柴胡皂苷b_2对LPS/GalN诱导的小鼠急性肝损伤炎症和能量代谢的影响
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摘要
该研究旨在探讨柴胡皂苷b2(SS-b2)对注射脂多糖/氨基半乳糖(LPS/Gal N)诱导的小鼠急性肝损伤炎症因子的调控和对能量代谢的影响。将小鼠随机分为空白对照组(等量生理盐水),模型组(100μg·kg~(-1)LPS+400 mg·kg~(-1)Gal N),SS-b2低、中、高剂量组(5,10,20 mg·kg~(-1)·d-1)和阳性对照组(地塞米松,10 mg·kg~(-1))。除空白对照组外,其余各组每天腹腔注射LPS/Gal N建立急性肝损伤小鼠模型。计算脏器指数;血清生化指标检测ALT和AST的含量和肝脏ATP酶(Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶)活性的变化;酶联免疫吸附(ELISA)法检测血清中TNF-α,IL-1β,IL-6的含量;微量酶标法测定肝脏LDH的含量; HE染色观察肝组织病理学变化;免疫组化法测定LDH-A的蛋白表达; Western blot法检测肝脏Sirt-6和NF-κB蛋白表达情况。结果显示,与模型组相比,SS-b2高剂量组小鼠脏器指数均有明显变化(P<0. 05); SS-b2中、高剂量组能明显降低肝损伤小鼠血清中ALT,AST,TNF-α,IL-1β,IL-6的水平及LDH活性(P<0. 01),随着SS-b2剂量的增加,小鼠肝脏病变范围减少,损伤程度减轻,各剂量组能够显著增强小鼠肝脏Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶的活性(P<0. 01); SS-b2中、高剂量组能够显著下调肝组织NF-κB的蛋白表达(P<0. 01),同时,各剂量组能够显著提高急性肝损伤小鼠肝脏中Sirt-6蛋白表达量(P<0. 01)。综上,SS-b2对LPS/Gal N诱导的小鼠急性肝损伤具有显著的保护作用,该作用可能与其下调NF-κB蛋白表达以及上调Sirt-6蛋白表达改善炎症损伤以及能量代谢有关。
To study the effects of saikosaponin b2( SS-b2) on inflammatory factors and energy metabolism against lipopolysaccharide/galactosamine( LPS/Gal N) induced acute liver injury in mice. Mice were randomly divided into normal group( equal amount of normal saline),model group( 100 g·kg~(-1) LPS and 400 mg·kg~(-1) Gal N),low,medium,high dose group of SS-b2( SS-b25,10,20 mg·kg~(-1)·d-1) and positive control group( dexamethasone,10 mg·kg~(-1)). All of the groups except for the normal group were treated with LPS/Gal N though intraperitoneally injection to establish the acute liver injury model. The organ indexes were calculated. The levels of serum transaminases( ALT and AST) and the activities of ATPase( Na+-K+-ATPase,Ca2+-Mg2+-ATPase) in liver were detected. The activity of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and interleukin-6( IL-6) were determined by the enzyme-linked immunosorbent assay( ELISA). The contents of lactate dehydrogenase( LDH) in liver were determined by micro-enzyme method. HE staining was used to observe the histopathological changes of the liver. Histochemical method was used to investigate the protein expression of liver lactate dehydrogenase-A( LDH-A). The protein expressions of Sirt-6 and NF-κB in the liver were detected by Western blot. According to the results,compared with the model group,there were significant changes in organ indexes in the high-dose group of SS-b2( P<0. 05). The level of ALT,AST,TNF-α,IL-1β,IL-6 and the activities of LDH in serum of mice with liver injury were significantly reduced in the medium and high dose groups of SS-b2( P<0. 01). With the increase of the concentration of SS-b2,the range of hepatic lesions and the damage in mice decreased. The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in liver of mice were significantly enhanced in each dose group( P<0. 01). The expression of NF-κB in liver tissues was significantly down-regulated in the medium and high dose group( P<0. 01). Meanwhile,the expression of Sirt-6 protein in the liver of mice with acute liver injury was significantly increased in each dose group( P<0. 01).In summary,SS-b2 has a significant protective effect on LPS/Gal N-induced acute liver injury in mice,which may be related to the down-regulation of NF-κB protein expression and up-regulation of Sirt-6 protein expression to improve inflammatory injury and energy metabolism.
To study the effects of saikosaponin b2( SS-b2) on inflammatory factors and energy metabolism against lipopolysaccharide/galactosamine( LPS/Gal N) induced acute liver injury in mice. Mice were randomly divided into normal group( equal amount of normal saline),model group( 100 g·kg~(-1) LPS and 400 mg·kg~(-1) Gal N),low,medium,high dose group of SS-b2( SS-b25,10,20 mg·kg~(-1)·d-1) and positive control group( dexamethasone,10 mg·kg~(-1)). All of the groups except for the normal group were treated with LPS/Gal N though intraperitoneally injection to establish the acute liver injury model. The organ indexes were calculated. The levels of serum transaminases( ALT and AST) and the activities of ATPase( Na+-K+-ATPase,Ca2+-Mg2+-ATPase) in liver were detected. The activity of tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and interleukin-6( IL-6) were determined by the enzyme-linked immunosorbent assay( ELISA). The contents of lactate dehydrogenase( LDH) in liver were determined by micro-enzyme method. HE staining was used to observe the histopathological changes of the liver. Histochemical method was used to investigate the protein expression of liver lactate dehydrogenase-A( LDH-A). The protein expressions of Sirt-6 and NF-κB in the liver were detected by Western blot. According to the results,compared with the model group,there were significant changes in organ indexes in the high-dose group of SS-b2( P<0. 05). The level of ALT,AST,TNF-α,IL-1β,IL-6 and the activities of LDH in serum of mice with liver injury were significantly reduced in the medium and high dose groups of SS-b2( P<0. 01). With the increase of the concentration of SS-b2,the range of hepatic lesions and the damage in mice decreased. The activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in liver of mice were significantly enhanced in each dose group( P<0. 01). The expression of NF-κB in liver tissues was significantly down-regulated in the medium and high dose group( P<0. 01). Meanwhile,the expression of Sirt-6 protein in the liver of mice with acute liver injury was significantly increased in each dose group( P<0. 01).In summary,SS-b2 has a significant protective effect on LPS/Gal N-induced acute liver injury in mice,which may be related to the down-regulation of NF-κB protein expression and up-regulation of Sirt-6 protein expression to improve inflammatory injury and energy metabolism.
引文
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[2]季宇彬,包晓威,单宇,等.覆盆子提取物对ConA致小鼠急性肝损伤的保护作用研究[J].中国中药杂志,2019,44(4):774.
[3]刘玉梅,周安,俞年军,等.HPLC-DAD双波长法同时测定柴胡中5种皂苷的含量[J].中国中药杂志,2018,43(2):363.
[4]王啸,林华,徐又佳.NF-κB通路在骨质疏松伴铁蓄积中的潜在作用[J].中华内分泌代谢杂志,2015,31(2):192.
[5]黄伟,孙蓉.柴胡皂苷类成分化学与药理和毒理作用研究进展[J].中药药理与临床,2010,26(3):71.
[6]崔哲,沈光海.柴胡皂苷B2(Saikosaponin B2)对四氯化碳损伤HepG2细胞的保护作用[J].中国医药指南,2014,12(22):82.
[7] Liu Y,Lou G,Li A,et al. AMSC-derived exosomes alleviate lipopolysaccharide/d-galactosamine-induced acute liver failure by miR-17-mediated reduction of TXNIP/NLRP3 inflammasome activation in macrophages[J]. EBioMedicine,2018,9(36):140.
[8] Handa P,Vemulakonda A L,Maliken B D,et al. Differences in hepatic expression of iron inflammation and stess-related genes in patients with nonalcoholic steatohepatitis[J]. Ann Hepatol,2017,16(1):77.
[9] Bie G V,Trautwein C. The innate immune response during liver inflammation and metabolic disease[J]. Trends Immunol,2013,34(9):446.
[10]朱平生,焦炎杰,付双楠,等.对乙酰氨基酚致大鼠肝损伤早期生物标志物水平的变化规律[J].中国实验方剂学杂志,2019,25(2):118.
[11]陈春,陈毅飞,曹后康,等.酢浆草对四氯化碳致急性肝损伤大鼠的保护作用及机制[J].中国实验方剂学杂志,2018,24(16):141.
[12]王凯,眭丹娟,王长锁,等.5种石斛对四氯化碳致小鼠急性肝损伤的保护作用[J].中国中药杂志,2017,42(10):1945.
[13]王巍,许立拔,张卓,等.金草消毒颗粒对D-GalN/LPS致小鼠急性肝损伤保护的影响[J].中国实验方剂学杂志,2018,24(7):108.
[14] Dominy J E Jr,Lee Y,Jedrychowski M P,et al. The deacetylase Sirt-6 activates the acetyltransferase GCN5 and suppresses hepatic gluconeogenesis[J].Mol Cell,2012,48:900.
[15] Kugel S,Mostoslavsky R. Chromatin and beyond:the multitasking roles for Sirt-6[J].Trends Biochem Sci,2014,39:72.
[16] Kim H S,Xiao C,Wang R H,et al. Hepatic-specific disruption of SIRT6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis[J]. Cell Metab,2010,12:224.
[17] Elhanati S,Kanfi Y,Varvak A,et al. Multiple regulatory layers of SREBP1/2 by SIRT6[J].Cell Rep,2013,4:905.
[18]董振,雷倩,刘力超.长寿蛋白Sirt-6在肿瘤发生发展中的作用[J].生物工程学报,2016,32(7):870.
[19] Bosch-PreseguéL,Vaquero A. The dual role of sirtuins in cancer[J].Genes Cancer,2011,2(6):648.
[20] Kenyon C J. The genetics of ageing[J]. Nature,2010,464(7288):504.
[21] Zhang N,Li Z,Mu W,et al. Calorie restriction-induced SIRT6activation delays aging by suppressing NF-κB signaling[J]. Cell Cycle,2016,15:1009.