摘要
背景
肝纤维化在国际疾病分类(ICD-10)中可作为一种病名(K74.001),但主要是一种组织病理学概念,指肝组织内细胞外基质(extracellular matrix,ECM)成分过度增生与异常沉积,导致肝脏结构或(和)功能异常的病理变化。慢性乙型病毒性肝炎(3亿)和丙型病毒性肝炎(1.7亿)在世界范围内的流行,以及不断增高的非酒精性脂肪性肝炎(nonalcohlic steatohepatitis,NASH)、脂肪肝病(nonalcoholic fatty liver disease,NAFLD)和酒精性脂肪性肝炎(alcoholicsteatohepatitis,ASH)构成慢性炎症性肝病的主要原因,引起肝实质的破坏并被疤痕组织代替,演变成肝纤维化。
我国属HBV感染高流行区,一般人群的HBsAg阳性率为9.09%;前瞻性研究表明,慢性乙型肝炎发展为肝硬化的估计年发生率为2.1%;HBeAg阴性慢性乙型肝炎患者进展为肝硬化的发生率为23%。肝炎病毒、酒精、药物与毒物、血吸虫、代谢和遗传、胆汁淤积、自身免疫性肝病等多种损伤因素长期慢性刺激肝脏,使肝窦内肝星状细胞的活化,胶原等ECM成分代谢失衡,生成大于降解,促使肝脏ECM沉积与组织结构重构,导致肝纤维化,是肝硬化形成的关键步骤。研究表明肝纤维化在去除损伤因素后尚有逆转的可能,因此抗肝纤维化成为防治慢性肝病的希望所在。
肝纤维化是一主动进展与动态变化的复杂病理过程,涉及多个环节与因素,治疗策略上应顾及肝纤维化发生和发展的各个方面。目前,尚无治疗HF的特效药,因此,充分利用中西医结合的优势,研发疗效可靠、作用靶点清楚、作用机理明确的中药是当务之急。
大黄蟅虫丸出自《金匮要略》,功能活血破瘀、通经消痞,是中国中西医结合学会肝病专业委员会制定的“肝纤维化中西医结合诊疗指南”推荐的防治肝纤维化的中成药。研究表明,大黄蟅虫丸对实验性肝纤维化有效并显著改善血清纤维化指标,但大黄蟅虫丸防治HF的作用机理我们知之甚少,大大限制了其临床应用及其国际化推广的可能性,因此,深入阐述其分子作用机理,明确其作用靶点,具有重要意义。
目的
观察大黄蟅虫丸对肝纤维化大鼠模型肝纤维化程度的影响;分析大黄蟅虫丸对肝纤维化血清指标的影响;检测大黄蟅虫丸对对肝纤维化大鼠细胞因子GM-CSF、IL-13、IL-18、IFN-γ和瘦素表达的影响;研究大黄蟅虫丸对丝裂原活化蛋白激酶通路和核因子-κB通路的影响,探讨丝裂原活化蛋白激酶及NF-κB调节细胞因子表达的可能机制。
方法
对肝组织进行病理学检查,HE染色观察其肝纤维化程度;应用放免法检测肝纤维化四项指标,透明质酸酶(Hyaluronidase,HA),层粘连蛋白(Laminin,LN),Ⅳ型胶原(Collagen TypeⅣ,Ⅳ—C),Ⅲ型前胶原(ProcollagenⅢ,PCⅢ);应用蛋白质液相芯片技术检测细胞因子GM-CSF、IFN-γ、IL-13、IL-18和瘦素水平;应用蛋白免疫印记分析上述药物对丝裂原激活的蛋白激酶的表达及磷酸化水平的影响;应用凝胶迁移率实验研究上述药物对NF-κB活化入核的影响,综合分析大黄蟅虫丸防治肝纤维化的作用通路及调控机理。
对实验数据采用SPSS 13.0进行统计分析,首先对数据进行方差齐性检验,方差齐则应用anova法进行检验,P＜0.05定义为有显著性差异,并用LSD法进行组间两两比较;如果数据方差不齐,则应用welch法进行检验,P＜0.05定义为有显著性差异,应用Games-Howell法进行两两比较。计数资料采用多个独立样本的非参数检验进行分析,P＜0.05为有显著性差异。
结果
应用光镜观察肝组织标本HE染色结果,发现正常组肝小叶结构清晰,细胞索排列整齐,肝索沿小叶中央静脉呈放射状排列,肝窦正常,细胞核圆而大,位于细胞中央,胞质丰富。汇管区无炎细胞浸润,无胶原纤维增生。模型组肝小叶界限不清,肝窦消失。正常肝小叶结构遭到到破坏,可见汇管区和小叶间有大量粗大增生的胶原纤维,组织切片中有大量假小叶形成,并且以小圆形为主。复方鳖甲软肝片组和秋水仙碱组大鼠肝细胞变性也以脂肪变性为主,汇管区扩张,有纤维隔,但多数尚未连接。大黄蟅虫丸组病理变化较模型组显著减轻,肝小叶结构基本清晰,细胞索、肝窦无明显异常。
应用放免法检测肝纤维化四项指标,与空白对照组相比,各模型组HA水平显著升高;与模型组相比,大黄蟅虫丸大、小剂量组、秋水仙碱均能降低肝纤维化大鼠的HA水平;大黄蟅虫丸大、小剂量组降低HA的作用优于秋水仙碱;但与复方鳖甲软肝片相比,无显著差异。大黄蟅虫大小剂量组均能降低肝纤维化大鼠的LN水平;与正常对照组相比,大黄蟅虫丸大、小剂量组、复方鳖甲软肝片组、秋水仙碱的LN水平高于正常组;与秋水仙碱组相比,大黄蟅虫丸降低LN水平;与复方鳖甲软肝片相比,无显著差异。与正常对照组相比,注射四氯化碳的肝纤维化大鼠Ⅳ-C显著升高;与模型组相比,大黄蟅虫大、小剂量组和复方鳖甲软肝片均能降低肝纤维化大鼠的Ⅳ-C水平;大黄蟅虫丸大、小剂量组降低Ⅳ-C作用优于秋水仙碱;与复方鳖甲软肝片相比,无显著差异。检测PCⅢ水平,大黄蟅虫大、小剂量组、复方鳖甲软肝片、秋水仙碱和模型组之间无显著差异。
应用液相蛋白质芯片检测GM-CSF、IFN-γ、IL-13、IL-18和瘦素水平,并进行统计学处理。不同处理组GM-CSF的水平不同,与正常对照组相比,腹腔注射四氯化碳制作肝纤维化模型提高大鼠GM-CSF水平,四个处理组虽能降低GM-CSF水平,但与正常对照组进行两两比较,存在差异;与模型组相比,大黄蟅虫大、小剂量组和复方鳖甲软肝片均能降低肝纤维化大鼠的GM-CSF水平;与复方鳖甲软肝片组相比,大黄蟅虫丸大、小剂量组在降低GM-CSF方面无显著差异。
不同处理组IFN-γ的水平不同,与正常对照组相比,四氯化碳降低大鼠IFN-γ水平,四个处理组提高了IFN-γ水平,但与正常对照组相比,存在差异;与模型组相比,大黄蟅虫大、小剂量组、复方鳖甲软肝片和秋水仙碱均能提高肝纤维化大鼠的IFN-γ水平;与秋水仙碱组和大黄蟅虫丸组相比,大黄蟅虫丸大、小剂量组在升高IFN-γ方面无显著差异。
对IL-13数据进行分析,提示不同处理组IL-13的水平不同,与正常对照组相比,肝纤维化模型大鼠IL-13水平升高,四个处理组均能降低IL-13水平,与正常对照组相比,存在差异;与模型组相比,大黄蟅虫大、小剂量组和复方鳖甲软肝片均能降低肝纤维化大鼠的IL-13水平;与复方鳖甲软肝片和秋水仙碱组相比,大黄蟅虫丸在降低IL-13水平方面,效果优于复方鳖甲软肝片和秋水仙碱。
不同处理组IL-18的水平不同,与正常对照组相比,模型制作的四氯化碳升高大鼠IL-18水平,四个处理组均能降低IL-13水平,与正常对照组相比,存在差异;与模型组相比,大黄蟅虫大、小剂量组均能降低肝纤维化大鼠的IL-18水平,而复方鳖甲软肝片和秋水仙碱作用效果不显著;与复方鳖甲软肝片和秋水仙碱组相比,大黄蟅虫丸在降低IL-18水平方面,效果优于复方鳖甲软肝片和秋水仙碱。
不同处理组瘦素的水平不同,与模型组相比,大黄蟅虫大、小剂量组和复方鳖甲软肝片均能降低肝纤维化大鼠的IL-18水平,而秋水仙碱作用效果不显著;与正常对照组相比,模型制作的四氯化碳升高大鼠瘦素水平,大黄蟅虫大、小剂量组和秋水仙碱组均能降低瘦素水平,与正常对照组相比存在差异。与秋水仙碱组相比,大黄蟅虫丸大剂量组在降低瘦素水平方面优于秋水仙碱,但大黄蟅虫丸大、小剂量组与复方鳖甲软肝片相比,在降低瘦素水平方面,无明显差异。
研究结果表明,模型组GM-CSF显著提高,而大黄蟅虫丸显著降低其表达,与前述的研究结果一致,表明大黄蟅虫丸能够通过抑制炎症形成和抑制成纤维细胞增殖干预肝纤维化;大黄蟅虫丸显著增高IFN-γ水平,抑制HSC活化,抑制成纤维细胞增殖,抑制胶原沉积;大黄蟅虫丸降低IL-18和IL-13水平,可能通过抑制枯否细胞激活、降低四氯化碳引起的炎症性损害;大黄蟅虫丸和复方鳖甲软肝片均能降低瘦素水平防治肝纤维化。
应用蛋白质免疫印记的方法(western blot)分析了大黄蟅虫丸对丝裂原活化的蛋白激酶家族成员p38、ERK和JNK及其磷酸化蛋白P-p38、P-ERK和P-JNK的表达。p38和磷酸化p38蛋白表达结果表明,不同处理组间蛋白表达水平差异有显著性意义。与模型组相比,大黄蟪虫丸大剂量、小剂量、复方鳖甲软肝片及秋水仙碱降低p38表达;模型组磷酸化p38表达增高,与模型组相比,大黄蟅虫丸大、小剂量、复方鳖甲软肝片及秋水仙碱降低p38表达。经单向方差检验,不同处理组间磷酸化ERK蛋白表达水平差异无显著性意义。JNK和磷酸化JNK蛋白表达结果经单向方差分析,表明不同处理组间蛋白表达水平差异有显著性意义。与模型组相比,大黄蟅虫丸大剂量、小剂量、复方鳖甲软肝片及秋水仙碱降低JNK表达;模型组磷酸化JNK表达增高,与模型组相比,大黄蟅虫丸大剂量、小剂量、复方鳖甲软肝片及秋水仙碱降低JNK表达,差异有显著性意义。
应用凝胶迁移率实验研究了NF-κB活化后进入细胞核的情况,研究发现模型组NF-κB活化入核显著增强。与模型组相比,大黄蟅虫丸大剂量组、小剂量组、复方鳖甲软肝片和秋水仙碱均能抑制NF-κB活化入核,差异有显著性意义。与正常对照组相比,模型制作的四氯化碳升高大鼠NF-κB活化水平,大黄蟅虫大、小剂量组和秋水仙碱组能降低NF-κB水平,与正常对照组相比存在差异。与复方鳖甲软肝片和秋水仙碱组相比,大黄蟅虫丸大、小剂量组在抑制NF-κB活化水平方面,无明显差异。
结论
1.大黄蟅虫丸能够减少炎细胞浸润、胶原纤维增生和肝细胞脂肪变性,改善肝小叶结构从而减轻肝纤维化的发生;
2.大黄蟅虫丸能降低肝纤维化大鼠的HA、LN、Ⅳ-C和PCⅢ水平,其中尤以HA和Ⅳ-C明显,提示即使剔除炎症因素的影响,大黄蟅虫丸仍表现出良好的降低血清肝纤维化指标的治疗效果;
3.大黄蟅虫丸能降低GM-CSF、IL-13、IL-18和瘦素表达,增加IFN-γ表达,提示可能是通过抑制炎症形成、抑制成纤维细胞增殖并抑制胶原沉积而干预肝纤维化;
4.大黄蟅虫丸能降低磷酸化p38和磷酸化JNK的表达,一方面能直接降低胶原基因的转录,降低其合成水平,同时能增加HSC凋亡;另一方面p38和JNK磷酸化水平的降低,可能通过降低NF-κB活化入核,调节炎症因子表达,从而达到防治肝纤维化的作用;
5.大黄蟅虫丸降低NF-κB活化入核,一方面可能通过降低GM-CSF、IL-13、IL-18转录,增加IFN-γ转录,减轻肝内炎症因子的表达,降低四氯化碳引起的炎症变化;另一方面,NF-κB活化入核减少,减少JNK磷酸化,增加HSC凋亡,从而达到减少成纤维细胞的增殖并减少胶原合成的作用,减少肝纤维化的发生和发展。
Background
Hepatic fibrosis (HF) is structural and functional pathologic changes of liver, which is due to excessive proliferation and abnormal deposition of extracellular matrix in hepatic tissue. Hepatic fibrosis is considered as a name of disease in international classification of Disease (ICD), but mainly as a concept of histopathology. Chronic hepatitis B (300 million), chronic hepatitis C (170 million), nonalcohlic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD) and alcoholic steatohepatitis (ASH ) are the main causes of chronic inflammation disease of liver, which leads to destroy and substitution of liver parenchyma to develop into HF.
HBV infections are widespread in China, and the positive rate of HBsAg in common population is 9.09%. According to perspective study, the occurrence rate of HF from chronic HBV is 2.1% in a year, and it is 23% from chronic HBV in which HBeAg is negative. Liver is stimulated long term by hepatitis virus, alcohol, drug, poison, blood fluke, metabolism and heredity, cholestasis, autoimmune liver disease and so on , which activate stellate cell of hepatic sinusoid, make ECM disequilibrium of metabolism and deposition and reconstruction, and lead to HF, which is committed step in the development of HF. According to Safadi, HF is still possible to recover when destroy are removed, so inhibiting HF is important therapeutic measure of chronic liver disease.
HF is a complex pathological process, and is related to many components and facors, so many aspects in the occurrence and development of HF should be valued in therapy. At present, there is still no specific medicine for HF, so it is urgent to develop effective traditional Chinese medicine by taking advantage of integrated Traditional and western medicine.
Dahuang zhechong pills from Golden Chamber, which can promot blood circulation , dredge channels and relieve oppression, is a Chinese patent medicine for preventing and curing HF recommended by Chinese association of integrative medicine. According to studies, Dahuang zhechong pills is effective for breadboard HF and can improve the index of fibrosis in serum, but we knew few of its mechanism of preventing and curing HF, which inhibits its application in clinic and international generalization, so it is significant to explain deeply its mechanism and target of molecular.
Objectives
To study the effects of Dahuang zhechong pillss on the occurrence and progress of HF in carbon tetrachloride (CCl_4) induced HF model rats. To study the effects of Dahuang zhechong pillss on the changes of HF related serum markers, such as Hyaluronidase (HA), Laminin (LN), Collagen TypeⅣ(Ⅳ-C), ProcollagenⅢ(PCⅢ) in CCl_4 treated rats. To detect the effects of Dahuang zhechong pills on the serum cytokine expression profiling including GM-CSF, TFN-γ, IL-13, IL-18 and leptin. To study the activation of NF-κB in HF liver tissue and the expression and phosphorylation of MAPK, including p38, ERK and JNK, and analyze the regulation mechanism among cytokine, MAPK and NF-κB.
Methods
Rats were divided into six groups and HF models were made by peritoneal injection of carbon tetrachloride (CCL_4). Dahuang zhechong pillss, Fu fang bie jia ruan gan pian and Colchicine were dissolved in puried water and rats were given Stomach Lavaging with the above drugs. We observed samples of liver tissue sections that stained with HE mehod by light microscope. We then detected the serum fibrosis markers with radio-immuno assay. We studied the changes of serum cytokine such as GM-CSF, IFN-γ, IL-13, IL-18 and leptin with liquichip. The expression and phosphorylation of MAPK, including p38, ERK and JNK, were assayed with western blot and the activation of NF-κB detected by electrophoretic mobility shift assay (EMSA). The data were analyzed with SPSS 13.0: first, data were tested via homogeneity test for variance first, if variance is homogenous, data were analyzed by one-way ANOVA, and groups were compared by LSD; if not, data would be analyzed by Welch method, the defference among groups were anzlyzed by Games-Howell. Numeration data were analyzed by non-parametric test. Difference is significant when P<0.05.
Results
Liver tissue sections that stained with HE mehod were observedby light microscope. In control group, the sections were found that liver lobules were clear in structure, cell chords arrangements were in order, hepatic cords arranged in radiation, hepatic sinusoids were normal, cellular nucleus were big and ground and in centre, cytoplasm was affluent, and there was no infiltration and proliferation in portal area. In model group, hepatic lobules were not clear he structure and destroyed in structure, hepatic sinusoids were disappeared, a lot of collagen fibers were arranged between portal area and hepatic lobules, and there were many pseudolobules. In Fufang biejia ruanganpian and colchicine groups, the major changes were fatty degeneration of liver cell, and portal areas were expanded, but not connected yet. In Dahuang zhechong pills group, fibrosis changes were milder than that of model group, hepatic cords, hepatic sinusoid were not appeareed apparent abnormality. Compared to model group, Dahuang zhechong pills high and low high dosage, and colchicine can decrease HA, but compared with blank group, there were still differences, and the role of Dahuang zhechong pills high and low dosage were superior to colchicine in decreasing HA, while compared with Fufang biejia ruanganpian, there were no differences.
Both Dahuang zhechong pills both high dosage and low dosage can decrease LN in rats of HF, and compared with the normal group, the level of LN in Dahuang zhechong pills high and low dosage, Fufang biejia ruanganpian and Colchicine groups were higher. Compared with colchicine group, Dahuang zhechong pills was more effective in decreasing the level of LN, while compared with Fufang biejia ruanganpian, there were no differences.
Compared with the model group, Dahuang zhechong pills high and low dosage, Fufang biejia ruanganpian all decreased IV-C in rats of HF, but compared with the normal group, there were differences. Dahuang zhechong pills both high and low dosage were superior to colchicine in decreasing IV-C, there were no differences where compared with Fufang biejia ruanganpian.
The expression of GM-CSF, IFN-γ, IL-13, IL-18 and leptin were measured by liquichip. The level of GM-CSF of group was different. Compared with the model group, Dahuang zhechong pills high and low dosage, Fufang biejia ruanganpian all decreased GM-CSF in rat of HF. Compared with the normal group, CCl_4 rised the level of GM-CSF, while the four treatment groups can degrade GM-CSF, and there were still differences. Compared with Colchicine group and Dahuang zhechong pills groups, there was no difference in depressing GM-CSF both Dahuang zhechong pills high and low dosage.
The levels of IFN-γof among groups were different. Compared with the model group, Dahuang zhechong pills high dosage and low dosage, Fufang biejia ruanganpian and Colchicine all lowered IFN-γin rats of HF. Compared to the normal group, CC14 can decrease the level of IFN-γ, while the four treatment groups raised the level of GM-CSF with significant difference. Compared to Dahuang zhechong pills and Colchicine groups, there was no difference in rising of IFN-γin both Dahuang zhechong pills high dosage and low dosage.
IL-13 levels were different among experimental groups. Compared with the model group, Dahuang zhechong pills high and low dosage, Fufang biejia ruanganpian all decreased IL-13 in rats of HF. Compared with the normal group, CCl_4 can raise the level of IL-13, while the four treatment groups decreased the level of IL-13, and there were still differences. Compared with Fufang biejia ruanganpian and Colchicine, Dahuang zhechong pills both high and low dosage were more superior in decreasing IL-13.
The level of IL-18 was different accord to statistical analysis. Compared with the model group, Dahuang zhechong pills high and low dosage decreased IL-18 in rat of HF, while there was no significant effect in Fufang biejia ruanganpian and Colchicine groups. Compared with the normal group, CCl_4 could raise the level of IL-18, while the four treatment groups decreased the level of IL-13, and there were still differences. Compared with Fufang biejia ruanganpian and colchicine groups, Dahuang zhechong pills was superior to them in decreasing IL-18.
The different groups have different levels of leptin. Compared with the model group, Dahuang zhechong pills high and low dosage and Fufang biejia ruanganpian could reduce the levels of IL-18 in rat of HF while the effect of colchicine was not significant. Compared with the normal group, CCl_4 can rise the level of leptin, while Dahuang zhechong pills high and low dosage and colchicine could reduce the levels of leptin, and there were differences. Compared with colchicine, Dahuang zhechong pills high dosage group was more superior in reducing leptin, but compared Dahuang zhechong pills high dose and low dose with Fufang biejia ruanganpian groups, there was no significant difference in degrading level of leptin.
Expression of p38 and phospho-p38 had significant difference in different groups by one-way ANOVA analysis. Compared with the model group, Dahuang zhechong pills high dosage and low dosage, Fufang biejia ruanganpian and colchicine decreased the expression of p38. Expression of phospho-p38 increased in model groupwhile Dahuang zhechong pills high dosage and low dosage, Fufang biejia ruanganpian and colchicine lowered the phosphorylation of p38 when compared with the model group.
Expression of ERK and phosphorylation level of ERK had no difference in different groups by one-way ANOVA. Expression of JNK and phospho-JNK had significant difference in different groups by one-way ANOVA anslysis. Compared with model group, Dahuang zhechong pills high dose and low dose, Fufang biejia ruanganpian and colchicine reduced expression of JNK. Expression of phospho-JNK increased in model group; Dahuang zhechong pills high dose and low dose, Fufang biejia ruanganpian and colchicine reduced phosphorylation level of JNK when compared with model group.
We observed the activation of NF-κB by EMSA, and found that the activation of NF-κB was obviously enhanced in the model group. Compared with the model group, Dahuang zhechong pills high and low dosages, Fufang biejia ruanganpian and colchicine could inhibit NF-κB from being activated into the nuclear, and differences were significant. Compared with the normal group, CCl_4 could promote the activation of NF-κB in rats, while Dahuang zhechong pills high and low dosages and colchicine could inhibit the activation of NF-κB, and there were differences. Compared with Fufang biejia ruanganpian and colchicine groups, Dahuang zhechong pillss two groups had no significant difference in inhibiting the activation of NF-κB.
Conclusions
1. Dahuang zhechong pills could dedrease the infiltration of inflammatory cells, the proliferation of collagen fibres and the fatty degeneration of liver cells to improve the structure of liver lobules and alleviate progression of HF.
2. Dahuang zhechong pills could lowered the level of hyaluronidase (HA), laminin (LN) and collagen type IV (IV-C), especially HA and IV-C, which hint us that Dahuang zhechong pills could lowered the serum hepatic fibosis markers even if eliminate its anti-inflammation effects.
3. We established the methods to measure cytokine level by liquichip and analyzed the effects of Dahuang zhechong pillss on the expression of cytokine profile. Dahuang zhechong pillss could lower the level of GM-CSF, IL-13, IL-18 and leptin and increased the level of IFN-γ.
4. Dahuang zhechong pills could partially inhibit the activation of NF-κB, which may decrease the transcription of GM-CSF, IL-13, IL-18 and leptin and increased the level of IFN-γ.
5. Dahuang zhechong pillss could partially inhibit the phosphorylation of p38 and JNK that may decrease the activation of NF-κB, which accordingly may inhibit the expression of JNK to activate the apoptosis of HSC and decrease the synthesis of collagen to prevent HF.
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