复方狗肝菜汤对四氯化碳致急性肝损伤大鼠的保护作用及机制
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摘要
目的:探究复方狗肝菜汤(FGD)对四氯化碳(CCl_4)致急性肝损伤大鼠的保护作用,及其相关的作用机制。方法:将60只大鼠随机分为正常组,模型组,水飞蓟素(120 mg·kg~(-1))组和FGD(475,950,1 900 mg·kg~(-1))组。正常组和模型组给予等体积生理盐水,其余各组按剂量连续灌胃给药10 d,末次用药2 h后,用12%的CCl_4花生油溶液腹腔注射(5 m L·kg~(-1))建立急性肝损伤模型,收集血清和肝脏组织。生化法检测血清中天门冬氨酸氨基转移酶(AST),丙氨酸氨基转移酶(ALT),碱性磷酸酶(ALP),总胆红素(TBIL),丙二醛(MDA),超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)水平;酶联免疫吸附测定(ELISA)检测肝组织中肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)的含量;蛋白免疫印迹法(Western blot)检测肝脏组织中核转录因子-κB(NF-κB)与过氧化物酶体增殖物激活受体-γ(PPAR-γ)的蛋白表达;苏木素-伊红(HE)染色观察肝组织病理学改变。结果:与正常组比较,模型组血清中AST,ALT,ALP活性与TBIL,MDA含量显著升高(P<0.01),肝组织中IL-1β,IL-6和TNF-α含量明显升高(P<0.01),血清中SOD,GSH-Px活性明显降低(P<0.01),肝组织中NF-κB表达明显上调(P<0.01),PPAR-γ表达明显下调(P<0.01),表明急性肝损伤模型建立成功;与模型组比较,FGD可降低血清中AST,ALT,ALP活性与TBIL,MDA含量(P<0.05,P<0.01),并减少肝组织中IL-1β,IL-6和TNF-α含量(P<0.05,P<0.01),且下调NF-κB表达(P<0.05,P<0.01),但可增强SOD,GSH-Px活性与PPAR-γ表达(P<0.05,P<0.01);镜下观察显示肝组织病变得到不同程度改善。结论:FGD对CCl_4致急性肝损伤大鼠有保护作用,其机制可能与抗炎、抗氧化和调节PPAR-γ和NF-κB信号通路有关。
Objective:To explore the protective effect of formula of Gougancai decoction(FGD)on acute liver injury induced by carbon tetrachloride(CCl_4)in rats,in order to provide basis for the development of pharmaceutical preparations or healthcare products.Method:Sixty rats were randomly divided into normal group,Silymarin group(120 mg·kg~(-1))and FGD groups(475,950,1 900 mg·kg~(-1)).The normal group and the model group were given equal volume of saline by gavage,while the other groups were administered with the corresponding dose of drugs according to the body weight.After 10 days,the acute liver injury model was established with 12%carbon tetrachloride peanut oil solution(5 m L·kg~(-1)),except the normal group.All of the rats were put to death to collect serum and liver tissues.The contents of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),total bilirubin(TBIL),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were detected by biochemical methods,the levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in liver tissues were determined by enzyme-linked immunosorbnent assay(ELISA).Nuclear factor-κB(NF-κB)and peroxisome proliferator-activated receptor-γ(PPAR-γ)protein expression in liver tissues were detected by Western blot,and htoxylin eosin(HE)staining was used to observe the variation of liver histopathological.Result:Compared with the normal group,the serum activities of AST,ALT,ALP and the content of TBIL,MDA in the model group were significantly increased(P<0.01),the levels of TNF-α,IL-1β,IL-6 in liver tissue were remarkably increased(P<0.01),but the serum activities of SOD,GSH-Px were significantly decreased(P<0.01),the expression of NF-κB was enhanced in liver tissue(P<0.01),and PPAR-γwas down-regulated(P<0.01),indicating the successful modeling of acute liver injury.Compared with the model group,FGD could reduce the activities of AST,ALT,ALP and the contents of TBIL,MDA(P<0.05,P<0.01),decease the level of TNF-α,IL-1β,IL-6(P<0.05,P<0.01),and down-regulate the expression of NF-κB(P<0.05,P<0.01),but up-regulate the activities of SOD,GSH-Px and the expression of PPAR-γ(P<0.05,P<0.01).The liver tissue lesions were alleviated to varying degrees.Conclusion:FGD has a protective effect on CCl_4-induced acute liver injury in rats,and its mechanism may be related to the activation of PPAR-γand the inhibition of NF-κB signaling pathway,with anti-inflammatory and antioxidative effects.
Objective:To explore the protective effect of formula of Gougancai decoction(FGD)on acute liver injury induced by carbon tetrachloride(CCl_4)in rats,in order to provide basis for the development of pharmaceutical preparations or healthcare products.Method:Sixty rats were randomly divided into normal group,Silymarin group(120 mg·kg~(-1))and FGD groups(475,950,1 900 mg·kg~(-1)).The normal group and the model group were given equal volume of saline by gavage,while the other groups were administered with the corresponding dose of drugs according to the body weight.After 10 days,the acute liver injury model was established with 12%carbon tetrachloride peanut oil solution(5 m L·kg~(-1)),except the normal group.All of the rats were put to death to collect serum and liver tissues.The contents of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),total bilirubin(TBIL),malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were detected by biochemical methods,the levels of tumor necrosis factor-alpha(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in liver tissues were determined by enzyme-linked immunosorbnent assay(ELISA).Nuclear factor-κB(NF-κB)and peroxisome proliferator-activated receptor-γ(PPAR-γ)protein expression in liver tissues were detected by Western blot,and htoxylin eosin(HE)staining was used to observe the variation of liver histopathological.Result:Compared with the normal group,the serum activities of AST,ALT,ALP and the content of TBIL,MDA in the model group were significantly increased(P<0.01),the levels of TNF-α,IL-1β,IL-6 in liver tissue were remarkably increased(P<0.01),but the serum activities of SOD,GSH-Px were significantly decreased(P<0.01),the expression of NF-κB was enhanced in liver tissue(P<0.01),and PPAR-γwas down-regulated(P<0.01),indicating the successful modeling of acute liver injury.Compared with the model group,FGD could reduce the activities of AST,ALT,ALP and the contents of TBIL,MDA(P<0.05,P<0.01),decease the level of TNF-α,IL-1β,IL-6(P<0.05,P<0.01),and down-regulate the expression of NF-κB(P<0.05,P<0.01),but up-regulate the activities of SOD,GSH-Px and the expression of PPAR-γ(P<0.05,P<0.01).The liver tissue lesions were alleviated to varying degrees.Conclusion:FGD has a protective effect on CCl_4-induced acute liver injury in rats,and its mechanism may be related to the activation of PPAR-γand the inhibition of NF-κB signaling pathway,with anti-inflammatory and antioxidative effects.
引文
[1]黄思茂,高雅,曹后康,等.过墙风总黄酮对四氯化碳致急性肝损伤小鼠的保护作用及机制研究[J].天然产物研究与开发,2018,30(2):212-217.
[2]曹后康,韦日明,张可锋,等.黄花倒水莲多糖对四氯化碳致急性肝损伤小鼠的保护作用[J].中药材,2018,41(1):203-206.
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[4]DING L,ZHANG X,LI L,et al. Qingchangligan formula alleviates acut Je liver injury by attenuating extracellular histone-associated inflammation[J]. Cardiovasc Toxicol,2018,118:1-9.
[5]杨春梅,胡源霖,侯俊玲,等. SPG复方对CCl4致小鼠急性肝损伤的保护作用[J].中国新药杂志,2018,27(7):817-821.
[6]高雅,张可兰,黄思茂,等.狗肝菜多糖对二甲基亚硝胺诱导的肝纤维化大鼠TGF-β1/Smads信号通路的影响[J].中药药理与临床,2016,32(6):113-117.
[7]高雅,王刚,杜沛霖,等.白马骨水提物对急性肝损伤小鼠氧化应激及炎症反应的影响[J].中国实验方剂学杂志,2017,23(21):135-140.
[8]赵铁建,傅品悦,刘露露,等.中药莪术几种活性成分对肝脏疾病防治作用的机制[J].世界华人消化杂志,2017,25(27):2433-2440.
[9]刘若轩,李阿荣,郭洁文,等.白花蛇舌草对肝内胆汁淤积模型大鼠的保护作用[J].中药材,2018,41(6):1476-1479.
[10]陈婧,张天洪,万雪梅,等.杠板归总黄酮提取物对α-萘异硫氰酸脂诱导小鼠胆汁淤积性肝损伤保护作用研究[J].重庆医科大学学报,2018,43(1):32-35.
[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):141-145.
[12]徐叔云,卞如濂,陈修,等.药理实验方法学[M]. 3版.北京:人民卫生出版社,2002:1350-1351.
[13]Pérez T R. Is cirrhosis of the liver experimentally produced by CCl4and adequate model of human cirrhosis?[J]. Hepatology,1983,3(1):112-120.
[14]OU Y,ZHENG S,LIN L,et al. Protective effect of Cphycocyanin against carbon tetrachloride-induced hepatocyte damage in vitro and in vivo[J]. Chem Biol Int,2010,185(2):94-100.
[15]ZHANG K F, GAO Y, ZHONG M L, et al.Hepatoprotective effects of Dicliptera chinensis polysaccharideson dimethylnitrosamine-induced hepatic fibrosis rats and its underlying mechanism[J]. J Ethnopharmacol,2016,179:38-44.
[16]Eastmond D A. Postulated carbon tetrachloride mode of action:a review[J]. J Environ Sci Heal C,2007,25(3):185-209.
[17]Roman P,Budziński G,Suszka-S'witek A,et al. Caspase-3 expression and ALT,AST,and GGT activity after 24hours of porcine liver cold storage,depending on the type of transgenesis[J]. Transpl P,2016,48(5):1829-1832.
[18]Akihara R,Homma T,Lee J,et al. Ablation of aldehyde reductase aggravates carbon tetrachloride-induced acute hepatic injury involving oxidative stress and endoplasmic reticulum stress[J]. Biochem Biophl Res Co,2016,478(2):765-771.
[19]Ramachandran A,Jaeschke H. Oxidative stress and acute hepatic injury[J]. Curr Opin Toxicol,2018,7:17-21.
[20]Ibrahim M,Mikail M A,Ahmed I A,et al. Comparison of the effects of three different Baccaurea angulata whole fruit juice doses on plasma,aorta and liver MDA levels,antioxidant enzymes and total antioxidant capacity[J].Eur J Nutr,2018,57(5):1817-1828.
[21]REN F,ZHOU L,ZHANG X,et al. Endoplasmic reticulum stress-activated glycogen synthase kinase 3βaggravates liver inflammation and hepatotoxicity in mice with acute liver failure[J]. Inflammation,2015,38(3):1151-1165.
[22]LI L,DUAN C,ZHAO Y,et al. Preventive effects of interleukin-6 in lipopolysaccharide/D-galactosamine induced acute liver injury via regulating inflammatory response in hepatic macrophages[J]. Int Immunopharmacol,2017,51:99-106.
[23]Pol J G,Lekbaby B,Redelsperger F,et al. Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-αstimulated signaling pathways and limits the extent of liver inflammation[J]. FASEB J,2015,29(5):1879-1889.
[24]Alcaraz-Quiles J, Titos E, Casulleras M, et al.Polymorphisms in the IL-1 gene cluster influence systemic inflammation in patients at risk for acute on chronic liver failure[J]. Hepatology,2017,65(1):202-216.
[25]ZHOU D,YU T,CHEN G,et al. Effects of NF-κB1(p50)targeted gene disruption on ionizing radiationinduced NF-κB activation and TNFα,IL-1α,IL-1βand IL-6 mRNA expression in vivo[J]. Int J Radiat Biol Relat Stud Phys Chem Med,2001,77(7):763-772.
[26]HUANG C,YANG Y,LI W X,et al. Hyperin attenuates inflammation by activating PPAR-γin mice with acute liver injury(ALI)and LPS-induced RAW264. 7 cells[J]. Int Immunopharmacol,2015,29(2):440-447.
[27]Niyazoglu M,Baykara O,Koc A,et al. Association of PARP-1,NF-κB,NF-κBIA and IL-6,IL-1βand TNF-αwith graves disease and graves ophthalmopathy[J].Gene,2014,547(2):226-232.
[2]曹后康,韦日明,张可锋,等.黄花倒水莲多糖对四氯化碳致急性肝损伤小鼠的保护作用[J].中药材,2018,41(1):203-206.
[3]Wamd F S,FAN J G,ZHANG Z,et al. The global burden of liver disease:the major impact of China[J].Hepatology,2014,60(6):2099-2108.
[4]DING L,ZHANG X,LI L,et al. Qingchangligan formula alleviates acut Je liver injury by attenuating extracellular histone-associated inflammation[J]. Cardiovasc Toxicol,2018,118:1-9.
[5]杨春梅,胡源霖,侯俊玲,等. SPG复方对CCl4致小鼠急性肝损伤的保护作用[J].中国新药杂志,2018,27(7):817-821.
[6]高雅,张可兰,黄思茂,等.狗肝菜多糖对二甲基亚硝胺诱导的肝纤维化大鼠TGF-β1/Smads信号通路的影响[J].中药药理与临床,2016,32(6):113-117.
[7]高雅,王刚,杜沛霖,等.白马骨水提物对急性肝损伤小鼠氧化应激及炎症反应的影响[J].中国实验方剂学杂志,2017,23(21):135-140.
[8]赵铁建,傅品悦,刘露露,等.中药莪术几种活性成分对肝脏疾病防治作用的机制[J].世界华人消化杂志,2017,25(27):2433-2440.
[9]刘若轩,李阿荣,郭洁文,等.白花蛇舌草对肝内胆汁淤积模型大鼠的保护作用[J].中药材,2018,41(6):1476-1479.
[10]陈婧,张天洪,万雪梅,等.杠板归总黄酮提取物对α-萘异硫氰酸脂诱导小鼠胆汁淤积性肝损伤保护作用研究[J].重庆医科大学学报,2018,43(1):32-35.
[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):141-145.
[12]徐叔云,卞如濂,陈修,等.药理实验方法学[M]. 3版.北京:人民卫生出版社,2002:1350-1351.
[13]Pérez T R. Is cirrhosis of the liver experimentally produced by CCl4and adequate model of human cirrhosis?[J]. Hepatology,1983,3(1):112-120.
[14]OU Y,ZHENG S,LIN L,et al. Protective effect of Cphycocyanin against carbon tetrachloride-induced hepatocyte damage in vitro and in vivo[J]. Chem Biol Int,2010,185(2):94-100.
[15]ZHANG K F, GAO Y, ZHONG M L, et al.Hepatoprotective effects of Dicliptera chinensis polysaccharideson dimethylnitrosamine-induced hepatic fibrosis rats and its underlying mechanism[J]. J Ethnopharmacol,2016,179:38-44.
[16]Eastmond D A. Postulated carbon tetrachloride mode of action:a review[J]. J Environ Sci Heal C,2007,25(3):185-209.
[17]Roman P,Budziński G,Suszka-S'witek A,et al. Caspase-3 expression and ALT,AST,and GGT activity after 24hours of porcine liver cold storage,depending on the type of transgenesis[J]. Transpl P,2016,48(5):1829-1832.
[18]Akihara R,Homma T,Lee J,et al. Ablation of aldehyde reductase aggravates carbon tetrachloride-induced acute hepatic injury involving oxidative stress and endoplasmic reticulum stress[J]. Biochem Biophl Res Co,2016,478(2):765-771.
[19]Ramachandran A,Jaeschke H. Oxidative stress and acute hepatic injury[J]. Curr Opin Toxicol,2018,7:17-21.
[20]Ibrahim M,Mikail M A,Ahmed I A,et al. Comparison of the effects of three different Baccaurea angulata whole fruit juice doses on plasma,aorta and liver MDA levels,antioxidant enzymes and total antioxidant capacity[J].Eur J Nutr,2018,57(5):1817-1828.
[21]REN F,ZHOU L,ZHANG X,et al. Endoplasmic reticulum stress-activated glycogen synthase kinase 3βaggravates liver inflammation and hepatotoxicity in mice with acute liver failure[J]. Inflammation,2015,38(3):1151-1165.
[22]LI L,DUAN C,ZHAO Y,et al. Preventive effects of interleukin-6 in lipopolysaccharide/D-galactosamine induced acute liver injury via regulating inflammatory response in hepatic macrophages[J]. Int Immunopharmacol,2017,51:99-106.
[23]Pol J G,Lekbaby B,Redelsperger F,et al. Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-αstimulated signaling pathways and limits the extent of liver inflammation[J]. FASEB J,2015,29(5):1879-1889.
[24]Alcaraz-Quiles J, Titos E, Casulleras M, et al.Polymorphisms in the IL-1 gene cluster influence systemic inflammation in patients at risk for acute on chronic liver failure[J]. Hepatology,2017,65(1):202-216.
[25]ZHOU D,YU T,CHEN G,et al. Effects of NF-κB1(p50)targeted gene disruption on ionizing radiationinduced NF-κB activation and TNFα,IL-1α,IL-1βand IL-6 mRNA expression in vivo[J]. Int J Radiat Biol Relat Stud Phys Chem Med,2001,77(7):763-772.
[26]HUANG C,YANG Y,LI W X,et al. Hyperin attenuates inflammation by activating PPAR-γin mice with acute liver injury(ALI)and LPS-induced RAW264. 7 cells[J]. Int Immunopharmacol,2015,29(2):440-447.
[27]Niyazoglu M,Baykara O,Koc A,et al. Association of PARP-1,NF-κB,NF-κBIA and IL-6,IL-1βand TNF-αwith graves disease and graves ophthalmopathy[J].Gene,2014,547(2):226-232.