吡咯烷二硫代氨基甲酸酯在二甲基亚硝胺诱导肝纤维化大鼠中的作用及机制
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摘要
目的观察吡咯烷二硫代氨基甲酸酯(pyrrolidine dithiocarbamate,PDTC)对二甲基亚硝胺(dimethylnitrosamine,DMN)诱导肝纤维化大鼠肝组织核转录因子-κB(nuclear factor-κB,NF-κB)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)及Ⅲ型胶原(collagen typeⅢ,Col-Ⅲ)表达的影响,探讨PDTC抗肝纤维化的可能机制。方法 22只SD大鼠随机分为对照组6只,模型组8只和治疗组8只。模型组和治疗组腹腔注射质量分数0.5%DMN溶液2mL/kg,每2d注射1次,连续4周,诱导大鼠肝纤维化模型;对照组腹腔注射等量生理盐水。治疗组造模期间给予PDTC溶液150mg/(kg·d)灌胃,对照组、模型组给予等量蒸馏水灌胃。实验结束后处死大鼠,取肝左叶组织光镜下观察肝组织病理改变,采用电泳迁移率试验检测肝组织NF-κBp65活性,实时荧光定量PCR法检测肝组织NF-κBp65、TGF-β1和Col-ⅢmRNA表达,免疫组织化学法检测肝组织NF-κBp65着色细胞数目和Col-Ⅲ着色面积。结果对照组肝小叶结构正常,细胞排列规律,未见肝细胞变性、坏死;模型组肝细胞融合性坏死,胶原纤维明显增生,假小叶形成;治疗组肝细胞肿胀、变性及炎症细胞浸润程度均较模型组轻;模型组、治疗组肝组织NF-κBp65活性[(76.6±6.5)%、(51.2±4.6)%],NF-κBp65mRNA(0.019 3±0.005 0、0.009 5±0.002 1)、TGF-β1mRNA(0.017 5±0.001 2、0.009 7±0.001 1)及Col-ⅢmRNA表达(0.077 5±0.030 5、0.030 1±0.003 3)均高于对照组((28.5±3.3)%、0.003 0±0.001 4、0.004 7±0.000 9、0.015 2±0.005 5)(P<0.05),NF-κBp65着色细胞数目[(68.60±4.65)、(49.65±3.60)个]均较对照组[(14.68±2.98)个]多(P<0.05),Col-Ⅲ着色面积[(10.39±1.53)、(7.36±1.17)μm2]均较对照组[(2.46±0.31)μm2]大(P<0.05);模型组肝组织NF-κBp65活性,NF-κBp65mRNA、TGF-β1mRNA及Col-ⅢmRNA表达均较治疗组高,NF-κBp65着色细胞数目较治疗组多,Col-Ⅲ着色面积较治疗组大(P<0.05);Pearson相关分析结果显示,肝组织NF-κBp65活性与NF-κBp65mRNA、TGF-β1mRNA、Col-ⅢmRNA表达,NF-κBp65着色细胞数目,Col-Ⅲ着色面积均呈正相关(r=0.884,P<0.001;r=0.976,P<0.001;r=0.769,P<0.001;r=0.934,P<0.001;r=0.942,P<0.001),TGF-β1 mRNA与Col-ⅢmRNA表达及Col-Ⅲ着色面积均呈正相关(r=0.831,P<0.001;r=0.918,P<0.001)。结论PDTC可能通过抑制NF-κB活性,下调TGF-β1表达,抑制以Col-Ⅲ为主要成分的细胞外基质过度沉积,来发挥抗肝纤维化作用。
Objective To observe the influences of pyrrolidine dithiocarbamate(PDTC)on the expressions of nuclear factor-κB(NF-κB),transforming growth factor-β1(TGF-β1)and collagen type Ⅲ(Col-Ⅲ)to explore the possible mechanism of anti-hepatic fibrosis in rats with dimethylnitrosamine(DMN)-induced liver fibrosis.Methods Twenty-two male SD rats were randomly divided into model group(n=8),treatment group(n=8)and control group(n=6).Hepatic fibrosis rat models were induced by injecting intraperitoneally 2 mL/kg of 0.5% DMN solution once every two days for four weeks in model group and treatment group.Control group was intraperitoneally injected with equivalent volume of normal saline.Treatment group received lavage by 150 mg/(kg·d)of PDTC solution,and control group and model group received lavage by the same amount of distilled water.At the end of the experiment,the rats were sacrificed and the tissue from the left liverwas obtained.The pathological changes of the liver tissue were observed under light microscope.The activity of NF-κBp65 of the liver tissue was detected by electrophoretic mobility shift assay.The mRNA expressions of NF-κBp65,TGF-β1 and Col-Ⅲ were detected by real-time PCR.The number of cells positively expressing NF-κBp65 and the area of cells positively expressing Col-Ⅲ in the liver tissue were detected by immunohistochemical staining technique.Results Control group was found normal hepatic lobule structure,regular arrangement of cells and no liver cell degeneration or necrosis.Model group was found liver cell fusion necrosis,obvious collagen fiber hyperplasia,and formation of pseudolobule.Treatment group was found swelling and degeneration of liver cells and the infiltration of inflammatory cells,which was lighter than model group.The activities of NF-κBp65((76.6±6.5)%,(51.2±4.6)%),NF-κBp65 mRNA(0.019 3±0.005 0,0.009 5±0.002 1),TGF-β1 mRNA(0.017 5±0.001 2,0.009 7±0.001 1)and Col-Ⅲ mRNA(0.077 5±0.030 5,0.030 1±0.003 3)in model group and treatment group were significantly higher than those in control group((28.5±3.3)%,0.003 0±0.001 4,0.004 7±0.000 9,0.015 2±0.005 5)(P<0.05),the number of cells positively expressing NF-κBp65 was significantly more in model group(68.60±4.65)and treatment group(49.65±3.60)than that in control group(14.68±2.98)(P<0.05),and the area of cells positively expressing Col-Ⅲ was significantly larger in model group((10.39±1.53)μm2)and treatment group((7.36±1.17)μm2)than that in control group((2.46±0.31)μm2)(P<0.05),and all above indexes were significantly higher in model group than those in treatment group(P<0.05).Pearson correlation analysis showed that the activity of NF-κBp65 in liver tissue was positively correlated with the mRNA expressions of NF-κBp65,TGF-β1 and Col-Ⅲ(r=0.884,P<0.001;r=0.976,P<0.001;r=0.769,P<0.001),the number of cells positively expressing NF-κBp65(r=0.934,P<0.001)and the area of cells positively expressing Col-Ⅲ(r=0.942,P<0.001).The mRNA expression of TGF-β1 was positively correlated with the expression of Col-Ⅲ mRNA and the area of cells positively expressing Col-Ⅲ(r=0.831,P<0.001;r=0.918,P<0.001).Conclusion PDTC protects against hepatic fibrosis by inhibiting the activity of NF-κBp65,down-regulatingthe expression of TGF-β1,and inhibiting the over-deposition of extracellular matrix which mainly contains Col-Ⅲ.
Objective To observe the influences of pyrrolidine dithiocarbamate(PDTC)on the expressions of nuclear factor-κB(NF-κB),transforming growth factor-β1(TGF-β1)and collagen type Ⅲ(Col-Ⅲ)to explore the possible mechanism of anti-hepatic fibrosis in rats with dimethylnitrosamine(DMN)-induced liver fibrosis.Methods Twenty-two male SD rats were randomly divided into model group(n=8),treatment group(n=8)and control group(n=6).Hepatic fibrosis rat models were induced by injecting intraperitoneally 2 mL/kg of 0.5% DMN solution once every two days for four weeks in model group and treatment group.Control group was intraperitoneally injected with equivalent volume of normal saline.Treatment group received lavage by 150 mg/(kg·d)of PDTC solution,and control group and model group received lavage by the same amount of distilled water.At the end of the experiment,the rats were sacrificed and the tissue from the left liverwas obtained.The pathological changes of the liver tissue were observed under light microscope.The activity of NF-κBp65 of the liver tissue was detected by electrophoretic mobility shift assay.The mRNA expressions of NF-κBp65,TGF-β1 and Col-Ⅲ were detected by real-time PCR.The number of cells positively expressing NF-κBp65 and the area of cells positively expressing Col-Ⅲ in the liver tissue were detected by immunohistochemical staining technique.Results Control group was found normal hepatic lobule structure,regular arrangement of cells and no liver cell degeneration or necrosis.Model group was found liver cell fusion necrosis,obvious collagen fiber hyperplasia,and formation of pseudolobule.Treatment group was found swelling and degeneration of liver cells and the infiltration of inflammatory cells,which was lighter than model group.The activities of NF-κBp65((76.6±6.5)%,(51.2±4.6)%),NF-κBp65 mRNA(0.019 3±0.005 0,0.009 5±0.002 1),TGF-β1 mRNA(0.017 5±0.001 2,0.009 7±0.001 1)and Col-Ⅲ mRNA(0.077 5±0.030 5,0.030 1±0.003 3)in model group and treatment group were significantly higher than those in control group((28.5±3.3)%,0.003 0±0.001 4,0.004 7±0.000 9,0.015 2±0.005 5)(P<0.05),the number of cells positively expressing NF-κBp65 was significantly more in model group(68.60±4.65)and treatment group(49.65±3.60)than that in control group(14.68±2.98)(P<0.05),and the area of cells positively expressing Col-Ⅲ was significantly larger in model group((10.39±1.53)μm2)and treatment group((7.36±1.17)μm2)than that in control group((2.46±0.31)μm2)(P<0.05),and all above indexes were significantly higher in model group than those in treatment group(P<0.05).Pearson correlation analysis showed that the activity of NF-κBp65 in liver tissue was positively correlated with the mRNA expressions of NF-κBp65,TGF-β1 and Col-Ⅲ(r=0.884,P<0.001;r=0.976,P<0.001;r=0.769,P<0.001),the number of cells positively expressing NF-κBp65(r=0.934,P<0.001)and the area of cells positively expressing Col-Ⅲ(r=0.942,P<0.001).The mRNA expression of TGF-β1 was positively correlated with the expression of Col-Ⅲ mRNA and the area of cells positively expressing Col-Ⅲ(r=0.831,P<0.001;r=0.918,P<0.001).Conclusion PDTC protects against hepatic fibrosis by inhibiting the activity of NF-κBp65,down-regulatingthe expression of TGF-β1,and inhibiting the over-deposition of extracellular matrix which mainly contains Col-Ⅲ.
引文
[1]陈琴,陈连云,金欢欢,等.肝脏树突状细胞在肝纤维化中的作用[J].中国药理学通报,2015,31(8):1053-1056.
[2]宋新文,高海丽,王宏伟,等.PDTC对肝纤维化大鼠核转录因子-κB、α-平滑肌肌动蛋白和Ⅰ型胶原表达影响研究[J].免疫学杂志,2013,29(3):216-221.
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[6]KIM K H,LEE W R,KANG Y N,et al.Inhibitory effect of nuclear factor-κB decoy oligodeoxynucleotide on liver fibrosis through regulation of the epithelial-mesenchymal transition[J].Hum Gene Ther,2014,25(8):721-729.
[7]WANG Y,WANG R,WANG Y J,et al.Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38 MAPK,NF-κB/IκBα,and Bcl-2/Bax signaling[J].Drug Des Devel Ther,2015,9:6303-6317.
[8]BOOPALAN T,ARUMUGAM A,PARADA J,et al.Receptor activator for nuclear factor-κB ligand signaling promotes progesterone-mediated estrogen-induced mammary carcinogenesis[J].Cancer Sci,2015,106(1):25-33.
[9]KONO Y,KAWAKAMI S,HIGUCHI Y,et al.Antitumor effect of nuclear factor-κB decoy transfer by mannose-modified bubble lipoplex into macrophages in mouse malignant ascites[J].Cancer Sci,2014,105(8):1049-1055.
[10]周宇,陈科全,叶石才,等.核因子-κBp65反义寡核苷酸对肝纤维化作用的实验研究[J].中华消化杂志,2009,29(4):254-257.
[11]黄强.银杏叶提取物对大鼠纤维化肝脏NF-κB活性的抑制作用[J].中国中医急症,2010,19(12):2102-2103.
[12]闫静波.AcSDKP对硅肺纤维化大鼠胶原含量及NF-κB p65表达的影响[J].重庆医学,2013,42(26):3139-3141.
[13]余晓红,吕宏迪,杨旸,等.转化生长因子-β1和Smad7信号通路与大鼠肝纤维化的相关性[J].中华实用诊断与治疗杂志,2015,29(7):648-650.
[14]ZHOU J,LIANG Y,PAN J X,et al.Protein extracts of Crassostrea gigas alleviate CCL4-induced hepatic fibrosis in rats by reducing the expression of CTGF,TGF-β1and NF-κB in liver tissues[J].Mol Med Rep,2015,11(4):2913-2920.
[15]康谊,曾艳丽,魏君锋,等.罗格列酮抑制大鼠肝纤维化机制研究[J].中华实用诊断与治疗杂志,2013,27(7):676-679.
[16]彭云龙,陈燕花,翁雨雄,等.转化生长因子-β1促进失神经骨骼肌肌源性干细胞表达胶原纤维的研究[J].中华手外科杂志,2012,28(1):51-54.
[17]李彩,林丽馨,肖绪华,等.荔枝核总黄酮抑制人肝星状细胞增殖作用机制的研究[J].安徽医科大学学报,2016,51(2):171-175.
[2]宋新文,高海丽,王宏伟,等.PDTC对肝纤维化大鼠核转录因子-κB、α-平滑肌肌动蛋白和Ⅰ型胶原表达影响研究[J].免疫学杂志,2013,29(3):216-221.
[3]王丽娜,刘成海,陈园,等.一种改良的二甲基亚硝胺肝纤维化模型诱导方法及其病理特点[J].中国实验动物学报,2007,15(2):90-94.
[4]王辉,王春,葛莹,等.内翻性乳头状瘤中角蛋白免疫组化方法研究[J].大连医科大学学报,2014,36(4):318-321.
[5]ALGANDABY M M,EL-HALAWANY A M,ABDALLAH H M,et al.Gingerol protects against experimental liver fibrosis in rats via suppression of pro-inflammatory and profibrogenic mediators[J].Naunyn Schmiedebergs Arch Pharmacol,2016,389(4):419-428.
[6]KIM K H,LEE W R,KANG Y N,et al.Inhibitory effect of nuclear factor-κB decoy oligodeoxynucleotide on liver fibrosis through regulation of the epithelial-mesenchymal transition[J].Hum Gene Ther,2014,25(8):721-729.
[7]WANG Y,WANG R,WANG Y J,et al.Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38 MAPK,NF-κB/IκBα,and Bcl-2/Bax signaling[J].Drug Des Devel Ther,2015,9:6303-6317.
[8]BOOPALAN T,ARUMUGAM A,PARADA J,et al.Receptor activator for nuclear factor-κB ligand signaling promotes progesterone-mediated estrogen-induced mammary carcinogenesis[J].Cancer Sci,2015,106(1):25-33.
[9]KONO Y,KAWAKAMI S,HIGUCHI Y,et al.Antitumor effect of nuclear factor-κB decoy transfer by mannose-modified bubble lipoplex into macrophages in mouse malignant ascites[J].Cancer Sci,2014,105(8):1049-1055.
[10]周宇,陈科全,叶石才,等.核因子-κBp65反义寡核苷酸对肝纤维化作用的实验研究[J].中华消化杂志,2009,29(4):254-257.
[11]黄强.银杏叶提取物对大鼠纤维化肝脏NF-κB活性的抑制作用[J].中国中医急症,2010,19(12):2102-2103.
[12]闫静波.AcSDKP对硅肺纤维化大鼠胶原含量及NF-κB p65表达的影响[J].重庆医学,2013,42(26):3139-3141.
[13]余晓红,吕宏迪,杨旸,等.转化生长因子-β1和Smad7信号通路与大鼠肝纤维化的相关性[J].中华实用诊断与治疗杂志,2015,29(7):648-650.
[14]ZHOU J,LIANG Y,PAN J X,et al.Protein extracts of Crassostrea gigas alleviate CCL4-induced hepatic fibrosis in rats by reducing the expression of CTGF,TGF-β1and NF-κB in liver tissues[J].Mol Med Rep,2015,11(4):2913-2920.
[15]康谊,曾艳丽,魏君锋,等.罗格列酮抑制大鼠肝纤维化机制研究[J].中华实用诊断与治疗杂志,2013,27(7):676-679.
[16]彭云龙,陈燕花,翁雨雄,等.转化生长因子-β1促进失神经骨骼肌肌源性干细胞表达胶原纤维的研究[J].中华手外科杂志,2012,28(1):51-54.
[17]李彩,林丽馨,肖绪华,等.荔枝核总黄酮抑制人肝星状细胞增殖作用机制的研究[J].安徽医科大学学报,2016,51(2):171-175.