老鹳草素对D-氨基半乳糖诱导的肝损伤小鼠的保护作用及机制
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摘要
目的:研究老鹳草素对D-氨基半乳糖(D-GalN)致急性肝损伤小鼠的保护作用及机制。方法:60只小鼠随机分为正常组,模型组,水飞蓟素组(180 mg·kg~(-1)),老鹳草素低,中,高剂量组(50,100,200 mg·kg~(-1)),灌胃给药每天1次,正常组及模型组小鼠灌胃生理盐水连续10 d。末次给药2 h后,除正常组外,其余各组腹腔注射D-GalN(500 mg·kg~(-1)),建立D-GalN致小鼠急性肝损伤模型。16 h后,收集血清和肝组织。生化法测定血清中丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),碱性磷酸酶(ALP),总胆红素(TBIL)和肝脏中丙二醛(MDA),总超氧化物歧化酶(T-SOD),谷胱甘肽过氧化物酶(GSH-Px)活性或含量;酶联免疫吸附测定(ELISA)检测血清中肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β),白细胞介素-6(IL-6)和γ-干扰素(IFN-γ)含量;蛋白免疫印迹法(Western blot)分析肝组织中Toll样受体-4(TLR-4)和核转录因子-κB(NF-κB)蛋白表达情况;苏木素-伊红(HE)染色观察肝组织病理学变化。结果:与正常组比较,模型组小鼠血清中ALT,AST,ALP,TBIL和肝脏MDA水平显著升高(P <0. 01),与模型组比较,老鹳草素各给药组能够明显降低肝损伤小鼠血清中ALT,AST,ALP,TBIL和肝脏MDA(P <0. 05,P <0. 01),且增强肝脏T-SOD和GSH-Px活性(P <0. 05,P <0. 01),同时抑制血清中TNF-α,IL-1β,IL-6和IFN-γ含量的升高和TLR-4,NF-κB的蛋白表达(P <0. 05,P <0. 01); HE染色结果显示其对肝组织损伤程度有明显改善作用。结论:综上所述,老鹳草素对D-GalN诱导的急性肝损伤小鼠具有显著的保护作用,其保肝作用机制可能与抑制氧化应激、炎症反应以及TLR-4/NF-κB信号通路有关。
Objective: To explore the hepatoprotective effect and its mechanism of the geraniin on mice with acute liver injury induced by D-galactosamine(D-GalN). Method: A total of 60 Kunming mice were randomly divided into normal group,model group,Silymarin group(positive group 180 mg·kg~(-1)),and low,medium and high-dose geraniin groups(50,100,200 mg·kg~(-1)). All the mice were given with saline or corresponding dose of drugs(10 m L·kg~(-1)) by gavage once a day for 10 d. After 2 h of the last administration,except the normal group,the mice of other groups were injected intraperitoneally with D-GalN(500 mg·kg~(-1)) to induce the acute liver injury. After 16 h,the eye balls of mice were removed to take blood,and all mice were put to death to collect samples of liver. Activity or content of alanine aminotransferase(ALT), aspartate aminotransferase(AST),alkaline phosphatase(ALP),total bilirubin(TBIL),malondialdehyde(MDA),total superoxide dismutase(T-SOD) and glutathione peroxidase(GSH-Px) in liver were determined by biochemical method. The serum levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),γ-interferon(IFN-γ) and interleukin-6(IL-6) were detected by enzyme-linked immunosorbent assay(ELISA),and expressions of Toll-like receptor-4(TLR-4) and nuclear factor(NF)-κB proteins were detected by Western blot. Liver histopathological changes were observed by hematoxylin-eosin(HE) staining. Result: Compared with the normal group,the serum levels of ALT,AST,ALP,TBIL and liver MDA in the model group were significantly increased(P < 0. 05,P <0. 01). Compared with the model group,geraniin can reduce activities or contents of ALT,AST,ALP,TBIL in serum and MDA in liver of mice(P < 0. 05,P < 0. 01),but increase activities of T-SOD and GSH-Px in liver(P < 0. 05,P < 0. 01),while inhibiting the contents of TNF-α,IL-1β,IL-6,IFN-γ and the expressions of TLR-4 and NF-κB proteins in serum(P < 0. 05,P < 0. 01). HE staining showed that acute liver injury of mice was alleviated obviously by geraniin. Conclusion: Geraniin has an obvious protective effective on acute liver injuries induced by D-GalN in mice. Its mechanism may be correlated with oxidative stress,inflammation and TLR-4/NF-κB signaling pathway.
Objective: To explore the hepatoprotective effect and its mechanism of the geraniin on mice with acute liver injury induced by D-galactosamine(D-GalN). Method: A total of 60 Kunming mice were randomly divided into normal group,model group,Silymarin group(positive group 180 mg·kg~(-1)),and low,medium and high-dose geraniin groups(50,100,200 mg·kg~(-1)). All the mice were given with saline or corresponding dose of drugs(10 m L·kg~(-1)) by gavage once a day for 10 d. After 2 h of the last administration,except the normal group,the mice of other groups were injected intraperitoneally with D-GalN(500 mg·kg~(-1)) to induce the acute liver injury. After 16 h,the eye balls of mice were removed to take blood,and all mice were put to death to collect samples of liver. Activity or content of alanine aminotransferase(ALT), aspartate aminotransferase(AST),alkaline phosphatase(ALP),total bilirubin(TBIL),malondialdehyde(MDA),total superoxide dismutase(T-SOD) and glutathione peroxidase(GSH-Px) in liver were determined by biochemical method. The serum levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),γ-interferon(IFN-γ) and interleukin-6(IL-6) were detected by enzyme-linked immunosorbent assay(ELISA),and expressions of Toll-like receptor-4(TLR-4) and nuclear factor(NF)-κB proteins were detected by Western blot. Liver histopathological changes were observed by hematoxylin-eosin(HE) staining. Result: Compared with the normal group,the serum levels of ALT,AST,ALP,TBIL and liver MDA in the model group were significantly increased(P < 0. 05,P <0. 01). Compared with the model group,geraniin can reduce activities or contents of ALT,AST,ALP,TBIL in serum and MDA in liver of mice(P < 0. 05,P < 0. 01),but increase activities of T-SOD and GSH-Px in liver(P < 0. 05,P < 0. 01),while inhibiting the contents of TNF-α,IL-1β,IL-6,IFN-γ and the expressions of TLR-4 and NF-κB proteins in serum(P < 0. 05,P < 0. 01). HE staining showed that acute liver injury of mice was alleviated obviously by geraniin. Conclusion: Geraniin has an obvious protective effective on acute liver injuries induced by D-GalN in mice. Its mechanism may be correlated with oxidative stress,inflammation and TLR-4/NF-κB signaling pathway.
引文
[1]赵欣,李贵节,胡园园,等.苦丁茶多酚提取物对四氯化碳诱导小鼠肝损伤的改善作用及机制研究[J].食品工业科技,2018,39(4):289-295.
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[13]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.
[14]黄思茂,许琼梅,王欢,等.火炭母总黄酮对D-氨基半乳糖诱导的肝损伤小鼠的保护作用及其机制[J].中药药理与临床,2018,34(2):32-35.
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[16]赵海梅,周步高,王馨,等.二至丸预防和治疗性给药对大鼠损伤后肝细胞再生障碍的保护作用[J].中国实验方剂学杂志,2017,23(16):128-132.
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[20]LI X,WU Y,ZHANG W,et al.Pre-conditioning with tanshinoneⅡAattenuates the ischemia/reperfusion injury caused by liver grafts via regulation of HMGB1 in rat Kupffer cells[J].Biomed Pharmacother,2017,89:1392-1400.
[21]ZHANG K F,GAO Y,ZHONG M L,et al.Hepatoprotective effects of Dicliptera chinensis polysaccharides on dimethylnitrosamine-induced hepatic fibrosis rats and its underlying mechanism[J].Ethnopharmacol,2016,179:38-44.
[22]Elshitany N A,Eid B.Proanthocyanidin protects against cisplatin-induced oxidative liver damage through inhibition of inflammation and NF-κB/TLR-4 pathway[J].Environ Toxicol,2017,32(7):1952-1963.
[23]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.
[24]JIANG M P,XU C,GUO Y W,et al.β-arrestin 2attenuates lipopolysaccharide-induced liver injury via inhibition of TLR4/NF-κB signaling pathway-mediated inflammation in mice[J].World J Gastroenterol,2018,24(2):216-225.
[25]吴庆,曹文富,张永越,等.益气化瘀化痰法制剂对CCl4所致肝纤维化大鼠肝组织KLF15、NF-κB及下游炎症因子的影响[J].中药新药与临床药理,2018,29(5):557-563.
[2]屠梦珏,魏进歌,陈鑫,等.二氢杨梅素对APAP诱导小鼠急性肝损伤的保护作用[J].中国药理学通报,2018,34(12):1707-1712.
[3]李倩,买吾拉江·阿不都热衣木,徐芳,等.老鹳草素药理研究进展[J].中国中医药信息杂志,2016,23(8):125-128.
[4]LU C,GAO S,XU G.Geraniin inhibits TNF-α-induced impairments of osteogenesis through NF-κB and p38MAPK signalling pathways in bone marrow stem cells[J].Stroke Vasc Neurol,2017,2(2):47-52.
[5]谷静逸,尹海波,赵容,等.辽宁省5种老鹳草中5种活性成分的含量测定[J].中国实验方剂学杂志,2016,22(6):26-29.
[6]赵露琴,张露元,杨永红.叶下珠多酚部位提取工艺研究[J].中药材,2017,40(11):2654-2656.
[7]马俊飞,张石蕾,吕梅霞,等.睡莲花烟花苷对半乳糖胺致小鼠急性肝损伤的保护作用[J].癌变·畸变·突变,2017,29(5):370-373,378.
[8]WANG Y,WAN D,ZHOU R,et al.Geraniin inhibits migration and invasion of human osteosarcoma cancer cells through regulation of PI3K/Akt and ERK1/2signaling pathways[J].Anticancer Drugs,2017,28(9):959-966.
[9]罗燕平,戚微岩,吴小东.老鹳草素的研究进展[J].药物生物技术,2016,23(3):279-282.
[10]Londhe J S,Devasagayam T P,Foo L Y,et al.Geraniin and amariin,ellagitannins from Phyllanthus amarus,protect liver cells against ethanol induced cytotoxicity[J].Fitoterapia,2012,83(8):1562-1568.
[11]杜晓鸣,郭永沺.老鹳草素(Geraniin)及其抗氧化作用[J].国外医药:植物药分册,1990,5(2):57-62,69.
[12]张帆,任真,高雅,等.基于TLR-4/NF-κB信号通路研究老鹳草素对四氯化碳致肝损伤小鼠的保肝作用[J].中药材,2018,41(12):2654-2658.
[13]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.
[14]黄思茂,许琼梅,王欢,等.火炭母总黄酮对D-氨基半乳糖诱导的肝损伤小鼠的保护作用及其机制[J].中药药理与临床,2018,34(2):32-35.
[15]吴甜,刘宪,闵剑斌,等.二至丸煎剂及配4方颗粒对小鼠急性肝损伤的保护作用[J].中药新药与临床药理,2018,29(3):277-281.
[16]赵海梅,周步高,王馨,等.二至丸预防和治疗性给药对大鼠损伤后肝细胞再生障碍的保护作用[J].中国实验方剂学杂志,2017,23(16):128-132.
[17]Elhosseiny L S,Alqurashy N N,Sheweita S A.Oxidative stress alleviation by sage essential oil in co-amoxiclav induced hepatotoxicity in rats[J].Int J Biomed Sci,2016,12(2):71-78.
[18]Hishinuma I,Nagakawa J,Hirota K,et al.Involvement of tumor necrosis factor-alpha in development of hepatic injury in galactosamine-sensitized mice[J].Hepatology,1990,12(5):1187-1191.
[19]Anne C,Mireille B,Elizabeth B,et al.Inflammatory cas1990cades driven by tumor necrosis factor-alpha play a major role in the progression of acute liver failure and its neurological complications[J].PLo S One,2012,7(11):e49670.
[20]LI X,WU Y,ZHANG W,et al.Pre-conditioning with tanshinoneⅡAattenuates the ischemia/reperfusion injury caused by liver grafts via regulation of HMGB1 in rat Kupffer cells[J].Biomed Pharmacother,2017,89:1392-1400.
[21]ZHANG K F,GAO Y,ZHONG M L,et al.Hepatoprotective effects of Dicliptera chinensis polysaccharides on dimethylnitrosamine-induced hepatic fibrosis rats and its underlying mechanism[J].Ethnopharmacol,2016,179:38-44.
[22]Elshitany N A,Eid B.Proanthocyanidin protects against cisplatin-induced oxidative liver damage through inhibition of inflammation and NF-κB/TLR-4 pathway[J].Environ Toxicol,2017,32(7):1952-1963.
[23]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.
[24]JIANG M P,XU C,GUO Y W,et al.β-arrestin 2attenuates lipopolysaccharide-induced liver injury via inhibition of TLR4/NF-κB signaling pathway-mediated inflammation in mice[J].World J Gastroenterol,2018,24(2):216-225.
[25]吴庆,曹文富,张永越,等.益气化瘀化痰法制剂对CCl4所致肝纤维化大鼠肝组织KLF15、NF-κB及下游炎症因子的影响[J].中药新药与临床药理,2018,29(5):557-563.