忍冬木层孔菌总酚通过调控肥大细胞抗四氯化碳所致大鼠肝纤维化
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摘要
探讨忍冬木层孔菌总酚(Phellinus lonicerinus total phenolics,PLTP)调控肥大细胞(Mast Cell,MC)改善四氯化碳(CCl4)诱导的大鼠肝纤维化的作用.将50只SD大鼠随机分为正常组、模型组、秋水仙碱组(0.2mg/kg)、PLTP低剂量组(250mg/kg)、PLTP高剂量组(500mg/kg),每组10只.除正常组注射橄榄油外,其余各组腹腔注射2mL/kg 50%CCl4橄榄油溶液,2次/w,共8w.首次注射后各给药组每天灌胃对应剂量的药物,正常组与模型组灌胃相应体积的生理盐水,1次/d,共8w.末次给药后禁食24h,取大鼠血和肝脏,检测血清中谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)、碱性磷酸酶(alkaline phosphatase,ALP)及透明质酸(haluronic acid,HA)、Ⅲ型前胶原(procollagen typeⅢ,PCⅢ)、Ⅳ型胶原(typeⅣcollagen,Ⅳ-C)的含量;免疫组化测定α-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)的表达量,同时切片观察肝脏组织学变化及肥大细胞的数量及脱颗粒变化.结果显示,与模型组比较,PLTP能降低大鼠血清AST、ALT及ALP的活性,降低血清中HA、PCⅢ、Ⅳ-C的含量;病理学检查结果表明PLTP能明显减轻CCl4引起的大鼠肝损伤及纤维化;甲苯胺蓝染色(TB)可见CCl4模型组大鼠肝脏血管周围及纤维间隔内大量正在脱颗粒和已经脱颗粒的充满蓝紫色颗粒的MC,MC数目较正常组明显升高,PLTP组MC数量明显减少.同时,免疫组织化学染色结果显示PLTP下调肝组织中α-SMA的表达.综上,PLTP对CCl4致大鼠肝纤维化有防治作用,其作用机制可能与减少MC的数量及其脱颗粒、下调肝组织中α-SMA的表达有关.
To investigate the protective effect of total phenolics acid of Phellinus lonicerinus(Bond.)Bond.et Sing(PLTP)in resisting hepatic fibrosis by regulating mast cells.50 rats were randomly divided into normal group,model group,colchicine group(0.2 mg/kg),PLTP-high and low dose group(500,250 mg/kg).Rat hepatic fibrosis was induced by intraperitoneal injection of 50% CCl4-olive oil solution at a dose of 2 mL/kg twice weekly for 8 w.After the first injection,the normal group and model group were given equivoluminal physiological saline,and other groups were given the corresponding does of drugs once per day for8 w.After the last administration,the rats were fasting for 24 h,and all the rats were killed with blood samples and liver tissues collected The serum contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),hyaluronic acid(HA),precollagen typeⅢ(PCⅢ)and typeⅣcollagen(Ⅳ-C)were measured biochemical method.Theα-smooth muscle actin(α-SMA)were detected by immunohistochemical assays.Meanwhile,the histopathological changes and mast cell(MC)of the liver tissue were observed.The results showed that the PLTP could significantly improve the liver tissue lesion,decrease the levels of ALT,AST,ALP,HA,PCⅢandⅣ-C.The microexamination results of histological sections showed that PLTP significantly reduced the rat liver injury and fibrosis induced by CCl4.The CCl4 model group exhibited flatty degeneration and fibrosis with many degranulating and degranulated mast cells filled with purple granula located around the liver blood vessels and in fiber-interval,while those of PLTP-treated group decreased significantly.Immunohistochemical staining suggested that the expression ofα-SMA was down-regulated with PLTP treatment.In summary,the PLTP possessed preventive and therapeutic effects on hepatic fibrosis induced by CCl4 in rats,and the mechanism might be related to the decrease of the number of mast cell and the expression ofα-SMA in liver tissues.
To investigate the protective effect of total phenolics acid of Phellinus lonicerinus(Bond.)Bond.et Sing(PLTP)in resisting hepatic fibrosis by regulating mast cells.50 rats were randomly divided into normal group,model group,colchicine group(0.2 mg/kg),PLTP-high and low dose group(500,250 mg/kg).Rat hepatic fibrosis was induced by intraperitoneal injection of 50% CCl4-olive oil solution at a dose of 2 mL/kg twice weekly for 8 w.After the first injection,the normal group and model group were given equivoluminal physiological saline,and other groups were given the corresponding does of drugs once per day for8 w.After the last administration,the rats were fasting for 24 h,and all the rats were killed with blood samples and liver tissues collected The serum contents of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),hyaluronic acid(HA),precollagen typeⅢ(PCⅢ)and typeⅣcollagen(Ⅳ-C)were measured biochemical method.Theα-smooth muscle actin(α-SMA)were detected by immunohistochemical assays.Meanwhile,the histopathological changes and mast cell(MC)of the liver tissue were observed.The results showed that the PLTP could significantly improve the liver tissue lesion,decrease the levels of ALT,AST,ALP,HA,PCⅢandⅣ-C.The microexamination results of histological sections showed that PLTP significantly reduced the rat liver injury and fibrosis induced by CCl4.The CCl4 model group exhibited flatty degeneration and fibrosis with many degranulating and degranulated mast cells filled with purple granula located around the liver blood vessels and in fiber-interval,while those of PLTP-treated group decreased significantly.Immunohistochemical staining suggested that the expression ofα-SMA was down-regulated with PLTP treatment.In summary,the PLTP possessed preventive and therapeutic effects on hepatic fibrosis induced by CCl4 in rats,and the mechanism might be related to the decrease of the number of mast cell and the expression ofα-SMA in liver tissues.
引文
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[9]昝立峰,包海鹰,李丹花.“桑黄”类真菌中多酚物质及其生物活性研究进展[J].天然产物研究与开发,2016,28:147-155.ZAN L F,BAO H Y,LI D H.Review on polyphenol components from medicinal fungi“Sanghuang”and their biological activity[J].Nat Prod Res Dev,2016,28:147-155.(Ch).
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[14]姜相君,林惠忠,解祥军.血清HA、PCⅢ、Ⅳ-C和LN水平对诊断肝纤维化的临床意义[J].标记免疫分析与临床,2002,9(4):207-208.JIANG X J,LIN H Z,JIE X J.The diagnostic value of serum HA,PCⅢ,Ⅳ-C and LN for hepatofibrosis[J].Labeled Immunoassays and Clin Med,2002,9(4):207-208.
[15]黄静,龙子江,李丽,等.水飞蓟宾胶囊对酒精性肝纤维化大鼠的保护作用[J].中成药,2016,38(2):229-234.HUNG J,LONG Z J,LI L,et al.Protective effects of silybin capsules on alcohol-induced hepatic fibrosis in rats[J].Chinese Traditional Patent Medicine,2016,38(2):229-234.(Ch).
[16]王磊,魏宁,徐浩,等.血清肝纤维化HA、Ⅳ-C、PCⅢ和LN联合检测在布加综合征导致的肝纤维化诊断中的价值[J].世界华人消化杂志,2012,20(36):3791-3794.WANG L,WEI N,XU H,et al.Detection of serum levels of HA,Ⅳ-C,PCⅢand LN in patients with Budd-Chiari syndrome[J].World Chinese Journal of Digestology,2012,20(36):3791-3794.(Ch).
[17]韩聚强,任永强,曹建彪.肥大细胞与肝炎病毒相关肝病研究进展[J].中华临床医师杂志(电子版),2011,5(18):5410-5411.HAN JQ,REN YQ,CAO JB.Advances in studies on mast cells and hepatopathy related to hepatitis virus[J].Chinese Journal of Clinicians(Electronic Edition),2011,5(18):5410-5411.(Ch).
[18] SHEN D Z.A target role for mast cell in the prevention and therapy of hepatic fibrosis[J].Med Hypotheses,2008,70(4):760-764.
[19] LU J,CHEN B,LI S,et al.Tryptase inhibitor APC 366prevents hepatic fibrosis by inhibiting collagen synthesis induced by tryptase/protease-activated receptor 2interactions in hepatic stellate cells[J].Int Immunopharmacol,2014,20(2):352-357.
[20] MASUBUCHI S,TAKAI S,JIN D,et al.Chymase inhibitor ameliorates hepatic steatosis and fibrosis on established non-alcoholic steatohepatitis in hamsters fed a methionineand choline-deficient diet[J].Hepatol Res,2013,43(9):970-978.
[21]叶立红,王翀奎,杨莉,等.软肝化坚颗粒对肝纤维化模型C57小鼠肝组织α-SMA、TGFβ1表达和I型胶原水平的影响[J].临床肝胆病杂志,2012,28(9):685-688.YE L H,WANG C K,YANG L,et al.Effects of Ruangan Huajian granule on hepatic expression ofα-SMA,TGF-β1,and COL-Ⅰin C57mice with experimental hepatic fibrosis[J].J Clini Hepatol,2012,28(9):685-688.
[2] ELPEK G.Cellular and molecular mechanisms in the pathogenesis of liver fibrosis:An update[J].World J Gastroenterol,2014,20(23):7260-7276.
[3] JARIDO V,KENNEDY L,HARGROVE L,et al.The emerging role of mast cells in liver disease[J].Am J Physiol Gastrointest Liver Physiol,2017,313(2):G89-G101.
[4] LI J L,CAI W S,SHEN F,et al.Protease-activated receptor-2modulates hepatic stellate cell collagen release and apoptotic status[J].Arch Biochem Biophys,2014,545:162-166.
[5] DOMITROVIC'R,POTOCˇNJAK I.A comprehensive overview of hepatoprotective natural compounds:mechanism of action and clinical perspectives[J].Arch Toxicol,2016,90(1):39-79.
[6]刘春静,戴玉成.中国锈革孔菌科一新记录种——忍冬木层孔菌[J].林业科学研究,2002,15(4):413-415.LIU C J,DAI Y C.A new polypore in China——Phellinus lonicericola[J].Forest Research,2002,15(4):413-415.(Ch).
[7]罗友成,汪鋆植,吴方方,等.忍冬木层孔菌醇提物对大鼠肝纤维化的影响[J].中药材,2013,36(8):1325-1328.LUO Y C,WANG J Z,WU F F,et al.Effects of ethanol extracts of Phellinus Lonicerinus(Bond.)Bond.et sing on hepatic fibrosis[J].Journal of Chinese Medicinal Materials,2013,36(8):1325-1328.(Ch).
[8]汪鋆植,武玲,罗友成,等.忍冬木层孔菌醇提物对肝纤维化大鼠肝星状细胞的影响[J].中药材,2015,38(8):1681-1685.WANG J Z,WU L,LUO Y C,et al.Effects of ethanol extracts of Phellinus Lonicerinus on hepatic stellate cells of fibrosis liver in rats[J].Journal of Chinese Medicinal Materials,2015,38(8):1681-1685.(Ch).
[9]昝立峰,包海鹰,李丹花.“桑黄”类真菌中多酚物质及其生物活性研究进展[J].天然产物研究与开发,2016,28:147-155.ZAN L F,BAO H Y,LI D H.Review on polyphenol components from medicinal fungi“Sanghuang”and their biological activity[J].Nat Prod Res Dev,2016,28:147-155.(Ch).
[10]周俊,孙文军,张玲,等.忍冬木层孔菌总酚的HPLC分离及其指示性成分分析[J].三峡大学学报(自然科学版),2016,28:147-155.ZHOU J,SUN W J,ZHANG L,et al.The HPLC separation and structural analysis of index composition for the total phenols in Phellinus Lonicerinus[J].Journal of China Three Gorges University(Natural Sciences),2016,28:147-155.(Ch).
[11] LEE I K,HAN M S,LEE M S,et al.Styrylpyrones from the medicinal fungus Phellinus baumii and their antioxidant properties[J].Bioorg Med Chem Lett,2010,20:5459-5461.(Ch).
[12] FRANCIS H,MEININGER CJ.A review of mast cells and liver disease:What have we learned?[J].Dig Liver Dis.,2010,42(8):529-536.
[13] NIE Y,REN D,LU X,et al.Differential protective effects of polyphenol extracts from apple peels and fleshes against acute CCl4-induced liver damage in mice[J].Food Funct,2015,6(2):513-524.
[14]姜相君,林惠忠,解祥军.血清HA、PCⅢ、Ⅳ-C和LN水平对诊断肝纤维化的临床意义[J].标记免疫分析与临床,2002,9(4):207-208.JIANG X J,LIN H Z,JIE X J.The diagnostic value of serum HA,PCⅢ,Ⅳ-C and LN for hepatofibrosis[J].Labeled Immunoassays and Clin Med,2002,9(4):207-208.
[15]黄静,龙子江,李丽,等.水飞蓟宾胶囊对酒精性肝纤维化大鼠的保护作用[J].中成药,2016,38(2):229-234.HUNG J,LONG Z J,LI L,et al.Protective effects of silybin capsules on alcohol-induced hepatic fibrosis in rats[J].Chinese Traditional Patent Medicine,2016,38(2):229-234.(Ch).
[16]王磊,魏宁,徐浩,等.血清肝纤维化HA、Ⅳ-C、PCⅢ和LN联合检测在布加综合征导致的肝纤维化诊断中的价值[J].世界华人消化杂志,2012,20(36):3791-3794.WANG L,WEI N,XU H,et al.Detection of serum levels of HA,Ⅳ-C,PCⅢand LN in patients with Budd-Chiari syndrome[J].World Chinese Journal of Digestology,2012,20(36):3791-3794.(Ch).
[17]韩聚强,任永强,曹建彪.肥大细胞与肝炎病毒相关肝病研究进展[J].中华临床医师杂志(电子版),2011,5(18):5410-5411.HAN JQ,REN YQ,CAO JB.Advances in studies on mast cells and hepatopathy related to hepatitis virus[J].Chinese Journal of Clinicians(Electronic Edition),2011,5(18):5410-5411.(Ch).
[18] SHEN D Z.A target role for mast cell in the prevention and therapy of hepatic fibrosis[J].Med Hypotheses,2008,70(4):760-764.
[19] LU J,CHEN B,LI S,et al.Tryptase inhibitor APC 366prevents hepatic fibrosis by inhibiting collagen synthesis induced by tryptase/protease-activated receptor 2interactions in hepatic stellate cells[J].Int Immunopharmacol,2014,20(2):352-357.
[20] MASUBUCHI S,TAKAI S,JIN D,et al.Chymase inhibitor ameliorates hepatic steatosis and fibrosis on established non-alcoholic steatohepatitis in hamsters fed a methionineand choline-deficient diet[J].Hepatol Res,2013,43(9):970-978.
[21]叶立红,王翀奎,杨莉,等.软肝化坚颗粒对肝纤维化模型C57小鼠肝组织α-SMA、TGFβ1表达和I型胶原水平的影响[J].临床肝胆病杂志,2012,28(9):685-688.YE L H,WANG C K,YANG L,et al.Effects of Ruangan Huajian granule on hepatic expression ofα-SMA,TGF-β1,and COL-Ⅰin C57mice with experimental hepatic fibrosis[J].J Clini Hepatol,2012,28(9):685-688.