清肠利肝方对急性肝衰竭小鼠肝细胞凋亡的影响
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摘要
目的:探讨中药组方清肠利肝方对D-氨基半乳糖(D-GalN)/脂多糖(LPS)所致急性肝衰竭小鼠肝细胞凋亡的影响。方法:将野生型健康C57BL/6雄性小鼠随机分为正常对照组、急性肝衰竭模型组和清肠利肝方干预组;模型组小鼠腹腔注射D-GalN(700 mg/kg)/LPS(10μg/kg)诱导肝衰竭,清肠利肝方干预组小鼠在D-GalN/LPS给药前3d给予清肠利肝方灌胃,1次/d。D-Gal/LPS给药6h后收集小鼠肝脏组织和血清。观察各组小鼠存活率,采用末端转移酶介导的缺口末端标记法(TUNEL)检测肝细胞凋亡,western blot检测肝细胞中caspase-3的含量,试剂盒检测肝组织caspase-3的活性。结果:模型组小鼠24h存活率为20%,清肠利肝方干预组小鼠存活率为70%。模型组小鼠肝细胞凋亡率为(67.6±3.01)%,caspase-3活性升高,cleaved caspase-3表达量也显著增加;清肠利肝方干预组凋亡率为(16.0±1.76)%,显著低于模型组(P<0.0001),caspase-3活性和cleaved caspase-3表达量均较模型组低,差异有统计学意义(P<0.05)。结论:清肠利肝方可降低D-GalN/LPS所致急性肝衰竭小鼠肝细胞caspase-3表达,减轻肝细胞凋亡。
Objective:To investigate the effect of Qingchangligan formula on hepatocyte apoptosis in acute liver failure mice induced by D-galactosamine/lipopolysaccharide.Methods:Wild-type healthy C57 BL/6 male mice were randomly divided into normal control group,acute liver failure model group,and Qingchangligan formula intervention group.The model group was intraperitoneally injected with D-GalN(700 mg/kg)/LPS(10 μg/kg)induced liver failure,Qingchangligan intervention group: Qingchangligan formula was given intragastrically three days before D-GalN/LPS administration,once a day.6 h after D-Gal/LPS administration,mouse li-ver tissue and serum were collected.The survival rate of each group of mice was observed.Hepatocyte apoptosis was detected by terminal transferase-mediated nick end labeling(TUNEL).Western blot was used to detect the content of caspase-3 in liver cells.The kit was used to detect the activity of caspase-3 in liver tissue.Results:The 24-hour survival rate of the mice in the model group was 20%,and the survival rate of the Qingchangligan formula treated mice was 70%.The apoptotic rate of mice in the model group was 67.6±3.01,caspase-3 activity was increased,and the expression of cleaved caspase-3 was also significantly increased in WB.The apoptotic rate of Qingchangligan formula was 16.0±1.76,which was significantly lower than that in the model group(P<0.0001),caspase-3 activity and cleaved caspase-3 expression were lower than the ALF group,the difference was statistically significant(P<0.05).Conclusion:Qingchangligan formula can reduce the expression of caspase-3 in hepatocytes of mice with acute liver failure induced by D-GalN/LPS and reduce hepatocyte apoptosis.
Objective:To investigate the effect of Qingchangligan formula on hepatocyte apoptosis in acute liver failure mice induced by D-galactosamine/lipopolysaccharide.Methods:Wild-type healthy C57 BL/6 male mice were randomly divided into normal control group,acute liver failure model group,and Qingchangligan formula intervention group.The model group was intraperitoneally injected with D-GalN(700 mg/kg)/LPS(10 μg/kg)induced liver failure,Qingchangligan intervention group: Qingchangligan formula was given intragastrically three days before D-GalN/LPS administration,once a day.6 h after D-Gal/LPS administration,mouse li-ver tissue and serum were collected.The survival rate of each group of mice was observed.Hepatocyte apoptosis was detected by terminal transferase-mediated nick end labeling(TUNEL).Western blot was used to detect the content of caspase-3 in liver cells.The kit was used to detect the activity of caspase-3 in liver tissue.Results:The 24-hour survival rate of the mice in the model group was 20%,and the survival rate of the Qingchangligan formula treated mice was 70%.The apoptotic rate of mice in the model group was 67.6±3.01,caspase-3 activity was increased,and the expression of cleaved caspase-3 was also significantly increased in WB.The apoptotic rate of Qingchangligan formula was 16.0±1.76,which was significantly lower than that in the model group(P<0.0001),caspase-3 activity and cleaved caspase-3 expression were lower than the ALF group,the difference was statistically significant(P<0.05).Conclusion:Qingchangligan formula can reduce the expression of caspase-3 in hepatocytes of mice with acute liver failure induced by D-GalN/LPS and reduce hepatocyte apoptosis.
引文
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[4] 中华医学会感染病学分会肝衰竭与人工肝学组,中华医学会肝病学分会重型肝病与人工肝学组.肝衰竭诊治指南(2012年版)[J].中华肝脏病杂志,2013,2l(3):177-183.
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[8] Mouratidis PX,Colston KW,Dalgleish AG.Doxycycline induces caspase-dependent apoptosis in human pancreatic cancer cells[J].Int J Cancer,2007,120(4):743-752.
[9] Nowak M,Moldawer LL.LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-α and the TNF-p55 receptor[J].Am J Physiol Regulat Integrat Comp Physiol,2000,278:1202-1209.
[10] Brenner C,Galluzzi L,Kepp O,et al.Decoding cell death signals in liver inflammation[J].J Hepatol,2013,59(3):583-594.
[11] Ding WX,Yin XM.Dissection of the multiple mechanisms of TNF-α induced apoptosis in liver injury[J].J Cell Mol Med,2004,8:445-454.
[12] Seki E,Brenner DA,Karin M.A liver full of JNK:signaling in regulation of cell function and disease pathogenesis,and clinical approaches[J].Gastroenterology,2012,143(2):307-320.
[13] Takamura M,Matsuda Y,Yamagiwa S,et al.An inhibitor of c-Jun NH2-terminal kinase,SP600125,protects mice from D-galactosamine/lipopolysaccharide-induced hepatic failure by modulating BH3-only proteins[J].Life Sci,2007,80(14):1335-1344.
[2] Lee WM,Squires Jr RH,Nyberg SL,et al.Acute liver failure:Summary of a workshop[J].Hepatology,2008,47(4):1401-1415.
[3] Michael D,Josephs FRB,Fukuzuka K,et al.Lipopolysaccharide and D-galactosamine-induced hepatic injury is mediated by TNF-α and not by Fas ligand[J].Am J Physiol Regulat Integrat Comp Physiol,2000,278:1196-1201.
[4] 中华医学会感染病学分会肝衰竭与人工肝学组,中华医学会肝病学分会重型肝病与人工肝学组.肝衰竭诊治指南(2012年版)[J].中华肝脏病杂志,2013,2l(3):177-183.
[5] Zong L,Yu QH,Du YX,et al.PMC3982944Edaravone protects endotoxin-induced liver injury by inhibiting apoptosis and reducing proinflanunatory cytokines[J].Braz J Med Biol Res,2014,47(3):231-236.
[6] Chen LV,Yang BS,Zhou L,et al.Promotion of mitochondrial energy metabolism during hepatocyte apoptosis in a rat model of acute liver failure[J].Mol Med Rep,2015,12(4):5035-5041.
[7] Luo M,Zhao A,Li J,et al.Acute liver injury attenuation of a novel recombinant sTNFR through blocking hepatic apoptosis[J].Immunopharmacol Immunotoxicol,2015,37(3):295-300.
[8] Mouratidis PX,Colston KW,Dalgleish AG.Doxycycline induces caspase-dependent apoptosis in human pancreatic cancer cells[J].Int J Cancer,2007,120(4):743-752.
[9] Nowak M,Moldawer LL.LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-α and the TNF-p55 receptor[J].Am J Physiol Regulat Integrat Comp Physiol,2000,278:1202-1209.
[10] Brenner C,Galluzzi L,Kepp O,et al.Decoding cell death signals in liver inflammation[J].J Hepatol,2013,59(3):583-594.
[11] Ding WX,Yin XM.Dissection of the multiple mechanisms of TNF-α induced apoptosis in liver injury[J].J Cell Mol Med,2004,8:445-454.
[12] Seki E,Brenner DA,Karin M.A liver full of JNK:signaling in regulation of cell function and disease pathogenesis,and clinical approaches[J].Gastroenterology,2012,143(2):307-320.
[13] Takamura M,Matsuda Y,Yamagiwa S,et al.An inhibitor of c-Jun NH2-terminal kinase,SP600125,protects mice from D-galactosamine/lipopolysaccharide-induced hepatic failure by modulating BH3-only proteins[J].Life Sci,2007,80(14):1335-1344.