BZL方对非酒精性脂肪性肝炎模型小鼠肠黏膜损伤的保护作用
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摘要
目的:观察中药组分复方BZL方(白术多糖、栀子苷、绿原酸)对高脂饮食诱导的小鼠脂肪性肝炎(NASH)及其肠黏膜损伤的作用。方法:C57BL/6J雄性小鼠27只,随机分为正常、模型和BZL方组,每组9只。除正常组外,其余各组均采用高脂饮食诱导NASH,第14周开始,BZL方组灌胃BZL方,其余各组灌胃等量饮用水,至17周末处死取材。观察各组小鼠体质量、进食量、肝体比,肝组织病理(HE和天狼猩红染色,SAF评分),检测血浆ALT、GGT活性、LPS水平,肝组织TG含量;肝组织胶原I型和IV型、内毒素受体CD14、TLR1、TLR2、TLR4、TLR9及炎性反应因子IL-1β、TNF-αmRNA表达(real-time PCR法);肠组织病理(HE染色);大肠组织紧密连接zo-1、occludin、claudin mRNA表达(real-time PCR法)及蛋白表达(免疫荧光染色)。结果:高脂饮食诱导NASH模型中,模型组血浆ALT、GGT、LPS及肝组织TG含量较正常组显著升高(P <0. 01);肝组织可见脂肪沉积、炎性反应、气球样变及纤维化,SAF评分显著高于正常组,肝组织胶原Col I、Col IV mRNA显著升高,CD14、TLR1、TLR2、TLR4、TLR9及IL-1β、TNF-αmRNA表达升高(P <0. 05,P <0. 01);肠组织病理变化光镜下不明显,大肠组织紧密连接zo-1、occludin、claudin mRNA表达降低(P <0. 01),zo-1、occludin免疫荧光阳性染色减少。BZL方可降低血浆ALT、GGT、LPS、肝组织TG含量(P <0. 05,P <0. 01),改善SAF评分,降低肝组织胶原Col I、Col IV mRNA表达(P <0. 01),降低CD14、TLR2、TLR4、TLR9及IL-1β、TNF-αmRNA表达(P <0. 05,P <0. 01),恢复大肠组织紧密连接zo-1、occludin mRNA表达(P <0. 01),恢复zo-1、occludin免疫荧光阳性染色。结论:BZL方有效减轻高脂饮食诱导的小鼠NASH,该作用与其保护肠黏膜、抑制LPS肠渗漏及肝组织中TLRs及炎性反应因子密切相关。
Objective: To observe the effects of Chinese Medicine BZL recipe( composed of Atractylodes macrocephala polysaccharide,chlorogenic acid,and geniposide) on non-alcoholic steatohepatitis( NASH) and intestinal mucosal injury induced by high-fat diet in mice. Methods: Twenty seven male C57 BL/6 J mice were randomly divided into the normal group,the model group,and BZL group,with 9 rats in each group. Except the normal group,the other groups were induced by NASH. From the 14 th week,mice in BZL group were administrated with BZL recipe by gavage daily,and the other mice were administrated with same dose distilled water. Till the end of 17 th week,the mice were killed for collection materials. The body weight,food intake,liver-to-body ratio,liver histopathology( HE and Sirius red staining,SAF score) were observed in each group. Plasma ALT,GGT activity,LPS level,liver tissue TG content,liver tissue collagen type I and type IV,endotoxin receptor CD14,TLR1,TLR2,TLR4,TLR9 and inflammatory factors IL-1β,TNF-α mRNA expression( real-time PCR),intestinal histopathology( HE staining),large intestine tissue tight junction zo-1,occludin,claudin mRNA expression( real-time PCR) and protein expression( immunofluorescence staining) were detected. Results: In the high-fat diet induced NASH model,plasma ALT,GGT,LPS and liver tissue TG levels in the model group were significantly higher than those in the normal group( P < 0. 01); liver tissue showed fat deposition,inflammation,balloon-like changes and fibrosis. SAF score was significantly higher than the normal group. Liver tissue collagen Col I,Col IV mRNA increased significantly,and CD14,TLR1,TLR2,TLR4,TLR9 and IL-1β,TNF-α mRNA expression increased( P < 0. 05,P < 0. 01); The pathological changes of intestinal tissue were not obvious under light microscope. The expression of zo-1,occludin and claudin mRNA in the large intestine was decreased( P < 0. 01),and the immunofluorescence staining of zo-1 and occludin was decreased. BZL recipe can reduce plasma ALT,GGT,LPS,liver tissue TG content( P < 0. 05,P < 0. 01),improve SAF score,reduce liver tissue collagen Col I,Col IV mRNA expression( P < 0. 01),reduce CD14,TLR2,TLR4,TLR9,IL-1β and TNF-α mRNA expression( P <0. 05,P < 0. 01),restored the expression of zo-1 and occludin mRNA in the large intestine,and restored the immunofluorescence staining of zo-1 and occludin. Conclusion: BZL recipe can effectively alleviate NASH induced by high-fat diet,which is closely related to the protection of intestinal mucosa,inhibition of LPS intestinal leakage and TLRs and inflammatory factors in liver tissue.
Objective: To observe the effects of Chinese Medicine BZL recipe( composed of Atractylodes macrocephala polysaccharide,chlorogenic acid,and geniposide) on non-alcoholic steatohepatitis( NASH) and intestinal mucosal injury induced by high-fat diet in mice. Methods: Twenty seven male C57 BL/6 J mice were randomly divided into the normal group,the model group,and BZL group,with 9 rats in each group. Except the normal group,the other groups were induced by NASH. From the 14 th week,mice in BZL group were administrated with BZL recipe by gavage daily,and the other mice were administrated with same dose distilled water. Till the end of 17 th week,the mice were killed for collection materials. The body weight,food intake,liver-to-body ratio,liver histopathology( HE and Sirius red staining,SAF score) were observed in each group. Plasma ALT,GGT activity,LPS level,liver tissue TG content,liver tissue collagen type I and type IV,endotoxin receptor CD14,TLR1,TLR2,TLR4,TLR9 and inflammatory factors IL-1β,TNF-α mRNA expression( real-time PCR),intestinal histopathology( HE staining),large intestine tissue tight junction zo-1,occludin,claudin mRNA expression( real-time PCR) and protein expression( immunofluorescence staining) were detected. Results: In the high-fat diet induced NASH model,plasma ALT,GGT,LPS and liver tissue TG levels in the model group were significantly higher than those in the normal group( P < 0. 01); liver tissue showed fat deposition,inflammation,balloon-like changes and fibrosis. SAF score was significantly higher than the normal group. Liver tissue collagen Col I,Col IV mRNA increased significantly,and CD14,TLR1,TLR2,TLR4,TLR9 and IL-1β,TNF-α mRNA expression increased( P < 0. 05,P < 0. 01); The pathological changes of intestinal tissue were not obvious under light microscope. The expression of zo-1,occludin and claudin mRNA in the large intestine was decreased( P < 0. 01),and the immunofluorescence staining of zo-1 and occludin was decreased. BZL recipe can reduce plasma ALT,GGT,LPS,liver tissue TG content( P < 0. 05,P < 0. 01),improve SAF score,reduce liver tissue collagen Col I,Col IV mRNA expression( P < 0. 01),reduce CD14,TLR2,TLR4,TLR9,IL-1β and TNF-α mRNA expression( P <0. 05,P < 0. 01),restored the expression of zo-1 and occludin mRNA in the large intestine,and restored the immunofluorescence staining of zo-1 and occludin. Conclusion: BZL recipe can effectively alleviate NASH induced by high-fat diet,which is closely related to the protection of intestinal mucosa,inhibition of LPS intestinal leakage and TLRs and inflammatory factors in liver tissue.
引文
[1]中华医学会肝病学分会脂肪肝和酒精性肝病学组.非酒精性脂肪性肝病防治指南(2018更新版)[J].现代医药卫生,2018,34(5):641-649.
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[3]Rinella M E,Sanyal A J.Management of NAFLD:a stage-based approach[J].Nat Rev Gastroenterol Hepatol,2016,13(4):196-205.
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[7]海亚美,黄甫,冷静,等.祛湿化瘀方对非酒精性脂肪肝小鼠肠粘膜损伤的保护作用[J].中国中西医结合杂志,2017,58(5):361-365.
[8]孟胜喜,胡义扬,冯琴,等.中药组分复方“BZL”的筛选及其对实验性脂肪肝大鼠的作用[J].世界科学技术-中医药现代化,2014,16(1):45-51.
[9]Meng S X,Liu Q,Tang Y J,et al.A Recipe Composed of Chinese Herbal Active Components Regulates Hepatic Lipid Metabolism of NAFLDIn Vivoand In Vitro[J].Bio Med Research International,2016,(2016):1-12.
[10]黄甫,彭景华,李晓飞,等.有效组分复方BZL对高脂饮食诱导小鼠非酒精性脂肪肝的干预作用[J].中华中医药杂志,2015,30(3):713-717.
[11]Pierre Bedossa,Poitou C,Veyrie N,et al.Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients[J].Hepatology,2012,56(5):1751-1759.
[12]Ekstedt M,Hagstr9m H,Nasr P,et al.Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up[J].Hepatology,2015,61(5):1547-1554.
[13]Angulo P,Kleiner D E,Damlarsen S,et al.Liver Fibrosis,but no Other Histologic Features,Associates with Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease[J].Gastroenterology,2015,149(2):389-397.
[14]Kleiner D E,Makhlouf H R.Histology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis in Adults and Children[J].Clinics in Liver Disease,2016,20(2):293-312.
[15]Odenwald M A,Turner J R.The intestinal epithelial barrier:a therapeutic target?[J].Nature Reviews Gastroenterology&Hepatology,2016,14(1):9.
[16]Rahman K,Desai C,Iyer S S,et al.Loss of Junctional Adhesion Molecule A Promotes Severe Steatohepatitis in Mice on a Diet High in Saturated Fat,Fructose,and Cholesterol[J].Gastroenterology,2016,151(4):733-746.e12.
[17]Yizhe Cui,Qiuju Wang,Renxu Chang,et al.Intestinal Barrier Function-NAFLD Interactions and Possible Role of Gut Microbiota[J].Journal of Agricultural and Food Chemistry,2019,doi:10.1021/acs.jafc.9b00080.
[18]Safari Z,Philippe Gérard.The links between the gut microbiome and non-alcoholic fatty liver disease(NAFLD)[J].Cellular and Molecular Life Sciences CMLS,2019,doi:10.1007/s00018-019-03011-w.
[19]Feng Q,Liu W,Baker S S,et al.Multi-targeting therapeutic mechanisms of the Chinese herbal medicine QHD in the treatment of nonalcoholic fatty liver disease[J].Oncotarget,2017,8(17):27820-27838.
[20]Cai J,Xu M,Zhang X,et al.Innate Immune Signaling in Nonalcoholic Fatty Liver Disease and Cardiovascular Diseases[J].Annual Review of Pathology Mechanisms of Disease,2019,14(1):153-184.
[21]Woolbright B L,Hartmut J.Mechanisms of Inflammatory Liver Injury and Drug-Induced Hepatotoxicity[J].Current Pharmacology Reports,2018,4(5):346-357.
[22]Narayanankutty Arunaksharan,Toll like receptors as a novel therapeutic target for natural products against chronic diseases[J].Curr Drug Targets,2019,doi:10.2174/1389450120666 190222181506.
[23]Greuter T,Malhi H,Gores G J,et al.Therapeutic opportunities for alcoholic steatohepatitis and nonalcoholic steatohepatitis:exploiting similarities and differences in pathogenesis[J].Jci Insight,2017,2(17):1-18.
[24]Zhou Huiting,Coveney Andrew P,Wu Ming et al.Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection[J].Front Immunol,2018,9:3082.
[25]Armstrong M J,Gaunt P,Aithal G P,et al.Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis(LEAN):a multicentre,double-blind,randomised,placebo-controlled phase 2 study[J].Lancet,2016,387(10019):679-690.
[26]Cusi K,Orsak B,Bril F,et al.Long-Term Pioglitazone Treatment for Patients With Nonalcoholic Steatohepatitis and Prediabetes or Type 2Diabetes Mellitus:A Randomized,Controlled Trial[J].Annals of Internal Medicine,2016,165(5):305.
[27]Sanyal AJ,Chalasani N,Kowdley KV,et al.Pioglitazone,vitamin E,or placebo for nonalcoholic steatohepatitis[J].N Engl J Med,2010,362(12):1185-1186.
[2]Younossi Z M,Koenig A B,Abdelatif D,et al.Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence,incidence,and outcomes[J].Hepatology,2016,64(1):73-84.
[3]Rinella M E,Sanyal A J.Management of NAFLD:a stage-based approach[J].Nat Rev Gastroenterol Hepatol,2016,13(4):196-205.
[4]Singh S,Allen A M,Zhen W,et al.Fibrosis Progression in Nonalcoholic Fatty Liver versus Nonalcoholic Steatohepatitis:A Systematic Review and Meta-analysis of Paired-Biopsy Studies[J].Clinical Gastroenterology&Hepatology,2015,13(4):643-654.
[5]Lindenmeyer C C,Mccullough A J.The Natural History of Nonalcoholic Fatty Liver Disease-An Evolving View[J].Clinics in Liver Disease,2018,22(1):11-21.
[6]Wiest R,Albillos A,Trauner M,et al.Targeting the gut-liver axis in liver disease[J],Journal of Hepatology,2017,67(5):1084-1103.
[7]海亚美,黄甫,冷静,等.祛湿化瘀方对非酒精性脂肪肝小鼠肠粘膜损伤的保护作用[J].中国中西医结合杂志,2017,58(5):361-365.
[8]孟胜喜,胡义扬,冯琴,等.中药组分复方“BZL”的筛选及其对实验性脂肪肝大鼠的作用[J].世界科学技术-中医药现代化,2014,16(1):45-51.
[9]Meng S X,Liu Q,Tang Y J,et al.A Recipe Composed of Chinese Herbal Active Components Regulates Hepatic Lipid Metabolism of NAFLDIn Vivoand In Vitro[J].Bio Med Research International,2016,(2016):1-12.
[10]黄甫,彭景华,李晓飞,等.有效组分复方BZL对高脂饮食诱导小鼠非酒精性脂肪肝的干预作用[J].中华中医药杂志,2015,30(3):713-717.
[11]Pierre Bedossa,Poitou C,Veyrie N,et al.Histopathological algorithm and scoring system for evaluation of liver lesions in morbidly obese patients[J].Hepatology,2012,56(5):1751-1759.
[12]Ekstedt M,Hagstr9m H,Nasr P,et al.Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up[J].Hepatology,2015,61(5):1547-1554.
[13]Angulo P,Kleiner D E,Damlarsen S,et al.Liver Fibrosis,but no Other Histologic Features,Associates with Long-term Outcomes of Patients With Nonalcoholic Fatty Liver Disease[J].Gastroenterology,2015,149(2):389-397.
[14]Kleiner D E,Makhlouf H R.Histology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis in Adults and Children[J].Clinics in Liver Disease,2016,20(2):293-312.
[15]Odenwald M A,Turner J R.The intestinal epithelial barrier:a therapeutic target?[J].Nature Reviews Gastroenterology&Hepatology,2016,14(1):9.
[16]Rahman K,Desai C,Iyer S S,et al.Loss of Junctional Adhesion Molecule A Promotes Severe Steatohepatitis in Mice on a Diet High in Saturated Fat,Fructose,and Cholesterol[J].Gastroenterology,2016,151(4):733-746.e12.
[17]Yizhe Cui,Qiuju Wang,Renxu Chang,et al.Intestinal Barrier Function-NAFLD Interactions and Possible Role of Gut Microbiota[J].Journal of Agricultural and Food Chemistry,2019,doi:10.1021/acs.jafc.9b00080.
[18]Safari Z,Philippe Gérard.The links between the gut microbiome and non-alcoholic fatty liver disease(NAFLD)[J].Cellular and Molecular Life Sciences CMLS,2019,doi:10.1007/s00018-019-03011-w.
[19]Feng Q,Liu W,Baker S S,et al.Multi-targeting therapeutic mechanisms of the Chinese herbal medicine QHD in the treatment of nonalcoholic fatty liver disease[J].Oncotarget,2017,8(17):27820-27838.
[20]Cai J,Xu M,Zhang X,et al.Innate Immune Signaling in Nonalcoholic Fatty Liver Disease and Cardiovascular Diseases[J].Annual Review of Pathology Mechanisms of Disease,2019,14(1):153-184.
[21]Woolbright B L,Hartmut J.Mechanisms of Inflammatory Liver Injury and Drug-Induced Hepatotoxicity[J].Current Pharmacology Reports,2018,4(5):346-357.
[22]Narayanankutty Arunaksharan,Toll like receptors as a novel therapeutic target for natural products against chronic diseases[J].Curr Drug Targets,2019,doi:10.2174/1389450120666 190222181506.
[23]Greuter T,Malhi H,Gores G J,et al.Therapeutic opportunities for alcoholic steatohepatitis and nonalcoholic steatohepatitis:exploiting similarities and differences in pathogenesis[J].Jci Insight,2017,2(17):1-18.
[24]Zhou Huiting,Coveney Andrew P,Wu Ming et al.Activation of Both TLR and NOD Signaling Confers Host Innate Immunity-Mediated Protection Against Microbial Infection[J].Front Immunol,2018,9:3082.
[25]Armstrong M J,Gaunt P,Aithal G P,et al.Liraglutide safety and efficacy in patients with non-alcoholic steatohepatitis(LEAN):a multicentre,double-blind,randomised,placebo-controlled phase 2 study[J].Lancet,2016,387(10019):679-690.
[26]Cusi K,Orsak B,Bril F,et al.Long-Term Pioglitazone Treatment for Patients With Nonalcoholic Steatohepatitis and Prediabetes or Type 2Diabetes Mellitus:A Randomized,Controlled Trial[J].Annals of Internal Medicine,2016,165(5):305.
[27]Sanyal AJ,Chalasani N,Kowdley KV,et al.Pioglitazone,vitamin E,or placebo for nonalcoholic steatohepatitis[J].N Engl J Med,2010,362(12):1185-1186.