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山楂酸对高脂饮食诱导的非酒精性脂肪肝模型小鼠炎症反应及氧化应激的影响

作者:未知

  摘 要 目的:探討山楂酸对高脂饮食诱导的非酒精性脂肪肝(NAFLD)模型小鼠炎症反应及氧化应激的影响。方法:将72只C57BL/6小鼠随机分为正常组(生理盐水)、模型组(生理盐水)、辛伐他汀组(阳性对照,3 mg/kg)和山楂酸低、中、高剂量组(25、50、100 mg/kg),每组12只。正常组小鼠给予标准饲料喂养,其余各组小鼠给予高脂饲料喂养以诱导NAFLD模型。造模同时,各组小鼠灌胃相应剂量的药物,每天给药1次,共计给药12周。末次给药后12 h,称量各组小鼠体质量及肝质量,并计算肝指数;全自动生化分析仪测定其血清生化指标[天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)活性及高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、三酰甘油(TG)、总胆固醇(TC)含量]变化;苏木精-伊红染色后观察其肝组织病理形态变化;酶联免疫吸附试验法检测其肝组织中炎症反应指标[核转录因子κB(NF-κB)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)含量]和分光光度法测定其中氧化应激指标[丙二醛(MDA)含量及超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性]变化。结果:与正常组比较,模型组小鼠肝指数显著升高(P<0.05);血清中HDL-C含量显著降低,其余血清生化指标活性/含量均显著升高(P<0.05);肝小叶界限不清晰,肝组织发生明显病理性改变;肝组织中炎症反应指标和MDA含量均显著升高(P<0.05),SOD、GSH-Px活性显著降低(P<0.05)。与模型组比较,除山楂酸低剂量组小鼠肝组织中SOD活性升高和MDA含量降低不显著外(P>0.05),其余各给药组小鼠上述指标均显著改善(P<0.05);肝组织中球形脂滴及炎性细胞浸润减少。结论:山楂酸可通过抗炎及抑制氧化应激来减轻高脂饮食诱导的NAFLD模型小鼠的肝组织病变程度。
  关键词 山楂酸;炎症反应;氧化应激;非酒精性脂肪肝;高脂饮食;小鼠
  Effects of Crataegolic Acid on Inflammatory Response and Oxidative Stress in Non-alcoholic Fatty Liver Disease Model Mice Induced by High-fat Diet
  HE Feng,ZHANG Xuelian,WEN Xiangchen(Dept. of Gastroenterology, the First Affiliated Hospital of Chengdu Medical College, Chengdu 610500, China)
  ABSTRACT OBJECTIVE: To investigate the effects of crataegolic acid on inflammatory response and oxidative stress in non-alcoholic fatty liver disease (NAFLD) model mice induced by high-fat diet. METHODS: Totally 72 C57BL/6 mice were randomly divided into normal group (normal saline), model group (normal saline), simvastatin group (positive control, 3 mg/kg) and crataegolic acid low-dose, medium-dose and high-dose groups (25, 50 and 100 mg/kg), with 12 mice in each group. Normal group was standard feed, while other groups were given high-fat diet to induce NAFLD model . At the same time of modeling, rats in each group was given relevant dose of drugs intragastrically, once a day, for consecutive 12 weeks. 12 h after last administration, body weight and liver weight of mice were determined, and liver index was calculated. The changes of serum biochemical indexes (activities of AST, ALT and contents of HDL-C, LDL-C, TG, TC) were determined by fully automatic biochemical analyzer. The pathological changes of liver tissue were observed after HE staining. The changes of inflammatory response indexes (contents of NF-κB, TNF-α, IL-6) in liver tissue were determined by ELISA, and the changes of oxidant stress indexes (contents of MDA, SOD and activity of GSH-Px) were determined by spectrophotometry. RESULTS: Compared with normal group, the liver index of mice in model group was increased significantly (P<0.05). The serum content of HDL-C was decreased significantly, and the activities/contents of other serum biochemical indexes were increased significantly (P<0.05). The boundary of hepatic lobules was not clear, and the liver tissue had obvious pathological changes. Inflammatory response indexes and the contents of MDA were increased significantly in liver tissue (P<0.05), the activities of SOD and GSH-Px were decreased significantly (P<0.05). Compared with model group, except that the increase of SOD activity and the decrease of MDA content of liver tissue were not significantly in crataegolic acid low-dose group (P>0.05), while above indexes of mice in other administration groups were improved significantly (P<0.05). The globular lipid droplets and inflammatory cell infiltration were decreased in liver tissue. CONCLUSIONS: Crataegolic acid can effectively alleviate the degree of liver lesions in NAFLD model mice induced by high-fat die through anti-inflammatory and inhibiting oxidative stress.   KEYWORDS Crataegolic acid; Inflammatory response; Oxidative stress; Non-alcoholic fatty liver disease; High-fat diet;mice
  非酒精性脂肪肝(NAFLD)以肝脏过量脂肪沉积和肝细胞脂肪变性为主要特征,发病机制排除酒精和其他引起继发性肝脂肪变性因素[1]。随着人们饮食习惯的改变及生活水平的提高,肥胖、高血压、高脂血症、非胰岛素依赖性糖尿病等疾病的发病人数相继增多,使人群罹患NAFLD的风险逐年增加,且NAFLD还会进一步促使心脑血管事件的发生[2]。NAFLD对社会发展和人类健康构成了严重威胁,已成为新的重大健康问题。
  山楂酸不仅存在于山楂的叶子和果实中,也存在于橄榄、大花紫薇、水杨梅、藿香、枇杷叶、石榴花等天然植物中,为一种五环三萜酸,具有抗炎、抗氧化、抗2型糖尿病、抗病毒、抗肿瘤等多种药理活性[3]。研究表明,山楂酸可以通过抑制核转录因子κB(NF-κB)通路以降低致炎因子的释放,也可以通过增强清除氧自由基的能力,最终起到保护四氯化碳诱导的小鼠急性肝损伤的作用[4]。然而到目前为止,山楂酸对高脂饮食诱导的NAFLD的作用尚未见报道。同时,鉴于炎症反应及氧化应激在高脂饮食诱导的NAFLD的发生、发展环节中的重要作用[5-6],本研究以高脂饮食诱导的NAFLD模型小鼠为研究对象,重点探讨山楂酸对该模型小鼠炎症反应及氧化应激的影响,以期为山楂酸临床治疗高脂饮食诱导的NAFLD提供一种新的思路。
  1 材料
  1.1 仪器
  Multiskan GO 1510型全波长酶标仪(美国Thermo Fisher Scientific公司);EUR-04型高速组织匀浆器(美国Biospec公司);AB104型电子天平(瑞士Mettler-Toledo公司);CX5型全自动生化分析仪(美国Beckman Coulter公司);L535R型冷冻离心机(湖南湘仪实验仪器公司);CM1900型冰冻切片机(德国Leica公司);UV1900型双光束紫外-可见分光光度计[让奇(上海)仪器科技有限公司]。
  1.2 药品与试剂
  山楂酸对照品(长沙雅莹生物科技有限公司,批号:170324,纯度:>90%);辛伐他汀片(贵州天安药业股份有限公司,批号:20170125,规格:10 mg/片);NF-κB、肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)检测试剂盒(北京同立海源生物科技公司,批号:20170426、20170513、20170622);超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)检测试剂盒(上海源叶生物公司,批号:171011、170908、171215);苏木精-伊红(HE)染液(上海歌凡生物科技有限公司)。
  1.3 动物
  清洁级C57BL/6小鼠72只,♂,4周龄,购于北京维通利华公司,实验动物使用许可证号:SYXK(京)2017- 0033。小鼠饲养条件为相对湿度50%、温度23 ℃,按照国家实验动物饲养标准饲养。
  2 方法
  2.1 分组、造模与给药
  将C57BL/6小鼠适应性饲养1周后,随机分为正常组(生理盐水)、模型组(生理盐水)、辛伐他汀组(阳性对照,3 mg/kg)和山楂酸低、中、高剂量组(25、50和100 mg/kg),每组12只。给药剂量参考王颖等[4]的研究,并结合前期的预实验结果而设置。正常组小鼠给予标准饲料喂养,其余5组小鼠给予高脂饲料(标准饲料中含0.5%胆酸、2%胆固醇和15%猪油)喂养;同时,各组小鼠灌胃相应剂量的药物,每天给药1次,共计给药12周。
  2.2 体质量、肝质量和肝指数测定
  末次给药后12 h,称量各组小鼠体质量;摘眼球采血后,颈椎脱臼法处死小鼠,分离肝组织,精密称质量,计算肝指数[肝指数(%)=肝质量/体质量×100%]。
  2.3 血清生化指标测定
  取“2.2”项下小鼠血液,放于1.5 mL离心管中,离心(3 000 r/min、10 min),分离上清液,采用全自动生化分析仪检测血清中天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)活性及高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、三酰甘油(TG)、总胆固醇(TC)含量。
  2.4 肝组织病理形态观察
  取相同部位的小鼠肝组织,在10%甲醛溶液中浸泡24 h,先后经脱水、透明、浸蜡、石蜡包埋等步骤,随后在切片机上进行连续5 μm切片,烘干备用。常规方法进行HE染色,光镜下观察肝组织病理形态改變情况。
  2.5 肝组织炎症反应及氧化应激指标测定
  取相同部位的小鼠肝组织100 g,放入预冷至0 ℃的0.9 mL生理盐水中,冰水浴下匀浆(3 000 r/min、5 min),得10%肝组织匀浆液。通过酶联免疫吸附试验法测定肝组织中炎症反应指标(NF-κB、TNF-α及IL-6含量),分光光度法测定肝组织中氧化应激指标(SOD、GSH-Px活性及MDA含量),相关操作严格按照相应试剂盒说明书操作指南进行。
  2.6 统计学方法
  采用SPSS 20.0软件对所得数据进行统计分析。采用Shapiro-Wilk法检验正态性,Levene法检验方差齐性;符合正态分布且方差齐性的计量资料以x±s表示,组间比较采用t检验。P<0.05表明差异具有统计学意义。
  3 结果
  3.1 山楂酸对模型小鼠体质量、肝质量和肝指数的影响
  与正常组比较,模型组小鼠体质量、肝质量及肝指数均明显升高(P<0.05),提示模型建立成功;与模型组比较,山楂酸各剂量组及辛伐他汀组小鼠体质量差异均无统计学意义(P>0.05),而肝质量和肝指数均显著降低(P<0.05)。各组小鼠体质量、肝质量和肝指数测定结果见表1。   参考文献
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  (收稿日期:2018-12-14 修回日期:2019-01-12)
  (编辑:林 静)
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