANG J,LAN T,LI C,et al. Activation of Slit2- Robo1 signaling promotes liver fibrosis [J].J Hepatol,2015,63(6):1413- 1420.
[2] LEUNG V Y,SHEN J,WONG V W,et al. Quantitative elastography of liver fibrosis and spleen stiffness in chronic hepatitis B carriers [J].Radiology,2013,269(3):910- 918.
[3] LIN X,BAI F,NIE J,et al. Didymin Alleviates Hepatic Fibrosis Through Inhibiting ERK and PI3K/Akt Pathways via Regulation of Raf Kinase Inhibitor Protein [J].Cell Physiol Biochem,2016,40(6):1422- 1432.
[4] KNOP V,HOPPE D,WELZEL T,et al. Regression of fibrosis and portal hypertension in HCV- associated cirrhosis and sustained virologic response after interferon- free antiviral therapy [J].J Viral Hepat,2016,23(12):994- 1002.
[5] JIN S,LI H,HAN M,et al. Mesenchymal Stem Cells with Enhanced Bcl- 2 Expression Promote Liver Recovery in a Rat Model of Hepatic Cirrhosis [J].Cell Physiol Biochem,2016,40(5):1117- 1128.
[6] CHEN X,LIU C,LU Y,et al. Paeoniflorin regulates macrophage activation in dimethylnitrosamine- induced liver fibrosis in rats [J].BMC Complement Altern Med,2012,12(1):254.
[7] JASTER R,EMMRICH J. Crucial role of fibrogenesis in pancreatic diseases [J].Best Pract Res Clin Gastroenterol,2008,22(1):17- 29.
[8] PIAO R L,XIU M,BRIGSTOCK D R,et al. An immortalized rat pancreatic stellate cell line RP- 2 as a new cell model for evaluating pancreatic fibrosis,inflammation and immunity [J].Hepatobiliary Pancreat Dis Int,2015,14(6):651- 659.
[9] LIU C,CAI J,CHENG Z,et al. Xiayuxue decoction reduces renal injury by promoting macrophage apoptosis in hepatic cirrhotic rats [J].Genet Mol Res,2015,14(3):10760- 10773.
[10] ZHANGL J,SUN M Y,NING B B,et al. Xiayuxue Decoction attenuates hepatic stellate cell activation and sinusoidal endothelium defenestration in CCl4- induced fibrotic liver of mice [J].Chin J Integr Med,2014,20(7):516- 523.
[11] 都广礼,刘平,王磊,等. 猪血清肝纤维化大鼠肝组织基质金属蛋白酶- 9/13和基质金属蛋白酶组织抑制因子- 1/2表达的动态变化及下瘀血汤对其影响[J].中国实验方剂学杂志,2009,15(11):48- 51.
[12] 丁赛丹,陈必成,刘艳,等. 下瘀血汤干预肝硬化大鼠的蛋白质组学研究[J].中草药,2012,43(1):131- 138.
[13] FERRARIO C M. ACE2:more of Ang- (1- 7) or less AngⅡ? [J].Curr Opin Nephrol Hypertens,2011,20(1):1- 6.
[14] 加秀凤.经方下瘀血汤调控ACE2介导的RAS自稳抑制肝纤维化进展的实验研究 [D].武汉:华中科技大学,2014.
[15] MA W,TAO L,ZHANG W,et al. Xia- Yu- Xue Decoction Inhibits Intestinal Epithelial Cell Apoptosis in CCl4- Induced Liver Fibrosis [J].Cell Physiol Biochem,2017,44(1):333- 344.
[16] 慕永平,刘平,都广礼,等. 祛瘀和养阴不同功效中医经典方剂抑制CCl4诱导大鼠肝硬化的作用机制 [J].自然科学进展,2006,16(9):1101- 1108.
[17] 中国中西医结合学会肝病专业委员会. 肝纤维化中西医结合诊疗指南 [J].中华肝脏病杂志,2006,14(11):866- 870.
[18] 姚东升,孙明瑜,刘平,等. 黄芪汤治疗肝纤维化的研究进展 [J].山西中医学院学报,2012,13(3):146- 148.
[19] ZALDIVAR M M,PAUELS K,von HUNDELSHAUSEN P,et al. CXC chemokine ligand 4 (Cxcl4) is a platelet- derived mediator of experimental liver fibrosis [J].Hepatology,2010,51(4):1345- 1353.
[20] IREDALE J P. Models of liver fibrosis:exploring the dynamic nature of inflammation and repair in a solid organ [J].J Clin Invest,2007,117(3):539- 548.
[21] ALBILLOS A,NIETO M,UBEDA M,et al. The biological response modifier AM3 attenuates the inflammatory cell response and hepatic fibrosis in rats with biliary cirrhosis [J].Gut,2010,59(7):943- 952.
[22] LIU C,TAO Q,SUN M,et al. Kupffer cells are associated with apoptosis,inflammation and fibrotic effects in hepatic fibrosis in rats [J].Lab Invest,2010,90(12):1805- 1816.
[23] YANG L,ROH Y S,SONG J,et al. Transforming growth factor beta signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism in mice[J].Hepatology,2014,59(2):483- 495.
[2] LEUNG V Y,SHEN J,WONG V W,et al. Quantitative elastography of liver fibrosis and spleen stiffness in chronic hepatitis B carriers [J].Radiology,2013,269(3):910- 918.
[3] LIN X,BAI F,NIE J,et al. Didymin Alleviates Hepatic Fibrosis Through Inhibiting ERK and PI3K/Akt Pathways via Regulation of Raf Kinase Inhibitor Protein [J].Cell Physiol Biochem,2016,40(6):1422- 1432.
[4] KNOP V,HOPPE D,WELZEL T,et al. Regression of fibrosis and portal hypertension in HCV- associated cirrhosis and sustained virologic response after interferon- free antiviral therapy [J].J Viral Hepat,2016,23(12):994- 1002.
[5] JIN S,LI H,HAN M,et al. Mesenchymal Stem Cells with Enhanced Bcl- 2 Expression Promote Liver Recovery in a Rat Model of Hepatic Cirrhosis [J].Cell Physiol Biochem,2016,40(5):1117- 1128.
[6] CHEN X,LIU C,LU Y,et al. Paeoniflorin regulates macrophage activation in dimethylnitrosamine- induced liver fibrosis in rats [J].BMC Complement Altern Med,2012,12(1):254.
[7] JASTER R,EMMRICH J. Crucial role of fibrogenesis in pancreatic diseases [J].Best Pract Res Clin Gastroenterol,2008,22(1):17- 29.
[8] PIAO R L,XIU M,BRIGSTOCK D R,et al. An immortalized rat pancreatic stellate cell line RP- 2 as a new cell model for evaluating pancreatic fibrosis,inflammation and immunity [J].Hepatobiliary Pancreat Dis Int,2015,14(6):651- 659.
[9] LIU C,CAI J,CHENG Z,et al. Xiayuxue decoction reduces renal injury by promoting macrophage apoptosis in hepatic cirrhotic rats [J].Genet Mol Res,2015,14(3):10760- 10773.
[10] ZHANGL J,SUN M Y,NING B B,et al. Xiayuxue Decoction attenuates hepatic stellate cell activation and sinusoidal endothelium defenestration in CCl4- induced fibrotic liver of mice [J].Chin J Integr Med,2014,20(7):516- 523.
[11] 都广礼,刘平,王磊,等. 猪血清肝纤维化大鼠肝组织基质金属蛋白酶- 9/13和基质金属蛋白酶组织抑制因子- 1/2表达的动态变化及下瘀血汤对其影响[J].中国实验方剂学杂志,2009,15(11):48- 51.
[12] 丁赛丹,陈必成,刘艳,等. 下瘀血汤干预肝硬化大鼠的蛋白质组学研究[J].中草药,2012,43(1):131- 138.
[13] FERRARIO C M. ACE2:more of Ang- (1- 7) or less AngⅡ? [J].Curr Opin Nephrol Hypertens,2011,20(1):1- 6.
[14] 加秀凤.经方下瘀血汤调控ACE2介导的RAS自稳抑制肝纤维化进展的实验研究 [D].武汉:华中科技大学,2014.
[15] MA W,TAO L,ZHANG W,et al. Xia- Yu- Xue Decoction Inhibits Intestinal Epithelial Cell Apoptosis in CCl4- Induced Liver Fibrosis [J].Cell Physiol Biochem,2017,44(1):333- 344.
[16] 慕永平,刘平,都广礼,等. 祛瘀和养阴不同功效中医经典方剂抑制CCl4诱导大鼠肝硬化的作用机制 [J].自然科学进展,2006,16(9):1101- 1108.
[17] 中国中西医结合学会肝病专业委员会. 肝纤维化中西医结合诊疗指南 [J].中华肝脏病杂志,2006,14(11):866- 870.
[18] 姚东升,孙明瑜,刘平,等. 黄芪汤治疗肝纤维化的研究进展 [J].山西中医学院学报,2012,13(3):146- 148.
[19] ZALDIVAR M M,PAUELS K,von HUNDELSHAUSEN P,et al. CXC chemokine ligand 4 (Cxcl4) is a platelet- derived mediator of experimental liver fibrosis [J].Hepatology,2010,51(4):1345- 1353.
[20] IREDALE J P. Models of liver fibrosis:exploring the dynamic nature of inflammation and repair in a solid organ [J].J Clin Invest,2007,117(3):539- 548.
[21] ALBILLOS A,NIETO M,UBEDA M,et al. The biological response modifier AM3 attenuates the inflammatory cell response and hepatic fibrosis in rats with biliary cirrhosis [J].Gut,2010,59(7):943- 952.
[22] LIU C,TAO Q,SUN M,et al. Kupffer cells are associated with apoptosis,inflammation and fibrotic effects in hepatic fibrosis in rats [J].Lab Invest,2010,90(12):1805- 1816.
[23] YANG L,ROH Y S,SONG J,et al. Transforming growth factor beta signaling in hepatocytes participates in steatohepatitis through regulation of cell death and lipid metabolism in mice[J].Hepatology,2014,59(2):483- 495.