小柴胡颗粒激活Nrf2通路抗TAA致大鼠急性肝损伤的机制探讨
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摘要
目的:观察小柴胡颗粒对硫代乙酰胺(TAA)致大鼠急性肝损伤(ALI)的治疗作用,探讨核转录因子(NF)-E2相关因子2(Nrf2)抗氧化损伤通路在其中的作用。方法:SD大鼠随机分为空白组,模型组,小柴胡颗粒低、中、高剂量组(1,2,4 g·kg-1),大鼠给予250 mg·kg-1TAA腹腔注射2 d制备肝损伤模型,从第3天各组分别给予等量双蒸水和不同剂量小柴胡颗粒,灌胃2周。末次给药后24 h取材,肝组织行苏木素-伊红(HE)染色;比色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)活性及组织中总超氧化物歧化酶(T-SOD),丙二醛(MDA)水平;实时荧光定量PCR(Real-time PCR)检测Nrf2通路相关mRNA表达;蛋白免疫印迹法(Western blot)检测Nrf2通路相关蛋白的表达。结果:与空白组比较,模型组ALT,AST,MDA水平显著增高,T-SOD活性明显降低(P <0. 05,P <0. 01),病理学呈现大片炎性浸润表现,肝脏组织Nrf2,Kelch样环氧氯丙烷相关蛋白1(Keap1),醌氧化还原酶1(NQO1),血红素加氧酶-1(HO-1),谷氨酸半胱氨酸合成酶催化亚基(GCLC),谷氨酸半胱氨酸合成酶调节亚基(GCLM) mRNA及蛋白表达水平均明显下降(P <0. 05,P <0. 01);与模型组比较,小柴胡颗粒各剂量组ALT,AST,MDA水平均明显降低,T-SOD活性明显升高(P <0. 05,P <0. 01),病理结果示大鼠肝脏病变范围与严重程度均有不同程度改善,肝脏组织Nrf2,Keap1,NQO1,HO-1,GCLC,GCLM的mRNA及蛋白表达水平均明显升高(P <0. 05,P <0. 01)。结论:小柴胡颗粒可能通过上调Nrf2信号通路上下游分子的表达,对TAA致急性肝损伤大鼠起到治疗作用。
Objective: To observe the therapeutic effect of Xiaochaihu granule on acute liver injury( ALI) induced by thioacetamide( TAA) in rats,and to explore the role of transcription factor( NF)-E2 related factor 2( Nrf2) in the pathway of oxidative damage. Method: SD rats were randomly divided into control group,model group,Xiaochaihu granule low,middle and high dose groups( 1,2,4 g·kg-1). 250 mg·kg-1 TAA was given to the rats by ip. administration for 2 days to prepare the liver injury model,and from the 3 rdday,same amount of double distilled water or different doses of Xiaochaihu granule was given to corresponding groups by ig.administration for 2 weeks. 24 hours after the last administration,liver tissues were taken and stained with hematoxylin-eosin( HE) staining. The activities of alanine aminotransferase( ALT) and aspartate aminotransferase( AST) in serum were measured by colorimetry. The activities of total superoxide dismutase( T-SOD) and malondialdehyde( MDA) in liver tissues were measured by colorimetry. Real-time PCR was used to detect Nrf2 pathway related mRNA expression. Result: As compared with control group,ALT,AST and MDA in model group were significantly increased,while T-SOD was significantly decreased( P < 0. 05,P < 0. 01),and pathological features showed large inflammatory infiltration,both mRNA and protein expression levels of Nrf2,Kelch-like ECHassociated protein 1( Keap1),quinone oxidoreductase( NQO1),heme oxygenase-1( HO-1),glutamate-cysteine ligase catalytic subunit( GCLC) and glutamate-cysteine ligase modifier subunit( GCLM) were decreased to different degrees( P < 0. 01). As compared with model group,ALT,AST,MDA levels in Xiaochaihu granule treatment groups were decreased,while T-SOD was increased significantly( P < 0. 05,P < 0. 01). Pathological results showed that the range and severity of liver lesions in rats were improved to varying degrees after treatment,both mRNA and protein expression levels of Nrf2,Keap1,NQO1,HO-1,GCLC and GCLM were increased to varying degrees( P < 0. 05,P < 0. 01). Conclusion: Xiaochaihu granule may play a therapeutic role in TAA induced ALI in rats by up-regulating the expression of downstream molecules in Nrf2 signaling pathway.
Objective: To observe the therapeutic effect of Xiaochaihu granule on acute liver injury( ALI) induced by thioacetamide( TAA) in rats,and to explore the role of transcription factor( NF)-E2 related factor 2( Nrf2) in the pathway of oxidative damage. Method: SD rats were randomly divided into control group,model group,Xiaochaihu granule low,middle and high dose groups( 1,2,4 g·kg-1). 250 mg·kg-1 TAA was given to the rats by ip. administration for 2 days to prepare the liver injury model,and from the 3 rdday,same amount of double distilled water or different doses of Xiaochaihu granule was given to corresponding groups by ig.administration for 2 weeks. 24 hours after the last administration,liver tissues were taken and stained with hematoxylin-eosin( HE) staining. The activities of alanine aminotransferase( ALT) and aspartate aminotransferase( AST) in serum were measured by colorimetry. The activities of total superoxide dismutase( T-SOD) and malondialdehyde( MDA) in liver tissues were measured by colorimetry. Real-time PCR was used to detect Nrf2 pathway related mRNA expression. Result: As compared with control group,ALT,AST and MDA in model group were significantly increased,while T-SOD was significantly decreased( P < 0. 05,P < 0. 01),and pathological features showed large inflammatory infiltration,both mRNA and protein expression levels of Nrf2,Kelch-like ECHassociated protein 1( Keap1),quinone oxidoreductase( NQO1),heme oxygenase-1( HO-1),glutamate-cysteine ligase catalytic subunit( GCLC) and glutamate-cysteine ligase modifier subunit( GCLM) were decreased to different degrees( P < 0. 01). As compared with model group,ALT,AST,MDA levels in Xiaochaihu granule treatment groups were decreased,while T-SOD was increased significantly( P < 0. 05,P < 0. 01). Pathological results showed that the range and severity of liver lesions in rats were improved to varying degrees after treatment,both mRNA and protein expression levels of Nrf2,Keap1,NQO1,HO-1,GCLC and GCLM were increased to varying degrees( P < 0. 05,P < 0. 01). Conclusion: Xiaochaihu granule may play a therapeutic role in TAA induced ALI in rats by up-regulating the expression of downstream molecules in Nrf2 signaling pathway.
引文
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[17] XU S F,JIN T,LU Y F,et al. Effect of icariin on UDP-glucuronosyltransferases in mouse liver[J]. Planta Med,2014,80(5):387-392.
[18]王淑娟,艾群.小柴胡汤抑制肝纤维化及肝癌的作用机制[J].日本医学介绍,2004,25(1):42-44.
[19]胡琴,刘维,邵宏.药物性肝损伤的药物治疗研究进展[J].中国临床药理学与治疗学,2016,21(2):231-236.
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[21]胡勇,张爱华. PKC-Nrf2/Keap1-ARE抗氧化通路与肝脏疾病[J].癌变·畸变·突变,2012,24(2):151-153,156.
[22] Rohrer R R,Rudraiah S,Goedken M J, et al. Is nuclear factor erythroid 2-related factor 2 responsible for sex differences in susceptibility to acetaminopheninduced hepatotoxicity in mice?[J]. Drug Metab Dispos,2014,42(10):1663-1674.
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[2]吴百灵.丹参对CCl4所致急性肝损伤保护作用的研究[J].中华中医药学刊,2002,20(3):363-381.
[3]杨婷婷,王伟,李国全,等.水飞蓟宾对小鼠急性肝损伤的保护作用[J].中国实验方剂学杂志,2016,22(10):102-106.
[4]刘钰华,叶少武,陈伟坚,等.复方肝宁颗粒的制备及其对CCl4诱导小鼠急性肝损伤的保护作用[J].中国实验方剂学杂志,2016,22(14):18-22.
[5]刘佩莉,李楠,于云,等.复方枳椇子对四氯化碳致大鼠急性肝损伤的保护作用[J].中国实验方剂学杂志,2012,18(15):234-237.
[6]沈洪,张蓓.急性肝损伤研究进展[J].现代中西医结合杂志,2009,18(18):2211-2213.
[7]冯艺萍.天然药物抗急性肝损伤研究进展[J].现代医药卫生,2011,27(21):3282-3284.
[8]王越,姚叶涛,杨亚洋,等.硫代乙酰胺引起兔肝纤维化及肾毒性作用的研究[J].毒理学杂志,2018,32(5):403-407.
[9]马一铭,张盈.小柴胡汤内科临床应用[J].天津中医药大学学报,2016,35(3):207-210.
[10]谢斌,余大军,严小军.加味小柴胡汤对酒精所致急性肝损伤的保护作用[J].中国热带医学,2007,7(8):1298-1299.
[11] Nguyen T,Nioi P,Pickett C B. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress[J]. J Biol Chem. 2009,284(20):13291-13295.
[12]李晋,徐尚福,李远洋,等.小柴胡汤对肝纤维化大鼠肝脏MMP-2、TIMP-2表达的影响[J].辽宁中医杂志,2018,45(5):1066-1068,1119.
[13]李晋,徐尚福,金凤,等.小柴胡汤下调JAK2,STAT3的表达治疗大鼠肝纤维化[J].中国实验方剂学杂志,2014,20(18):118-122.
[14] LI J,HU R,XU S,et al. Xiaochaihutang attenuates liver fibrosis by activation of Nrf2 pathway in rats[J].Biomed Pharmacother,2017,96(1):847-853.
[15] SU G Y,YANG J Y,WANG F,et al. Xiaochaihutang prevents depressive-like behaviour in rodents by enhancing the serotonergic system[J]. J Pharm Pharmacol,2014,66(6):823-834.
[16]魏新智,付勇强,王珲,等.硫代乙酰胺建立大鼠急性肝损伤模型探讨[J].临床医学工程,2009,16(5):48-49.
[17] XU S F,JIN T,LU Y F,et al. Effect of icariin on UDP-glucuronosyltransferases in mouse liver[J]. Planta Med,2014,80(5):387-392.
[18]王淑娟,艾群.小柴胡汤抑制肝纤维化及肝癌的作用机制[J].日本医学介绍,2004,25(1):42-44.
[19]胡琴,刘维,邵宏.药物性肝损伤的药物治疗研究进展[J].中国临床药理学与治疗学,2016,21(2):231-236.
[20] Bellezza I,Giambanco I,Minelli A,et al. Nrf2-Keap1signaling in oxidative and reductive stress[J]. Biochim Biophys Acta Mol Cell Res,2018,1865(5):721-733.
[21]胡勇,张爱华. PKC-Nrf2/Keap1-ARE抗氧化通路与肝脏疾病[J].癌变·畸变·突变,2012,24(2):151-153,156.
[22] Rohrer R R,Rudraiah S,Goedken M J, et al. Is nuclear factor erythroid 2-related factor 2 responsible for sex differences in susceptibility to acetaminopheninduced hepatotoxicity in mice?[J]. Drug Metab Dispos,2014,42(10):1663-1674.
[23] XU S F,JI L L,WU Q,et al. Ontogeny and aging of Nrf2 pathway genes in livers of rats[J]. Life Sci,2018,doi:10. 1016/j. lfs. 2018. 04. 018.