绿茶水提物对蛋氨酸和胆碱缺乏饮食诱导的非酒精性脂肪肝炎小鼠的保护作用
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摘要
为探究绿茶水提物对蛋氨酸和胆碱缺乏(MCD)饮食诱导的非酒精性脂肪肝炎的药效作用及其作用机制,将40只C57BL/6小鼠随机分为空白组(MCS组)、MCD组、绿茶水提物低浓度组(0.15 g/kg)和高浓度组(0.30 g/kg)。小鼠饲喂MCD饲料,建立非酒精性脂肪肝炎模型,造模同时给予绿茶水提物进行干预。8周后,测定血清的谷丙酸转氨酶(ALT)、谷草转氨酶(AST)、白介素6(IL-6)和肿瘤坏死因子-α(TNF-α)的浓度以及肝组织的甘油三脂(TG)和胆固醇(TC)浓度;油红染色检测肝组织脂肪堆积情况和HE染色对肝组织病理等级评定;Western blot检测葡萄糖调节蛋白78(GRP78)和微管相关蛋白轻链3(LC3)表达水平。结果发现,与模型组相比,绿茶水提物显著降低小鼠血清AST、ALT、IL-6和TNF-α水平,其最大降幅分别约为38%、45%、46%、74%,并且可以降低肝脏TG、TC水平以及减少脂肪堆积面积,TG和TC的最大降幅分别约为44%和37%,同时改善肝组织的脂肪变性、炎症反应和细胞气球样变。Western blot显示,绿茶水提物降低GRP78蛋白水平,同时升高LC3-II的表达水平。因此,绿茶对MCD饮食诱导的非酒精性脂肪肝炎小鼠模型有明显保护作用,其作用机制可能与GRP78蛋白和LC3蛋白相关。
To evaluate the effect of green tea extract on the NASH mice-induced by MCD diet and its possible mechanism, forty C57 BL/6 mice were randomly divided into four groups: control group(MCS diet), MCD group(MCD diet), MCD diet supplemented with green tea extract(0.15 g/kg/day or 0.30 g/kg/day). After 8-weeks administration, blood and liver were harvested. The levels of serum ALT, AST, IL-6 and TNF-α were measured. The levels of hepatic TG and TC were also measured. The oil red stained was used to evaluate the lipid accumulation area in liver. Additionally, the liver tissues were stained with hematoxylin-eosin to evaluate the hepatic steatosis, inflammation and the ballooning according to the non-alcoholic fatty liver disease activity score. Western blot was used to analyze the protein expression levels of GRP78 and LC3. Results showed that, compared to MCD model, administration of green tea extract could significantly decrease the levels of serum ALT、AST、IL-6 and TNF-α, with the largest decrease rate of 38%, 45%, 46% and 74%, respectively. In addition, result of oil red O staining indicated that the extract also could obviously attenuate the accumulated fat in the liver. The extract could decrease the levels of hepatic TG and TC, with the largest decrease rate of 44% and 37%, when compared to MCD group. Result of H&E staining showed that the score of steatosis, lobular inflammation, hepatic ballooning was significantly reduced by green tea extract in C57/BL6 mice subjected to MCD diet. The expression level of GRP78 was markedly restrained while that of LC3-II was increased by administration of green tea extract. Green tea extract has beneficial effects against NASH-induced by MCD diet. The regulations of GRP78 and LC3-II might be involved in the mechanisms of action of green tea extract.
To evaluate the effect of green tea extract on the NASH mice-induced by MCD diet and its possible mechanism, forty C57 BL/6 mice were randomly divided into four groups: control group(MCS diet), MCD group(MCD diet), MCD diet supplemented with green tea extract(0.15 g/kg/day or 0.30 g/kg/day). After 8-weeks administration, blood and liver were harvested. The levels of serum ALT, AST, IL-6 and TNF-α were measured. The levels of hepatic TG and TC were also measured. The oil red stained was used to evaluate the lipid accumulation area in liver. Additionally, the liver tissues were stained with hematoxylin-eosin to evaluate the hepatic steatosis, inflammation and the ballooning according to the non-alcoholic fatty liver disease activity score. Western blot was used to analyze the protein expression levels of GRP78 and LC3. Results showed that, compared to MCD model, administration of green tea extract could significantly decrease the levels of serum ALT、AST、IL-6 and TNF-α, with the largest decrease rate of 38%, 45%, 46% and 74%, respectively. In addition, result of oil red O staining indicated that the extract also could obviously attenuate the accumulated fat in the liver. The extract could decrease the levels of hepatic TG and TC, with the largest decrease rate of 44% and 37%, when compared to MCD group. Result of H&E staining showed that the score of steatosis, lobular inflammation, hepatic ballooning was significantly reduced by green tea extract in C57/BL6 mice subjected to MCD diet. The expression level of GRP78 was markedly restrained while that of LC3-II was increased by administration of green tea extract. Green tea extract has beneficial effects against NASH-induced by MCD diet. The regulations of GRP78 and LC3-II might be involved in the mechanisms of action of green tea extract.
引文
[1]Patel Y A,Gifford E J,Glass L M,et al.Risk factors for biopsy-proven advanced non-alcoholic fatty liver disease in the veterans health administration[J].Alimentary Pharmacology&Therapeutics,2018,47(2):268-278
[2]Lallukka S,Yki-J Rvinen H.Non-alcoholic fatty liver disease and risk of type 2 diabetes[J].Best Practice&Research Clinical Endocrinology&Metabolism,2016,30(3):385-395
[3]Gaggini M,Morelli M,Buzzigoli E,et al.Non-alcoholic fatty liver disease(NAFLD)and its connection with insulin resistance,dyslipidemia,atherosclerosis and coronary heart disease[J].Nutrients,2013,5(5):1544-1560
[4]Ara Jo A R,Rosso N,Bedogni G,et al.Global epidemiology of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis:What we need in the future[J].Liver International,2018,38(S1):47-51
[5]Schohraya S,Edgard D,Jean-Michel B,et al.Oxidative stress as a critical factor in nonalcoholic fatty liver disease pathogenesis[J].Antioxidants&Redox Signaling,2017,26(10):519-541
[6]Ibrahim S H,Hirsova P,Malhi H,et al.Animal models of nonalcoholic steatohepatitis:Eat,delete,and inflame[J].Digestive Diseases and Sciences,2016,61(5):1325-1336
[7]Onakpoya I,Spencer E,Heneghan C,et al.The effect of green tea on blood pressure and lipid profile:A systematic review and meta-analysis of randomized clinical trials[J].Nutrition,Metabolism and Cardiovascular Diseases,2014,24(8):823-836
[8]Bogdanski P,Suliburska J,Szulinska M,et al.Green tea extract reduces blood pressure,inflammatory biomarkers,and oxidative stress and improves parameters associated with insulin resistance in obese,hypertensive patients[J].Nutrition Research,2012,32(6):421-427
[9]Park H J,Dinatale D A,Chung M-Y,et al.Green tea extract attenuates hepatic steatosis by decreasing adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice[J].The Journal of Nutritional Biochemistry,2011,22(4):393-400
[10]张莹,李惠琼,邹佳,等.凤冈富锌硒茶对ERp57蛋白酶的抑制作用及其有效成分的虚拟筛选[J].中国现代医学杂志,2018,28(11):22-26ZHANG Ying,LI Hui-qiong,ZOU Jia,et al.Inhibitory effect of Se-enriched and zinc-enriched green tea of Fenggang on ERp57 protease and virtual screening of its effective constituents[J].China Journal of Modern Medicine,2018,28(11):22-26
[11]Kleiner D E,Brunt E M,Van Natta M,et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease[J].Hepatology(Baltimore,Md),2005,41(6):1313-1321
[12]崔国祯,余汉濠,黄天养,等.胆碱和蛋氨酸缺乏饲料诱导大?小鼠非酒精脂肪性肝炎动物模型的比较研究[J].遵义医学院学报,2017,40(3):229-233CUI Guo-zhen,YU Han-hao,HUANG Tian-yang,et al.Comparison of NASH models-induced by methionine and choline deficient diets between rat and mouse[J].Journal of Zunyi Medical University,2017,40(3):229-233
[13]Miyake T,Kumagi T,Hirooka M,et al.Metabolic markers and ALT cutoff level for diagnosing nonalcoholic fatty liver disease:a community-based cross-sectional study[J].Journal of Gastroenterology,2012,47(6):696-703
[14]LI S,HONG M,TAN H-Y,et al.Insights into the role and interdependence of oxidative stress and inflammation in liver diseases[J].Oxidative Medicine and Cellular Longevity,2016,2016:4234061
[15]Tanida I,Ueno T,Kominami E.LC3 and Autophagy[M].DERETIC V.Autophagosome and Phagosome.Totowa,NJ;Humana Press.2008
[16]LIN C-W,ZHANG H,LI M,et al.Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice[J].Journal of Hepatology,2013,58(5):993-999
[17]Zhou J,Farah B L,Sinha R A,et al.Epigallocatechin-3-Gallate(EGCG),a green tea polyphenol,stimulates hepatic autophagy and lipid clearance[J].PLOSONE,2014,9(1):e87161
[18]Kim J Y,Garcia-Carbonell R,Yamachika S,et al.ER stress drives lipogenesis and steatohepatitis via caspase-2 activation of S1P[J].Cell,2018,175(1):133-145
[19]Muraki Y,Makita Y,Yamasaki M,et al.Elevation of liver endoplasmic reticulum stress in a modified choline-deficient l-amino acid-defined diet-fed non-alcoholic steatohepatitis mouse model[J].Biochemical and Biophysical Research Communications,2017,486(3):632-638
[20]Greene M W,Burrington C M,Ruhoff M S,et al.PKCδis activated in a dietary model of steatohepatitis and regulates endoplasmic reticulum stress and cell death[J].The Journal of Biological Chemistry,2010,285(53):42115-42129
[21]刘佳,李传飞,宁波,等.内质网应激通过SCAP/SREBP-1c调控L02肝细胞脂质合成代谢[J].第三军医大学学报,2015,37(5):443-448LIU Jia,LI Chuan-fei,NING Bo,et al.Endoplasmic reticulum stress regulates lipid synthesis metabolism in L02hepatocytes through SCAP/SREBP-1c[J].Journal of Third Military Medical University,2015,37(5):443-448
[2]Lallukka S,Yki-J Rvinen H.Non-alcoholic fatty liver disease and risk of type 2 diabetes[J].Best Practice&Research Clinical Endocrinology&Metabolism,2016,30(3):385-395
[3]Gaggini M,Morelli M,Buzzigoli E,et al.Non-alcoholic fatty liver disease(NAFLD)and its connection with insulin resistance,dyslipidemia,atherosclerosis and coronary heart disease[J].Nutrients,2013,5(5):1544-1560
[4]Ara Jo A R,Rosso N,Bedogni G,et al.Global epidemiology of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis:What we need in the future[J].Liver International,2018,38(S1):47-51
[5]Schohraya S,Edgard D,Jean-Michel B,et al.Oxidative stress as a critical factor in nonalcoholic fatty liver disease pathogenesis[J].Antioxidants&Redox Signaling,2017,26(10):519-541
[6]Ibrahim S H,Hirsova P,Malhi H,et al.Animal models of nonalcoholic steatohepatitis:Eat,delete,and inflame[J].Digestive Diseases and Sciences,2016,61(5):1325-1336
[7]Onakpoya I,Spencer E,Heneghan C,et al.The effect of green tea on blood pressure and lipid profile:A systematic review and meta-analysis of randomized clinical trials[J].Nutrition,Metabolism and Cardiovascular Diseases,2014,24(8):823-836
[8]Bogdanski P,Suliburska J,Szulinska M,et al.Green tea extract reduces blood pressure,inflammatory biomarkers,and oxidative stress and improves parameters associated with insulin resistance in obese,hypertensive patients[J].Nutrition Research,2012,32(6):421-427
[9]Park H J,Dinatale D A,Chung M-Y,et al.Green tea extract attenuates hepatic steatosis by decreasing adipose lipogenesis and enhancing hepatic antioxidant defenses in ob/ob mice[J].The Journal of Nutritional Biochemistry,2011,22(4):393-400
[10]张莹,李惠琼,邹佳,等.凤冈富锌硒茶对ERp57蛋白酶的抑制作用及其有效成分的虚拟筛选[J].中国现代医学杂志,2018,28(11):22-26ZHANG Ying,LI Hui-qiong,ZOU Jia,et al.Inhibitory effect of Se-enriched and zinc-enriched green tea of Fenggang on ERp57 protease and virtual screening of its effective constituents[J].China Journal of Modern Medicine,2018,28(11):22-26
[11]Kleiner D E,Brunt E M,Van Natta M,et al.Design and validation of a histological scoring system for nonalcoholic fatty liver disease[J].Hepatology(Baltimore,Md),2005,41(6):1313-1321
[12]崔国祯,余汉濠,黄天养,等.胆碱和蛋氨酸缺乏饲料诱导大?小鼠非酒精脂肪性肝炎动物模型的比较研究[J].遵义医学院学报,2017,40(3):229-233CUI Guo-zhen,YU Han-hao,HUANG Tian-yang,et al.Comparison of NASH models-induced by methionine and choline deficient diets between rat and mouse[J].Journal of Zunyi Medical University,2017,40(3):229-233
[13]Miyake T,Kumagi T,Hirooka M,et al.Metabolic markers and ALT cutoff level for diagnosing nonalcoholic fatty liver disease:a community-based cross-sectional study[J].Journal of Gastroenterology,2012,47(6):696-703
[14]LI S,HONG M,TAN H-Y,et al.Insights into the role and interdependence of oxidative stress and inflammation in liver diseases[J].Oxidative Medicine and Cellular Longevity,2016,2016:4234061
[15]Tanida I,Ueno T,Kominami E.LC3 and Autophagy[M].DERETIC V.Autophagosome and Phagosome.Totowa,NJ;Humana Press.2008
[16]LIN C-W,ZHANG H,LI M,et al.Pharmacological promotion of autophagy alleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditions in mice[J].Journal of Hepatology,2013,58(5):993-999
[17]Zhou J,Farah B L,Sinha R A,et al.Epigallocatechin-3-Gallate(EGCG),a green tea polyphenol,stimulates hepatic autophagy and lipid clearance[J].PLOSONE,2014,9(1):e87161
[18]Kim J Y,Garcia-Carbonell R,Yamachika S,et al.ER stress drives lipogenesis and steatohepatitis via caspase-2 activation of S1P[J].Cell,2018,175(1):133-145
[19]Muraki Y,Makita Y,Yamasaki M,et al.Elevation of liver endoplasmic reticulum stress in a modified choline-deficient l-amino acid-defined diet-fed non-alcoholic steatohepatitis mouse model[J].Biochemical and Biophysical Research Communications,2017,486(3):632-638
[20]Greene M W,Burrington C M,Ruhoff M S,et al.PKCδis activated in a dietary model of steatohepatitis and regulates endoplasmic reticulum stress and cell death[J].The Journal of Biological Chemistry,2010,285(53):42115-42129
[21]刘佳,李传飞,宁波,等.内质网应激通过SCAP/SREBP-1c调控L02肝细胞脂质合成代谢[J].第三军医大学学报,2015,37(5):443-448LIU Jia,LI Chuan-fei,NING Bo,et al.Endoplasmic reticulum stress regulates lipid synthesis metabolism in L02hepatocytes through SCAP/SREBP-1c[J].Journal of Third Military Medical University,2015,37(5):443-448