乳黄制剂对肝硬化模型大鼠肠源性内毒素血症的影响
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摘要
目的肠源性内毒素血症(IETM)在各种肝病,特别是肝硬化和重症肝炎时发生率高。肝硬化患者由于肠道菌群失调,屏障功能受损,细菌易位增多,门体分流,同时肝脏细胞功能、Kupffer细胞功能减退,对内毒素(LPS)降解能力下降,使肠道产生的内毒素大量进入外周循环。IETM导致肝损伤机制很复杂,可直接损害肝细胞,还可以通过激活Kupffer细胞、单核巨噬细胞释放促炎介质间接损害肝细胞,又可诱发全身代谢和血流动力学紊乱,引起全身器官功能衰竭,出现腹水、肝性脑病、肝肾综合征等种种并发症的发生和发展,严重影响患者的预后。中医理论中没有对肠源性内毒素血症的专门论述,但根据其致病特点,属于“内生毒邪”范畴。导师张赤志教授根据中医理论和临床经验,认为本病病因病机为是由于肝脾肾功能失调,水湿内停,瘀血阻滞,湿瘀互结,郁久化热成毒。毒邪再犯肝体,肝失疏泄,脾失健运,肠道传导失司,腑浊内生,病位在肝、肠。据此立“通下解毒法”,结合“肝与大肠相通,肝病宜疏通大肠”的学术思想,选用酒制大黄和乳酸菌素组成乳黄制剂防治肝硬化IETM。我们在前期防治急性肝性脑病的实验研究中发现乳黄制剂(湖北省卫生厅资助课题)能降低血浆内毒素水平,较实验性大鼠空白对照组,模型组,乳果糖对照组作用明显。为了进一步研究该制剂在防治肝硬化IETM的作用机理,我们将从细胞分子水平进行探讨。
方法SPF级雄性Wister大鼠88只,体重200～230克,随机分为6组:正常组、模型组、预防组、预防对照组、模型治疗组、模型治疗对照组。除正常组外,其余各组均采用CcL_4复合因素法制造肝硬化模型,饲养10周。正常组、模型组蒸馏水2ml/d灌胃;预防组、预防对照组在造模同时分别给予乳黄制剂、乳果糖2ml/d灌胃;造模第9周开始模型治疗组、模型治疗对照组分别给予乳黄制剂、乳果糖2ml/d灌胃连续2周。第10周末处死大鼠取材。(1)在无热源条件下采右心室血5ml检测血内毒素水平;(2)半定量逆转录-多聚酶链法(RT-PCR)检测大鼠肝组织LBPmRNA、CD14mRNA、TLR-4mRNA的表达;(3)检测血清D-乳酸、二胺氧化酶(DAO)水平了解对肠通透性的影响;(4)取小肠组织的提取液200μl,应用自动放免仪测定各组样品的肠黏膜分泌型免疫球蛋白A(S-IgA)浓度,了解对肠道免疫屏障功能的影响;(5)取肝右叶、脾组织各0.1g用生理盐水洗净后进行细菌培养,观察肠道细菌移位的情况。(6)取肝左叶作病检。
结果(1)乳黄制剂可明显降低肝硬化大鼠内毒素水平(P＜0.05);(2)乳黄制剂可下调肝硬化大鼠肝组织LBPmRNA、CD14mRNA、TLR4mRNA的表达水平,从而减少内毒素对肝脏的直接或间接的伤害,尤其是模型治疗组疗效更佳;(3)乳黄制剂可明显降低肝硬化大鼠血清D-乳酸、DAO水平(P＜0.05),降低肠通透性,尤以预防组效果明显;(4)乳黄制剂能有效提高肠黏膜S-IgA的分泌水平(P＜0.05),早期干预效果佳;(5)预防组经过乳黄制剂干预后没有出现肠道细菌移位情况;(6)乳黄制剂能改善预防组和模型治疗组肝组织的肝硬化以及纤维组织增生、炎性细胞浸润程度,其中预防组肝组织的病理变化改善明显。
结论乳黄制剂防治肝硬化大鼠内毒素血症的作用,可能有下列机制实现:①清除肠道内毒素,同时通过抑制LPS与脂多糖结合蛋白(LBP)以及相应受体CD14、TLR-4结合,降低肠道内毒素的吸收;②提高肠道局部免疫功能,有效阻止有害菌对肠黏膜的黏附,保护肠黏膜,防止细菌移位。③提高肠道血流灌注,促进肠细胞增生,减少肠黏膜绒毛上皮脱落变短,降低肠黏膜的通透性;④补充肠道的有益菌,抑制细菌过度生长,平衡肠道微生态环境,减轻内毒素血症。
Objective Intestinal endotoxemia(IETM)in various liver diseases,particularly severe cirrhosis of the liver and the high incidence of hepatitis.Patients with cirrhosis due to intestinal flora imbalance,impaired barrier function,increased bacterial translocation,the doors of segregation,and liver cells,Kupffer cell dysfunction, endotoxin(LPS)degradation ability to gut the inside Toxins into the large number of peripheral circulation.IETM lead to liver damage mechanism is very complicated and can damage the liver cells,can also activate Kupffer cells,macrophages single-core to release pro-inflammatory media indirect damage liver cells,can induce systemic metabolism and blood flow dynamics disorder,caused systemic organ Failure,there ascites,hepatic encephalopathy,liver and kidney syndrome and other complications of the occurrence and development,which has seriously affected the prognosis of patients.Chinese medicine theory,not gut endotoxemia the devoted,but according to their pathogenic features of the "intrinsic evil drug" areas.Mentor Professor Zhang Chizhi According to Chinese medicine theory and clinical experience that the pathogenesis of this disease is due to liver spleen and kidney dysfunction,endogenous dampness and water retention,block blood stasis,wet stasis among guitar,pathogen accumulation causing fire into drugs.Commit evil of liver toxicity,liver losing regulating,spleen losing transportation and transformation,stolen intestinal conduction Division,Fu cloud of Health,the disease of the liver and intestine.This legislation "-the antidote laws" in light of "the same liver and large intestine,liver disease to clear the large intestine," the academic thinking,wine selection system composed of lactic acid bacteria and RuHuang preparation of cirrhosis IETM.We in the early prevention and treatment of acute hepatic encephalopathy in the experimental study found that RuHuang agents(the Hubei Provincial Health Department funding issues)can reduce the plasma levels of toxins,more experimental rat control group,model,lactulose significant role in the control group.To further study the drug in the prevention and treatment of liver cirrhosis IETM mechanism,we will discuss cellular and molecular level.
Methods SPF level of 88 male Wister rats,weight 200 to 230 grams,were divided into six groups:the normal group,model group,prevention group,the prevention of the control group,model treatment group,the model of control Group.In addition to the normal group,other groups of factors are used CcL4 cirrhosis of manufacturing model,keeping 10 weeks.The normal group,the model group distilled water 2 ml/d gavage;prevention group,prevention model in the control group were also given RuHuang formulations,lactulose 2 ml/d gavage;model first nine weeks beginning of the treatment group model,the model for the control group were given RuHuang, lactulose 2 ml/d gavage for two weeks.Article 10 killed in weekend material.(1)in the absence of heat sources under the condition of the right ventricle-5 ml of blood testing blood levels of endotoxin(2)semi-quantitative reverse transcriptasepolymerase chain Act(RT-PCR)testing of liver tissue CD14mRNA,TLR-4mRNA, LBPmRNA The expression(3)of serum D-lactic acid,diamine oxidase(DAO)the level of understanding of the permeability of the intestine(4)from the small intestine extract of 200μl,RIA-automatic application of the sample group of enterovirus Mucosa secretory immunoglobulin A(S-IgA)concentration understanding of the intestinal barrier function of the immune(5)take 0.1 g of the right of liver,0.1 g of the spleen with saline wash after the bacterial culture to observe the intestinal bacteria Shift the situation.(6)take the left of liver disease to do the examination.
Results(1)RuHuang agents can significantly reduce the level of toxins in the liver cirrhosis in rats(P＜0.05);(2)RuHuang preparations can be lowered rat liver cirrhosis LBPmRNA,CD14mRNA,TLR4mRNA the expression level,thereby reducing the toxin directly on the liver Or indirect harm,in particular model effect of the treatment group better;(3)RuHuang agents can significantly reduce cirrhosis of serum D-lactic acid,DAO level(P＜0.05),lower intestinal permeability,especially in the prevention of significant effects of group;(4)RuHuang preparation can effectively improve the intestinal mucosa S-IgA secretion levels(P＜0.05),good effects of early intervention; (5)prevention group after RuHuang agents did not appear after the intervention of bacterial translocation situation;(6)RuHuang preparation can Improve prevention group and model treatment group of liver cirrhosis and fibrous tissue proliferation, inflammatory cells invasion,prevention group the pathological changes in liver tissue to improve significantly.
Conclusion RuHuang preparation of cirrhosis of the role of endotoxemia,there may be mechanisms to achieve the following:①remove intestinal toxins,and also by inhibiting LPS LPS-binding protein(LBP)and the corresponding receptor CD14, TLR-4 Combine to reduce the toxins in the intestinal absorption;②increase of intestinal immune function,effectively prevent the harmful bacteria to the intestinal mucosa of adhesion,the protection of the intestinal mucosa,to prevent bacterial translocation.③improve intestinal perfusion,and promote intestinal cell proliferation and reduce the intestinal mucosa villi epithelial shedding shorter,reducing the permeability of the intestinal mucosa;④added the profitable fungus of intestinal, curb the excessive growth of bacteria,balance the microbiology of the gut environment,Reduce endotoxemia.
方法SPF级雄性Wister大鼠88只,体重200～230克,随机分为6组:正常组、模型组、预防组、预防对照组、模型治疗组、模型治疗对照组。除正常组外,其余各组均采用CcL_4复合因素法制造肝硬化模型,饲养10周。正常组、模型组蒸馏水2ml/d灌胃;预防组、预防对照组在造模同时分别给予乳黄制剂、乳果糖2ml/d灌胃;造模第9周开始模型治疗组、模型治疗对照组分别给予乳黄制剂、乳果糖2ml/d灌胃连续2周。第10周末处死大鼠取材。(1)在无热源条件下采右心室血5ml检测血内毒素水平;(2)半定量逆转录-多聚酶链法(RT-PCR)检测大鼠肝组织LBPmRNA、CD14mRNA、TLR-4mRNA的表达;(3)检测血清D-乳酸、二胺氧化酶(DAO)水平了解对肠通透性的影响;(4)取小肠组织的提取液200μl,应用自动放免仪测定各组样品的肠黏膜分泌型免疫球蛋白A(S-IgA)浓度,了解对肠道免疫屏障功能的影响;(5)取肝右叶、脾组织各0.1g用生理盐水洗净后进行细菌培养,观察肠道细菌移位的情况。(6)取肝左叶作病检。
结果(1)乳黄制剂可明显降低肝硬化大鼠内毒素水平(P＜0.05);(2)乳黄制剂可下调肝硬化大鼠肝组织LBPmRNA、CD14mRNA、TLR4mRNA的表达水平,从而减少内毒素对肝脏的直接或间接的伤害,尤其是模型治疗组疗效更佳;(3)乳黄制剂可明显降低肝硬化大鼠血清D-乳酸、DAO水平(P＜0.05),降低肠通透性,尤以预防组效果明显;(4)乳黄制剂能有效提高肠黏膜S-IgA的分泌水平(P＜0.05),早期干预效果佳;(5)预防组经过乳黄制剂干预后没有出现肠道细菌移位情况;(6)乳黄制剂能改善预防组和模型治疗组肝组织的肝硬化以及纤维组织增生、炎性细胞浸润程度,其中预防组肝组织的病理变化改善明显。
结论乳黄制剂防治肝硬化大鼠内毒素血症的作用,可能有下列机制实现:①清除肠道内毒素,同时通过抑制LPS与脂多糖结合蛋白(LBP)以及相应受体CD14、TLR-4结合,降低肠道内毒素的吸收;②提高肠道局部免疫功能,有效阻止有害菌对肠黏膜的黏附,保护肠黏膜,防止细菌移位。③提高肠道血流灌注,促进肠细胞增生,减少肠黏膜绒毛上皮脱落变短,降低肠黏膜的通透性;④补充肠道的有益菌,抑制细菌过度生长,平衡肠道微生态环境,减轻内毒素血症。
Objective Intestinal endotoxemia(IETM)in various liver diseases,particularly severe cirrhosis of the liver and the high incidence of hepatitis.Patients with cirrhosis due to intestinal flora imbalance,impaired barrier function,increased bacterial translocation,the doors of segregation,and liver cells,Kupffer cell dysfunction, endotoxin(LPS)degradation ability to gut the inside Toxins into the large number of peripheral circulation.IETM lead to liver damage mechanism is very complicated and can damage the liver cells,can also activate Kupffer cells,macrophages single-core to release pro-inflammatory media indirect damage liver cells,can induce systemic metabolism and blood flow dynamics disorder,caused systemic organ Failure,there ascites,hepatic encephalopathy,liver and kidney syndrome and other complications of the occurrence and development,which has seriously affected the prognosis of patients.Chinese medicine theory,not gut endotoxemia the devoted,but according to their pathogenic features of the "intrinsic evil drug" areas.Mentor Professor Zhang Chizhi According to Chinese medicine theory and clinical experience that the pathogenesis of this disease is due to liver spleen and kidney dysfunction,endogenous dampness and water retention,block blood stasis,wet stasis among guitar,pathogen accumulation causing fire into drugs.Commit evil of liver toxicity,liver losing regulating,spleen losing transportation and transformation,stolen intestinal conduction Division,Fu cloud of Health,the disease of the liver and intestine.This legislation "-the antidote laws" in light of "the same liver and large intestine,liver disease to clear the large intestine," the academic thinking,wine selection system composed of lactic acid bacteria and RuHuang preparation of cirrhosis IETM.We in the early prevention and treatment of acute hepatic encephalopathy in the experimental study found that RuHuang agents(the Hubei Provincial Health Department funding issues)can reduce the plasma levels of toxins,more experimental rat control group,model,lactulose significant role in the control group.To further study the drug in the prevention and treatment of liver cirrhosis IETM mechanism,we will discuss cellular and molecular level.
Methods SPF level of 88 male Wister rats,weight 200 to 230 grams,were divided into six groups:the normal group,model group,prevention group,the prevention of the control group,model treatment group,the model of control Group.In addition to the normal group,other groups of factors are used CcL4 cirrhosis of manufacturing model,keeping 10 weeks.The normal group,the model group distilled water 2 ml/d gavage;prevention group,prevention model in the control group were also given RuHuang formulations,lactulose 2 ml/d gavage;model first nine weeks beginning of the treatment group model,the model for the control group were given RuHuang, lactulose 2 ml/d gavage for two weeks.Article 10 killed in weekend material.(1)in the absence of heat sources under the condition of the right ventricle-5 ml of blood testing blood levels of endotoxin(2)semi-quantitative reverse transcriptasepolymerase chain Act(RT-PCR)testing of liver tissue CD14mRNA,TLR-4mRNA, LBPmRNA The expression(3)of serum D-lactic acid,diamine oxidase(DAO)the level of understanding of the permeability of the intestine(4)from the small intestine extract of 200μl,RIA-automatic application of the sample group of enterovirus Mucosa secretory immunoglobulin A(S-IgA)concentration understanding of the intestinal barrier function of the immune(5)take 0.1 g of the right of liver,0.1 g of the spleen with saline wash after the bacterial culture to observe the intestinal bacteria Shift the situation.(6)take the left of liver disease to do the examination.
Results(1)RuHuang agents can significantly reduce the level of toxins in the liver cirrhosis in rats(P＜0.05);(2)RuHuang preparations can be lowered rat liver cirrhosis LBPmRNA,CD14mRNA,TLR4mRNA the expression level,thereby reducing the toxin directly on the liver Or indirect harm,in particular model effect of the treatment group better;(3)RuHuang agents can significantly reduce cirrhosis of serum D-lactic acid,DAO level(P＜0.05),lower intestinal permeability,especially in the prevention of significant effects of group;(4)RuHuang preparation can effectively improve the intestinal mucosa S-IgA secretion levels(P＜0.05),good effects of early intervention; (5)prevention group after RuHuang agents did not appear after the intervention of bacterial translocation situation;(6)RuHuang preparation can Improve prevention group and model treatment group of liver cirrhosis and fibrous tissue proliferation, inflammatory cells invasion,prevention group the pathological changes in liver tissue to improve significantly.
Conclusion RuHuang preparation of cirrhosis of the role of endotoxemia,there may be mechanisms to achieve the following:①remove intestinal toxins,and also by inhibiting LPS LPS-binding protein(LBP)and the corresponding receptor CD14, TLR-4 Combine to reduce the toxins in the intestinal absorption;②increase of intestinal immune function,effectively prevent the harmful bacteria to the intestinal mucosa of adhesion,the protection of the intestinal mucosa,to prevent bacterial translocation.③improve intestinal perfusion,and promote intestinal cell proliferation and reduce the intestinal mucosa villi epithelial shedding shorter,reducing the permeability of the intestinal mucosa;④added the profitable fungus of intestinal, curb the excessive growth of bacteria,balance the microbiology of the gut environment,Reduce endotoxemia.
引文
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