脱水穿心莲内酯对四氯化碳诱导的肝纤维化模型小鼠肝细胞凋亡的抑制作用及其机制

doi:10.13481/j.1671-587x.20190506

摘要/Abstract

摘要： 目的：探讨脱水穿心莲内酯（DA）对四氯化碳（CCl₄）诱导的肝纤维化模型小鼠肝细胞凋亡的抑制作用，并阐明其可能的作用机制。方法： 30只6~8周龄C57BL/6J雄性小鼠随机分为对照组、模型组和治疗组，每组10只。除正常对照组外其余2组小鼠采用20% CCl₄ 2.0 mL·kg^-1腹腔注射，每周3次，制备肝纤维化模型，对照组小鼠给予等量的橄榄油。治疗组小鼠按100 mg·kg^-1 DA灌胃给药，每周3次，对照组和模型组小鼠灌服等体积生理盐水。给药4周，末次给药后24 h摘眼球取血，断髓，取肝脏。检测各组小鼠血清中丙氨酸氨基转移酶（ALT）和天门冬氨酸氨基转移酶（AST）活性；检测各组小鼠肝组织中超氧化物歧化酶（SOD）活性和丙二醛（MDA）水平；HE染色和天狼猩红染色观察各组小鼠肝脏病理学表现；Western blotting法检测各组小鼠肝组织中8-氧代鸟嘌呤DNA糖基化酶1（Ogg1）、半胱氨酸蛋白酶-3（Caspase-3）、Bcl-2相关X蛋白（Bax）和B淋巴细胞瘤-2基因（Bcl-2）蛋白水平；免疫组织化学法检测各组小鼠肝组织中转化生长因子β1（TGF-β1）和α平滑肌肌动蛋白（α-SMA）蛋白表达水平。结果：与对照组比较，模型组小鼠血清中ALT和AST活性及肝组织中MDA水平均明显升高（P<0.05），肝组织中SOD活性明显下降（P<0.05）；与模型组比较，治疗组小鼠血清中ALT和AST活性及肝组织中MDA水平明显降低（P<0.05），肝组织中SOD活性明显升高（P<0.05）。HE染色和天狼猩红染色，与模型组比较，治疗组小鼠肝组织中炎性细胞浸润和胶原沉积程度明显改善。与对照组比较，模型组小鼠肝组织中Ogg1、Caspase-3、Bax、TGF-β1、α-SMA和Bcl-2蛋白表达水平明显升高（P<0.05）；与模型组比较，治疗组小鼠肝组织中Ogg1、Caspase-3、Bax、TGF-β1和α-SMA蛋白表达水平明显降低（P<0.05），Bcl-2蛋白表达水平明显升高（P<0.05）。结论： DA对CCl₄导致的肝纤维化小鼠具有保护作用，其机制可能与降低氧化应激损伤、抑制肝细胞凋亡和减少肝星状细胞活化有关。

关键词: 脱水穿心莲内酯, 肝纤维化, 细胞凋亡, 肝星状细胞

Abstract: Objective:To investigate the inhibitory effect of dehydroandrographolide (DA) on the hepatocyte apoptosis induced by carbon tetrachloride (CCl₄) in the hepatic fibrosis model mice, and to elucidate its possible mechanism. Methods:A total of 30 male C57BL/6J mice aged 6-8 weeks were randomly divided into control group, model group and treatment group;there were 10 mice in each group. Except the normal control group, the mice in other two groups were intraperitoneally injected with 20% CCl₄ 2.0 mL·kg^-1, three times a week;the hepatic fibrosis models were establised. The mice in control group were given the same volume of olive oil. The mice in treatment group were intragastrically given 100 mg·kg^-1 DA,three times a week; while the mice in control group and model group were given the equal volume of normal saline. Four weeks later, 24 h after the last administration, the eyeball blood was taken, the marrow was broken, and the liver was taken. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the mice in various groups were detected; the activities of superoxide dismutase (SOD) and the levels of malondialdehyde (MDA) in liver tissue of the mice in various groups were detected. The pathological performance of liver tissue of the mice in various groups were observed by HE staining and Sirius red staining. Western blotting method was used to detect the levels of 8-oxoguanine DNA glycosylase 1 (Ogg1), Caspase-3, Bcl-2-associated X protein(Bax), and B-cell lymphoma-2 (Bcl-2) proteins in liver tissue of the mice in various groups. The expression levels of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) proteins in liver tissue of the mice in various groups were observed by immunohistochemistry. Results:Compared with control group, the activities of serum ALT and AST and the level of MDA in liver tissue of the mice in model group were significantly increased (P<0.05), and the activity of SOD in liver tissue was significantly decreased (P<0.05). Compared with model group, the activities of ALT and AST in serum and the level of MDA in liver tissue of the mice in treatment group were significantly decreased (P<0.05), and the activity of SOD in liver tissue was significantly increased (P<0.05). The HE staining and Sirius red staining results showed that compared with model group, the inflammatory cell infiltration and collagen deposition in liver tissue of the mice in treatment group were significantly improved. Compared with control group, the expression levels of Ogg1, Caspase-3, Bax, TGF-β1,α-SMA and Bcl-2 proteins in liver tissue of the mice in treatment group were significantly increased (P<0.05); compared with model group, the expression levels of Ogg1, Caspase-3, Bax, TGF-β1 and α-SMA proteins in liver tissue of the mice in treatment group were significantly decreased (P<0.05), and the expression level of Bcl-2 protein was significantly increased (P<0.05). Conclusion:DA has a protective effect in the mice with hepatic fibrosis induced by CCl₄, which might be related to reducing oxidative stress injury, inhibiting hepatocyte apoptosis and reducing hepatic stellate cell activation.

Key words: dehydroandrographolide, hepatic fibrosis, apoptosis, hepatic stellate cells

中图分类号:

R657.3

谢婧, 李丽华. 脱水穿心莲内酯对四氯化碳诱导的肝纤维化模型小鼠肝细胞凋亡的抑制作用及其机制[J]. 吉林大学学报(医学版), 2019, 45(05): 1009-1014.

XIE Jing, LI Lihua. Inhibitory effect of dehydroandrographolide on hepatocyte apoptosis induced by carbon tetrachloride in hepatic fibrosis model mice and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1009-1014.

参考文献

[1] WANG R, YU X Y, GUO Z Y, et al. Inhibitory effects of salvianolic acid B on CCl(4)-induced hepatic fibrosis through regulating NF-κB/IκBα signaling[J]. J Ethnopharmacol, 2012, 144(3):592-598.
[2] TANG L Y, HELLER M, MENG ZJ, et al. Transforming growth factor-β (TGF-β) directly activates the JAK1-STAT3 axis to induce hepatic fibrosis in coordination with the SMAD pathway[J]. J Biol Chem, 2017, 292(10):4302-4312.
[3] CHEN YH, WU ZF, YUAN BY, et al. MicroRNA-146a-5p attenuates irradiation-induced and LPS-induced hepatic stellate cell activation and hepatocyte apoptosis through inhibition of TLR4 pathway[J]. Cell Death Dis, 2018, 9(2):22.
[4] AL-RASHEED N, FADDAH L, AL-RASHEED N, et al. Protective effects of silymarin, alone or in combination with chlorogenic acid and/or melatonin, against carbon tetrachloride-induced hepatotoxicity[J]. Pharmacogn Mag, 2016, 12(Suppl 3):S337-S345.
[5] TAO L, ZHANG L, GAO R, et al. Andrographolide alleviates acute brain injury in a rat model of traumatic brain injury:possible involvement of inflammatory signaling[J]. Front Neurosci, 2018, 12:657.
[6] LIANG ES, LIU X, DU ZH, et al. Andrographolide ameliorates diabetic cardiomyopathy in mice by blockage of oxidative damage and NF-κB-mediated inflammation[J]. Oxid Med Cell Longev, 2018, 2018:9086747.
[7] HSIEH Y L, SHIBU M A, LⅡ C K, et al. Andrographis paniculata extract attenuates pathological cardiac hypertrophy and apoptosis in high-fat diet fed mice[J]. J Ethnopharmacol, 2016, 192:170-177.
[8] CHEN Y Y, HSU M J, HSIEH C Y, et al. Andrographolide inhibits nuclear factor-κB activation through JNK-Akt-p65 signaling cascade in tumor necrosis factor-α-stimulated vascular smooth muscle cells[J]. Sci World J, 2014, 2014:130381.
[9] WENG ZY, CHI Y, XIE J, et al. Anti-inflammatory activity of dehydroandrographolide by TLR4/NF-κB signaling pathway inhibition in bile duct-ligated mice[J]. Cell Physiol Biochem, 2018, 49(3):1083-1096.
[10] SEO H Y, JUNG Y A, LEE S H, et al. Kahweol decreases hepatic fibrosis by inhibiting the expression of connective tissue growth factor via the transforming growth factor-beta signaling pathway[J]. Oncotarget, 2017, 8(50):87086-87094.
[11] DAS N, MANDALA A, NAAZ S, et al. Melatonin protects against lipid-induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice[J]. J Pineal Res, 2017, 62(4).DOI:10.1111/jpi.12404.
[12] CHEN S R, LI F, DING M Y, et al. Andrographolide derivative as STAT3 inhibitor that protects acute liver damage in mice[J]. Bioorg Med Chem, 2018, 26(18):5053-5061.
[13] LEE T Y, LEE K C, CHANG H H. Modulation of the cannabinoid receptors by andrographolide attenuates hepatic apoptosis following bile duct ligation in rats with fibrosis[J]. Apoptosis, 2010, 15(8):904-914.
[14] SHI G J, ZHANG Z J, ZHANG R, et al. Protective effect of andrographolide against concanavalin A-induced liver injury[J]. Naunyn Schmiedebergs Arch Pharmacol, 2012, 385(1):69-79.
[15] SHAH B, SHAH G. Antifibrotic effect of heparin on liver fibrosis model in rats[J]. World J Gastrointest Pharmacol Ther, 2012, 3(6):86-92.
[16] LI X X, ZHENG Q C, WANG Y, et al. Theoretical insights into the reductive metabolism of CCl₄ by cytochrome P450 enzymes and the CCl4-dependent suicidal inactivation of P450[J]. Dalton Trans, 2014, 43(39):14833-14840.
[17] XIE Y, HAO H P, WANG H, et al. Reversing effects of lignans on CCl4-induced hepatic CYP450 down regulation by attenuating oxidative stress[J]. J Ethnopharmacol, 2014, 155(1):213-221.
[18] TABET E, GENET V, TIAHO F, et al. Chlordecone potentiates hepatic fibrosis in chronic liver injury induced by carbon tetrachloride in mice[J]. Toxicol Lett, 2016, 255:1-10.
[19] WU H, QIU Y, SHU Z Y, et al. Protective effect of Trillium tschonoskii saponin on CCl4-induced acute liver injury of rats through apoptosis inhibition[J]. Can J Physiol Pharmacol, 2016, 94(12):1291-1297.
[20] 陆璐,武超,谢咚,等.八宝丹对急性肝衰竭大鼠肝性脑病的防治作用[J].临床肝胆病杂志,2018,34(12):2635-2641.
[21] WANG Y, WANG R, WANG Y, et al. Ginkgo biloba extract mitigates liver fibrosis and apoptosis by regulating p38 MAPK, NF-κB/IκBα, and Bcl-2/Bax signaling[J]. Drug Des Devel Ther, 2015, 9:6303-6317.
[22] MARIN J J, HERNANDEZ A, REVUELTA I E, et al. Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis:role of the Akt/mTOR survival pathway and Bcl-2 family proteins[J]. Free Radic Biol Med, 2013, 61:218-228.