铜负荷大鼠肝星状细胞凋亡p38MAPK信号通路探讨及通腑利湿化瘀散结方对其干预的代谢组学研究
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摘要
1.研究背景及目的
肝豆状核变性(Wilson disease, WD)是常染色体隐性遗传的铜代谢障碍疾病,wD好发于青少年,临床上以慢性肝脏损害、肾脏功能病变及神经系统障碍等症状为主要表现。研究表明,肝脏是wD最先累及的脏器之一,发病年龄越早,以肝脏病变为唯一临床表现的可能性越大,肝纤维化(Hepatic Fibrosis)几乎是每个wD患者肝脏损害的主要病理改变。wD作为铜代谢障碍性疾病,发生肝纤维化的根本原因是铜离子向胆道排泄困难,造成游离铜在肝组织内缓慢蓄积。研究证实肝星状细胞(hepatic stellate cells, HSC)的激活是肝纤维化发生的主要环节,激活的HSC大量增殖,发生表型改变、分泌过多的细胞外基质(Extra Cellular matrix, ECM)沉积于肝脏是肝纤维化形成的关键。
在HSC信号通路研究中,丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)是近年研究的热点。p38MAPK信号通路是MAPK介导的信号通路的重要分支,主要参与炎症、增殖、凋亡等多种生理过程,是目前调控细胞凋亡的关键通路之一。
代谢组学日益成为整体性研究生命体系功能变化的非常有力的分析手段。由于wD为铜代谢障碍性疾病,其代谢物谱的变化可能不同于其他肝脏疾病,因此wD的代谢组学研究对于wD的病机、治疗及预后显得尤为重要。
目前肝纤维化的防治特别是wD肝纤维化尚缺乏针对性较强的特效手段,而中医中药以其多靶点的整体调节和以人为本的个性化治疗方案,在肝纤维化的防治中具有独特的疗效。导师所在课题组在前期的临床实践中证明：通腑利湿、化瘀散结的中药专方肝豆灵可以改善wD患者的肝纤维化指标,促进wD者肝硬化及肝功能的恢复。
本实验主要开展以下研究：建立铜负荷大鼠模型,观察通腑利湿、化瘀散结方肝豆灵对肝纤维化HSC凋亡及p38MAPK信号通路蛋白表达的影响,运用代谢组学技术检测肝豆灵对铜负荷大鼠肝纤维化小分子代谢标志物变化的影响,初步探讨通腑利湿、化瘀散结方肝豆灵治疗wD肝纤维化的分子机制。
2.方法
2.1实验分为5组：分别为空白对照组,模型对照组,肝豆灵组,肝豆灵加青霉胺组,青霉胺组；
2.2按照文献复制铜负荷大鼠模型；
2.3使用通腑利湿、化瘀散结方肝豆灵对各治疗组铜负荷大鼠进行干预,选用青霉胺为阳性对照药物；
2.4光镜观察肝纤维化病理改变；
2.5采用免疫组化法、Western-Blot法检测p38MAPK蛋白的表达,TUNEL法检测细胞凋亡；
2.6运用1H-NMR技术检测肝纤维化大鼠血清、尿液的小分子代谢产物的变化。
3.结果
3.1通腑利湿化瘀散结方肝豆灵对铜负荷大鼠肝纤维化病理的影响
空白组肝小叶结构完整,肝细胞排列规则,小叶内无细胞变性,无炎性细胞浸润；铜负荷大鼠模型组HE染色汇管区可见明显纤维组织增生,各治疗组肝纤维化程度明显减轻；Masson染色光镜下为绿色为胶原纤维,正常对照组中央静脉管壁可见少量绿色的胶原纤维,模型组于汇管区和中央静脉管壁可见大量粗大的绿色的胶原纤维,与正常对照组相比有显著性意义(P<0.01),各治疗组肝纤维化程度明显减轻,其中肝豆灵加青霉胺组积分下降最为显著(P<0.01),肝豆灵组和青霉胺组病理积分亦明显下降(P<0.05)。
3.2通腑利湿化瘀散结方肝豆灵对铜负荷大鼠HSC的影响
TUNEL法检测细胞凋亡,空白组未见明显HSC增生,模型组可见大量HSC增生表达,各治疗组可见HSC凋亡细胞表达,各治疗组凋亡指数较模型组明显增高,其中肝豆灵加青霉胺组凋亡指数显著增高(P<0.01),肝豆灵组和青霉胺组凋亡指数亦明显升高(P<0.05)。
3.3通腑利湿化瘀散结方肝豆灵对铜负荷大鼠P38MAPK信号通路的影响
3.3.1免疫组化法检测p38MAPK信号通路表达
p38MAPK蛋白阳性信号呈棕黄色,模型组及各治疗组可见阳性细胞表达,模型组阳性细胞数表达较空白组显著增强(P<0.001),青霉胺加肝豆灵组、肝豆灵组及青霉胺组较模型组p38MAPK阳性细胞数表达显著减少(P<0.05),组间比较无显著差异。
3.3.2Western-Blot法检p38MAPK信号通路表达
p38MAPK蛋白水平在各组之间比较有明显差别,与空白组相比,模型组及各治疗组可见p38MAPK特异性蛋白条带表达,模型组较空白组阳性蛋白相对表达量显著增强(P<0.001),青霉胺加肝豆灵组、肝豆灵组及青霉胺组与模型组比较阳性蛋白相对表达量显著下降,具有统计学意义(P<0.01),组间比较无显著差异。
3.4通腑利湿化瘀散结方肝豆灵对铜负荷大鼠代谢组学的影响
3.4.1对血清特征性代谢物的影响
模型组较空白组含有较多的乳酸盐、糖蛋白、脂质、牛磺酸、丙酮酸盐,模型组较空白组含有较少的葡萄糖、胆碱、甜菜碱、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、肌醇,通腑利湿化瘀散结方肝豆灵能明显提高铜负荷大鼠血浆亮氨酸、异亮氨酸、胆碱、葡萄糖、3-羟基丁酸的含量,能降低糖蛋白、脂质、乳酸盐、丙酮酸盐的含量,通腑利湿化瘀散结方肝豆灵对铜负荷大鼠小分子代谢物有明显的调节效应,对wD肝纤维化的代谢异常有修复的作用。
3.4.2对尿液特征性代谢物的影响
模型组较空白组含有较多的乳酸盐、脂质、牛磺酸、三甲胺、肌氨酸、肌酐,含有较少的氧化三甲胺、马尿酸盐,通腑利湿化瘀散结方肝豆灵能明显提高铜负荷大鼠尿液氧化三甲胺、马尿酸盐的含量,降低乳酸盐、脂质、牛磺酸、肌氨酸、肌酐、三甲胺的含量。尿液的特征性代谢物的变化,不仅能够反应wD铜负荷大鼠肝脏本身损害,也可同时提示肾脏及其他脏器损害,与wD多脏器损害的病理特点相一致。
4.结论
4.1本研究采用铜负荷方法建立了铜过量负荷大鼠模型,模型组可见肝纤维化病理改变,该模型可反应铜过量沉积导致肝损伤的病理机理。
4.2本研究发现铜过量负荷大鼠模型可见HSc增生表达,各治疗组凋亡指数较模型组明显升高,提示通腑利湿、化瘀散结方肝豆灵对铜负荷大鼠有诱导HSc凋亡的作用,特别是和青霉胺合用作用更显著。
4.3本研究发现模型组有p38MAPK信号通路蛋白的阳性表达,各治疗组较模型组明显改善,而在空白对照组无相应改变。提示：铜负荷大鼠肝纤维化时p38MAPK被激活,可能参与了wD铜负荷大鼠肝纤维化发生、发展的过程,通腑利湿、化瘀散结方肝豆灵可能通过对p38MAPK信号通路的抑制作用而发挥抗纤维化改善肝功能的作用。
4.4采用基于1H-NMR的代谢组学方法研究通腑利湿化瘀散结方对铜负荷大鼠的血清和尿液的干预作用,得到了乳酸、牛磺酸、胆碱、脂质等特征代谢物,通过对这些特征代谢物的代谢途径的分析发现,铜不仅影响了糖、脂类和蛋白质的代谢,也导致了氨基酸等其它代谢系统的紊乱,通腑利湿化瘀散结方肝豆灵对铜负荷大鼠小分子代谢物是多途径、多靶点的代谢网络调节,对铜负荷大鼠小分子代谢物有明显的调节效应,对wD肝纤维化的代谢异常有修复的作用。
Objectives:Wilson disease is an autosomal recessive disorder of copper homeostasis, the incidence of which is30/1000,000. WD typically occurs in young people. The clinical manifestations are chronic hepatitis, renal and neurological abnormalities. Research shows liver is the first organ involved, and the smaller the age is, the greater possibility of the liver disease is the only clinical manifestation. Hepatic fibrosis is the main liver pathological change almost in every WD patient. As a copper metabolic aplastic disease, the basic cause of hepatic fibrosis is the difficulty of copper to get drained through bile, which causes free copper to accumulate slowly in liver tissue.
Researches prove that HSC is a key part of the happening of liver fibrosis, the activation of proliferation in HSC, phenotype change and excessive secretion in ECM play important roles in hepatic fibrosis.
Among the studies on HSC signal pathway in recent years, MAPK is one of the hot points. P38mitogen-activated protein kinases pathway is an important branch in MAPK mediated signal pathway, which is involved in inflammation, proliferation, apoptosis and many kinds of other physical processes, and has become one of the key pathways in cell apoptosis.
Metabolomics increasingly becomes a very powerful method in studying the functional change of life system. Since WD is a disorder of copper homeostasis,the changes of the liver metabolites spectrum may be different from other liver diseases, and studies of metabonomics in WD for pathogenesis, treatment and prognosis seem particularly important.
At present, there is no specially effective means in the prevention of hepatic fibrosis especially in WD, but traditional Chinese medicine has unique curative effect with its overall adjustment in many targets and personalized treatment in the prevention of hepatic fibrosis. It is proved by my tutor's research group through their clinical practice that catharsis and diuresis, expelling stasis and mass recipe, Gandouling, can improve the index of hepatic fibrosis, and promote the recovery of liver cirrhosis and liver function in WD.
This experiment is to carry out the following research:to establish copper overloaded rats, to observe the effects of catharsis and diuresis, expelling stasis and mass recipe, Gandoutang, on p38-MAPK pathway and the apoptosis of hepatic stellate cell, to use metabonomics to inspect the effect of Gandouling on changes of metabolites of hepatic fibrosis in copper overloaded rats, to discuss the molecular mechanism of catharsis and diuresis, expelling stasis and mass recipe in treating hepatic fibrosis in WD.
Methods:
1.The rats were divided into5groups:control group, model group, Gandouling group, Penieillamine group,Gandoutang and Penieillamine group.
2. Copper overloaded rats were modeled according to documents.
3. Copper overloaded rats in each group were intervened with catharsis and diuresis, expelling stasis and mass recipe, and Penieillamine was used as positive drugs.
4. Pathological changes of hepatic fibrosis were observed.
5. Expression of P38MAPK protein was observed with immunohistoche-mical and western-blot methods, and cell apoptosis of HSC was detected with TUNEL method.
6. Changes of small molecules metabolites from serum and urine of hepatic fibrosis in WD were analyzed with1H-NMR techniques.
Result:
1.The effect of the catharsis and diuresis, expelling stasis and mass recipe on pathology of hepatic fibrosis in copper overloaded rats.
Normal group had complete structure in hepatic lobule. liver cells were in order. There was no cell degeneration inside the lobule, and no infiltration of inflammatory cells. There was obvious fiber hyperplasia in portal area in model group, and the degree of hepatic fibrosis was significantly reduced in treatment groups. There were green collagen fibers under light microscopy in Masson dyeing. There were only a few green collagen fiber distributions in central venous wall in normal group. There were thick green collagen fiber distributing in portal area and central venous wall in model group, which was of significant meaning compared with normal group (P<0.01).The degree of hepatic fibrosis was significantly reduced in treatment groups, which was of significant meaning compared with normal group (P<0.01).There was a significant decrease of pat-hogenic score in the integrated treatment of Gandoutang and Penieillamine group (P<0.01).Pathogenic score decreased obviously in Gandoutang and Penieillamine groups(P<0.05).
2.The effect of the catharsis and diuresis, expelling stasis and mass recipe on HSC in copper overloaded rats
Cell apoptosis detected with TUNEL method,there were visible expressions of hepatic stellate cells in model group, and there were visible expressions of apoptotic cells in treatment groups.The apoptosis index of treatment groups was significantly higher than that of model group(P<0.05). The apoptosis index of Gandoutang and Penieillamine group was significantly higher(P<0.01).
3.The influence of the the catharsis and diuresis, expelling stasis and mass recipe on copper overloaded rats in p38MAPK signal pathway.
3.1The expression of p38MAPK signaling pathway was detected with immunohistochemical method
The positive signal of P38MAPK protein was tan-yellow in color, and there were visible positive expression in model group and treatment group. The data of model group ware significantly enhanced than those of normal group (P<0.001).The positive signal of P38MAPK protein of treatment groups decreased more than that of the model group(P<0.05), and there were no significant differences between the groups.
3.2The expression of p38MAPK signaling pathways was detected with western-Blot method.
There were significant differences in P38MAPK protein levels between the groups.Compared with the normal group, there were visible specific protein strip expressions of p38MAPK in model group and treatment groups, and positive protein expression increased remarkably in model group than in normal group(P<0.001).Positive protein expression decreased more obviously in treatment groups than in model group,and was of statistics significance(P<0.05), and there was no significant difference between the groups.
4.The metabolomic effect of the catharsis and diuresis, expelling stasis and mass recipe on copper overloaded rats
4.1The influence of the characteristic metabolites in serum
Model group contained more lactate,glycoprotein,lipid, taurine and pyruvate than normal group.Model group contained less glucose,choline, betaine,alanine,glycine,leucine, isoleucine and myoinositol than normal group.The catharsis and diuresis,expelling stasis and mass recipe could obviously increase the content of leucine, isoleucine, choline, glucose and3-hydroxybutyrate in serum of copper overloaded rats, and could reduce the content of glycoprotein, lipid, lactate and pyruvate. The catharsis and diuresis, expelling stasis and mass recipe had obvious adjustment effect on small molecules metabolites in copper overloaded rats, and played a repair role in metabolic abnormalities of hepatic fibrosis in copper overloaded rats.
4.2The influence of the characteristic metabolites of urine
Model group contained more lactate, lipid, taurine, TMA, creatine and creatinine than control group. Model group contained less TMAO and hippuratethan than control group. The catharsis and diuresis, expelling stasis and mass recipe could obviously increase the content of TMAO and hippuratethan in urine of copper overloaded rats, and could reduce the content of lactate, lipid, taurine, TMA, creatine and creatinine. The change of the characteristic metabolites in urine can not only reflect the liver damage itself, but also indicate the damage of kidney and other organs in copper overloaded rats, and are in accordance with the pathological characteristics of multiple organs damage of WD.
Conelusion:
1.In the study,the model of copper overloaded rat was esta blished by copper excess load in the rat. There were visible pathological changes of hepatic fibrosis in model group, and this model can reflect the pathogenesis of liver damage caused by sedimentation of excessive copper.
2.This study found visible hyperplasia expression of HSC in copper overloaded rats. The apoptosis index increased signi ficantly in treatment groups than in model group, indicating that the catharsis and diuresis, expelling stasis and mass recipe played the role of inducing apoptosis of HSC, and the effect was better when it was used with Penieillamine.
3.The study found there was positive expression of protein p38MAPK signal pathway in model group. The expression was obviously improved in treatment groups than in model group, but in normal group there were no corresponding change. The above results indicated the p38MAPK of hepatic fibrosis was activated in copper overloaded rats, and was possibly involved in the occurrence and development process of hepatic fibrosis in copper overloaded rats. The catharsis and diuresis, expelling stasis and mass recipe maybe play a role of inhibiting fibrosis in improving liver function through p38MAPK signaling pathway.
4.This study got some characteristics metabolites such as lactate,taurine and choline by exploring the effect of catha rsis and diuresis, expelling stasis and mass recipe on serum and urine in copper overloaded rats with 'H-NMR metabolomicmethod. Through analyzing the metabolic pathway of the chara cteristics metabolites,we found copper not only affected the metabolism of glucose, lipids and proteins,but also caused the disorders of amino acids and other metabolic system. We also found the catharsis and diuresis, expelling stasis and mass recipe adjusted small molecules metabolites of overloaded rats in many ways through many targets and metabolic network, had obvious adjustment effect on small molecules metabolites,and played a repair role in abnormal metabolism of hepatic fibrosis of WD.
肝豆状核变性(Wilson disease, WD)是常染色体隐性遗传的铜代谢障碍疾病,wD好发于青少年,临床上以慢性肝脏损害、肾脏功能病变及神经系统障碍等症状为主要表现。研究表明,肝脏是wD最先累及的脏器之一,发病年龄越早,以肝脏病变为唯一临床表现的可能性越大,肝纤维化(Hepatic Fibrosis)几乎是每个wD患者肝脏损害的主要病理改变。wD作为铜代谢障碍性疾病,发生肝纤维化的根本原因是铜离子向胆道排泄困难,造成游离铜在肝组织内缓慢蓄积。研究证实肝星状细胞(hepatic stellate cells, HSC)的激活是肝纤维化发生的主要环节,激活的HSC大量增殖,发生表型改变、分泌过多的细胞外基质(Extra Cellular matrix, ECM)沉积于肝脏是肝纤维化形成的关键。
在HSC信号通路研究中,丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)是近年研究的热点。p38MAPK信号通路是MAPK介导的信号通路的重要分支,主要参与炎症、增殖、凋亡等多种生理过程,是目前调控细胞凋亡的关键通路之一。
代谢组学日益成为整体性研究生命体系功能变化的非常有力的分析手段。由于wD为铜代谢障碍性疾病,其代谢物谱的变化可能不同于其他肝脏疾病,因此wD的代谢组学研究对于wD的病机、治疗及预后显得尤为重要。
目前肝纤维化的防治特别是wD肝纤维化尚缺乏针对性较强的特效手段,而中医中药以其多靶点的整体调节和以人为本的个性化治疗方案,在肝纤维化的防治中具有独特的疗效。导师所在课题组在前期的临床实践中证明：通腑利湿、化瘀散结的中药专方肝豆灵可以改善wD患者的肝纤维化指标,促进wD者肝硬化及肝功能的恢复。
本实验主要开展以下研究：建立铜负荷大鼠模型,观察通腑利湿、化瘀散结方肝豆灵对肝纤维化HSC凋亡及p38MAPK信号通路蛋白表达的影响,运用代谢组学技术检测肝豆灵对铜负荷大鼠肝纤维化小分子代谢标志物变化的影响,初步探讨通腑利湿、化瘀散结方肝豆灵治疗wD肝纤维化的分子机制。
2.方法
2.1实验分为5组：分别为空白对照组,模型对照组,肝豆灵组,肝豆灵加青霉胺组,青霉胺组；
2.2按照文献复制铜负荷大鼠模型；
2.3使用通腑利湿、化瘀散结方肝豆灵对各治疗组铜负荷大鼠进行干预,选用青霉胺为阳性对照药物；
2.4光镜观察肝纤维化病理改变；
2.5采用免疫组化法、Western-Blot法检测p38MAPK蛋白的表达,TUNEL法检测细胞凋亡；
2.6运用1H-NMR技术检测肝纤维化大鼠血清、尿液的小分子代谢产物的变化。
3.结果
3.1通腑利湿化瘀散结方肝豆灵对铜负荷大鼠肝纤维化病理的影响
空白组肝小叶结构完整,肝细胞排列规则,小叶内无细胞变性,无炎性细胞浸润；铜负荷大鼠模型组HE染色汇管区可见明显纤维组织增生,各治疗组肝纤维化程度明显减轻；Masson染色光镜下为绿色为胶原纤维,正常对照组中央静脉管壁可见少量绿色的胶原纤维,模型组于汇管区和中央静脉管壁可见大量粗大的绿色的胶原纤维,与正常对照组相比有显著性意义(P<0.01),各治疗组肝纤维化程度明显减轻,其中肝豆灵加青霉胺组积分下降最为显著(P<0.01),肝豆灵组和青霉胺组病理积分亦明显下降(P<0.05)。
3.2通腑利湿化瘀散结方肝豆灵对铜负荷大鼠HSC的影响
TUNEL法检测细胞凋亡,空白组未见明显HSC增生,模型组可见大量HSC增生表达,各治疗组可见HSC凋亡细胞表达,各治疗组凋亡指数较模型组明显增高,其中肝豆灵加青霉胺组凋亡指数显著增高(P<0.01),肝豆灵组和青霉胺组凋亡指数亦明显升高(P<0.05)。
3.3通腑利湿化瘀散结方肝豆灵对铜负荷大鼠P38MAPK信号通路的影响
3.3.1免疫组化法检测p38MAPK信号通路表达
p38MAPK蛋白阳性信号呈棕黄色,模型组及各治疗组可见阳性细胞表达,模型组阳性细胞数表达较空白组显著增强(P<0.001),青霉胺加肝豆灵组、肝豆灵组及青霉胺组较模型组p38MAPK阳性细胞数表达显著减少(P<0.05),组间比较无显著差异。
3.3.2Western-Blot法检p38MAPK信号通路表达
p38MAPK蛋白水平在各组之间比较有明显差别,与空白组相比,模型组及各治疗组可见p38MAPK特异性蛋白条带表达,模型组较空白组阳性蛋白相对表达量显著增强(P<0.001),青霉胺加肝豆灵组、肝豆灵组及青霉胺组与模型组比较阳性蛋白相对表达量显著下降,具有统计学意义(P<0.01),组间比较无显著差异。
3.4通腑利湿化瘀散结方肝豆灵对铜负荷大鼠代谢组学的影响
3.4.1对血清特征性代谢物的影响
模型组较空白组含有较多的乳酸盐、糖蛋白、脂质、牛磺酸、丙酮酸盐,模型组较空白组含有较少的葡萄糖、胆碱、甜菜碱、丙氨酸、甘氨酸、亮氨酸、异亮氨酸、肌醇,通腑利湿化瘀散结方肝豆灵能明显提高铜负荷大鼠血浆亮氨酸、异亮氨酸、胆碱、葡萄糖、3-羟基丁酸的含量,能降低糖蛋白、脂质、乳酸盐、丙酮酸盐的含量,通腑利湿化瘀散结方肝豆灵对铜负荷大鼠小分子代谢物有明显的调节效应,对wD肝纤维化的代谢异常有修复的作用。
3.4.2对尿液特征性代谢物的影响
模型组较空白组含有较多的乳酸盐、脂质、牛磺酸、三甲胺、肌氨酸、肌酐,含有较少的氧化三甲胺、马尿酸盐,通腑利湿化瘀散结方肝豆灵能明显提高铜负荷大鼠尿液氧化三甲胺、马尿酸盐的含量,降低乳酸盐、脂质、牛磺酸、肌氨酸、肌酐、三甲胺的含量。尿液的特征性代谢物的变化,不仅能够反应wD铜负荷大鼠肝脏本身损害,也可同时提示肾脏及其他脏器损害,与wD多脏器损害的病理特点相一致。
4.结论
4.1本研究采用铜负荷方法建立了铜过量负荷大鼠模型,模型组可见肝纤维化病理改变,该模型可反应铜过量沉积导致肝损伤的病理机理。
4.2本研究发现铜过量负荷大鼠模型可见HSc增生表达,各治疗组凋亡指数较模型组明显升高,提示通腑利湿、化瘀散结方肝豆灵对铜负荷大鼠有诱导HSc凋亡的作用,特别是和青霉胺合用作用更显著。
4.3本研究发现模型组有p38MAPK信号通路蛋白的阳性表达,各治疗组较模型组明显改善,而在空白对照组无相应改变。提示：铜负荷大鼠肝纤维化时p38MAPK被激活,可能参与了wD铜负荷大鼠肝纤维化发生、发展的过程,通腑利湿、化瘀散结方肝豆灵可能通过对p38MAPK信号通路的抑制作用而发挥抗纤维化改善肝功能的作用。
4.4采用基于1H-NMR的代谢组学方法研究通腑利湿化瘀散结方对铜负荷大鼠的血清和尿液的干预作用,得到了乳酸、牛磺酸、胆碱、脂质等特征代谢物,通过对这些特征代谢物的代谢途径的分析发现,铜不仅影响了糖、脂类和蛋白质的代谢,也导致了氨基酸等其它代谢系统的紊乱,通腑利湿化瘀散结方肝豆灵对铜负荷大鼠小分子代谢物是多途径、多靶点的代谢网络调节,对铜负荷大鼠小分子代谢物有明显的调节效应,对wD肝纤维化的代谢异常有修复的作用。
Objectives:Wilson disease is an autosomal recessive disorder of copper homeostasis, the incidence of which is30/1000,000. WD typically occurs in young people. The clinical manifestations are chronic hepatitis, renal and neurological abnormalities. Research shows liver is the first organ involved, and the smaller the age is, the greater possibility of the liver disease is the only clinical manifestation. Hepatic fibrosis is the main liver pathological change almost in every WD patient. As a copper metabolic aplastic disease, the basic cause of hepatic fibrosis is the difficulty of copper to get drained through bile, which causes free copper to accumulate slowly in liver tissue.
Researches prove that HSC is a key part of the happening of liver fibrosis, the activation of proliferation in HSC, phenotype change and excessive secretion in ECM play important roles in hepatic fibrosis.
Among the studies on HSC signal pathway in recent years, MAPK is one of the hot points. P38mitogen-activated protein kinases pathway is an important branch in MAPK mediated signal pathway, which is involved in inflammation, proliferation, apoptosis and many kinds of other physical processes, and has become one of the key pathways in cell apoptosis.
Metabolomics increasingly becomes a very powerful method in studying the functional change of life system. Since WD is a disorder of copper homeostasis,the changes of the liver metabolites spectrum may be different from other liver diseases, and studies of metabonomics in WD for pathogenesis, treatment and prognosis seem particularly important.
At present, there is no specially effective means in the prevention of hepatic fibrosis especially in WD, but traditional Chinese medicine has unique curative effect with its overall adjustment in many targets and personalized treatment in the prevention of hepatic fibrosis. It is proved by my tutor's research group through their clinical practice that catharsis and diuresis, expelling stasis and mass recipe, Gandouling, can improve the index of hepatic fibrosis, and promote the recovery of liver cirrhosis and liver function in WD.
This experiment is to carry out the following research:to establish copper overloaded rats, to observe the effects of catharsis and diuresis, expelling stasis and mass recipe, Gandoutang, on p38-MAPK pathway and the apoptosis of hepatic stellate cell, to use metabonomics to inspect the effect of Gandouling on changes of metabolites of hepatic fibrosis in copper overloaded rats, to discuss the molecular mechanism of catharsis and diuresis, expelling stasis and mass recipe in treating hepatic fibrosis in WD.
Methods:
1.The rats were divided into5groups:control group, model group, Gandouling group, Penieillamine group,Gandoutang and Penieillamine group.
2. Copper overloaded rats were modeled according to documents.
3. Copper overloaded rats in each group were intervened with catharsis and diuresis, expelling stasis and mass recipe, and Penieillamine was used as positive drugs.
4. Pathological changes of hepatic fibrosis were observed.
5. Expression of P38MAPK protein was observed with immunohistoche-mical and western-blot methods, and cell apoptosis of HSC was detected with TUNEL method.
6. Changes of small molecules metabolites from serum and urine of hepatic fibrosis in WD were analyzed with1H-NMR techniques.
Result:
1.The effect of the catharsis and diuresis, expelling stasis and mass recipe on pathology of hepatic fibrosis in copper overloaded rats.
Normal group had complete structure in hepatic lobule. liver cells were in order. There was no cell degeneration inside the lobule, and no infiltration of inflammatory cells. There was obvious fiber hyperplasia in portal area in model group, and the degree of hepatic fibrosis was significantly reduced in treatment groups. There were green collagen fibers under light microscopy in Masson dyeing. There were only a few green collagen fiber distributions in central venous wall in normal group. There were thick green collagen fiber distributing in portal area and central venous wall in model group, which was of significant meaning compared with normal group (P<0.01).The degree of hepatic fibrosis was significantly reduced in treatment groups, which was of significant meaning compared with normal group (P<0.01).There was a significant decrease of pat-hogenic score in the integrated treatment of Gandoutang and Penieillamine group (P<0.01).Pathogenic score decreased obviously in Gandoutang and Penieillamine groups(P<0.05).
2.The effect of the catharsis and diuresis, expelling stasis and mass recipe on HSC in copper overloaded rats
Cell apoptosis detected with TUNEL method,there were visible expressions of hepatic stellate cells in model group, and there were visible expressions of apoptotic cells in treatment groups.The apoptosis index of treatment groups was significantly higher than that of model group(P<0.05). The apoptosis index of Gandoutang and Penieillamine group was significantly higher(P<0.01).
3.The influence of the the catharsis and diuresis, expelling stasis and mass recipe on copper overloaded rats in p38MAPK signal pathway.
3.1The expression of p38MAPK signaling pathway was detected with immunohistochemical method
The positive signal of P38MAPK protein was tan-yellow in color, and there were visible positive expression in model group and treatment group. The data of model group ware significantly enhanced than those of normal group (P<0.001).The positive signal of P38MAPK protein of treatment groups decreased more than that of the model group(P<0.05), and there were no significant differences between the groups.
3.2The expression of p38MAPK signaling pathways was detected with western-Blot method.
There were significant differences in P38MAPK protein levels between the groups.Compared with the normal group, there were visible specific protein strip expressions of p38MAPK in model group and treatment groups, and positive protein expression increased remarkably in model group than in normal group(P<0.001).Positive protein expression decreased more obviously in treatment groups than in model group,and was of statistics significance(P<0.05), and there was no significant difference between the groups.
4.The metabolomic effect of the catharsis and diuresis, expelling stasis and mass recipe on copper overloaded rats
4.1The influence of the characteristic metabolites in serum
Model group contained more lactate,glycoprotein,lipid, taurine and pyruvate than normal group.Model group contained less glucose,choline, betaine,alanine,glycine,leucine, isoleucine and myoinositol than normal group.The catharsis and diuresis,expelling stasis and mass recipe could obviously increase the content of leucine, isoleucine, choline, glucose and3-hydroxybutyrate in serum of copper overloaded rats, and could reduce the content of glycoprotein, lipid, lactate and pyruvate. The catharsis and diuresis, expelling stasis and mass recipe had obvious adjustment effect on small molecules metabolites in copper overloaded rats, and played a repair role in metabolic abnormalities of hepatic fibrosis in copper overloaded rats.
4.2The influence of the characteristic metabolites of urine
Model group contained more lactate, lipid, taurine, TMA, creatine and creatinine than control group. Model group contained less TMAO and hippuratethan than control group. The catharsis and diuresis, expelling stasis and mass recipe could obviously increase the content of TMAO and hippuratethan in urine of copper overloaded rats, and could reduce the content of lactate, lipid, taurine, TMA, creatine and creatinine. The change of the characteristic metabolites in urine can not only reflect the liver damage itself, but also indicate the damage of kidney and other organs in copper overloaded rats, and are in accordance with the pathological characteristics of multiple organs damage of WD.
Conelusion:
1.In the study,the model of copper overloaded rat was esta blished by copper excess load in the rat. There were visible pathological changes of hepatic fibrosis in model group, and this model can reflect the pathogenesis of liver damage caused by sedimentation of excessive copper.
2.This study found visible hyperplasia expression of HSC in copper overloaded rats. The apoptosis index increased signi ficantly in treatment groups than in model group, indicating that the catharsis and diuresis, expelling stasis and mass recipe played the role of inducing apoptosis of HSC, and the effect was better when it was used with Penieillamine.
3.The study found there was positive expression of protein p38MAPK signal pathway in model group. The expression was obviously improved in treatment groups than in model group, but in normal group there were no corresponding change. The above results indicated the p38MAPK of hepatic fibrosis was activated in copper overloaded rats, and was possibly involved in the occurrence and development process of hepatic fibrosis in copper overloaded rats. The catharsis and diuresis, expelling stasis and mass recipe maybe play a role of inhibiting fibrosis in improving liver function through p38MAPK signaling pathway.
4.This study got some characteristics metabolites such as lactate,taurine and choline by exploring the effect of catha rsis and diuresis, expelling stasis and mass recipe on serum and urine in copper overloaded rats with 'H-NMR metabolomicmethod. Through analyzing the metabolic pathway of the chara cteristics metabolites,we found copper not only affected the metabolism of glucose, lipids and proteins,but also caused the disorders of amino acids and other metabolic system. We also found the catharsis and diuresis, expelling stasis and mass recipe adjusted small molecules metabolites of overloaded rats in many ways through many targets and metabolic network, had obvious adjustment effect on small molecules metabolites,and played a repair role in abnormal metabolism of hepatic fibrosis of WD.
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