摘要
一、目的
铁沉积与肝纤维化的关系密切,其在肝纤维化的发生、持续及进展中发挥着重要作用。本研究以肝纤维化发生、发展的中心环节一肝星状细胞(HSC)活化为切入点,通过建立铁沉积HSC细胞模型,研究铁沉积于HSC细胞对HSC细胞α-SMA蛋白表达和Ⅰ型胶原、TGF-β_1 mRNA的表达以及对HSC细胞凋亡的影响,开展铁沉积于HSC细胞对肝纤维化的影响及复方肝毒清干预作用的研究。用二甲基亚硝胺(DMN)建立大鼠肝纤维化模型,以大鼠血清铁蛋白、转铁蛋白及肝组织肝铁浓度作为大鼠铁负载的评价指标,并通过普鲁士蓝和α-SMA双染色,对HSC细胞铁沉积进行定位,明确HSC细胞铁沉积与肝纤维化之间的联系;进一步通过研究HSC细胞活化相关因子TGFβ_1 mRNA的表达,初步明确铁沉积于HSC细胞在大鼠肝纤维化中的作用。从组织病理学、细胞学及分子水平开展铁沉积于HSC细胞与肝纤维化的相关性研究,试图阐明铁沉积于HSC细胞促进肝纤维化的发病机理。体内外分别以中药汤剂复方肝毒清对铁沉积HSC细胞模型和DMN诱导的肝纤维化大鼠模型进行干预,试图阐明以扶正利湿解毒为治疗原则的复方肝毒清对铁沉积于HSC细胞促进肝纤维化的影响。
二、方法
(一)细胞实验:铁沉积于HSC细胞对肝纤维化的影响及复方肝毒清的干预作用
1.建立铁沉积HSC细胞模型
以HSC-T6细胞为模式细胞系,培养于6孔板,每孔中均置入1.8cm之正方形盖玻片(无菌处理),将细胞分为铁沉积模型组、铁沉积模型+去铁铵组、正常对照组共3组,每组重复2孔。根据细胞毒性实验,选择25μM的柠檬酸铁胺(FAC)作为实验浓度。待细胞贴壁长满后铁沉积模型组中加入FAC;铁沉积模型+去铁铵组中分别按照摩尔数1:1加入FAC和去铁铵;正常对照组为正常培养的HSC-T6细胞。继续培养24 h后行普鲁士蓝染色。
2.铁沉积于HSC细胞对其活化和转归的影响及去铁铵的干预作用
HSC-T6细胞分为正常对照组、铁沉积模型组、50μM去铁铵组、25μM去铁铵组。正常对照组为正常培养的HSC-T6细胞,铁沉积模型组为经25μM FAC处理并证实铁沉积的HSC-T6细胞,50μM去铁铵组、25μM去铁铵组为分别经50μM和25μM去铁铵处理的HSC-T6细胞。继续培养24 h后,免疫组化检测α-SMA的表达;定量PCR法检测细胞Ⅰ型胶原、TGF-β_1 mRNA的表达;TUNEL法检测HSC-T6细胞的凋亡;电镜观察HSC-T6细胞超微结构。
3.复方肝毒清对铁沉积诱导HSC细胞活化和转归的干预作用
HSC-T6细胞分为正常对照组、铁沉积模型组、铁沉积模型+中药组。正常对照组为正常培养的HSC-T6细胞,铁沉积模型组为经25μM FAC处理并证实铁沉积的HSC-T6细胞,铁沉积模型+中药组为复方肝毒清(按细胞毒性实验确定的最大无毒浓度)处理的铁沉积模型组HSC-T6细胞。温育24小时后,免疫组化检测α-SMA的表达;定量PCR法检测HSC-T6细胞TGF-β_1 mRNA的表达;TUNEL法检测HSC-T6细胞的凋亡;电镜观察HSC-T6细胞超微结构。
(二)动物实验:铁沉积于HSC细胞在DMN诱导大鼠肝纤维化中的作用及复方肝毒清的干预机理
75只SD大鼠随机分为空白对照组(12只)、模型组(15只)、模型+去铁铵组(12只)、模型+秋水仙碱组(12只)、模型+中药复方肝毒清大剂量组(12只)、模型+中药复方肝毒清小剂量组(12只)共6组。铁沉积大鼠肝纤维化模型造模方法为:用生理盐水配制1:100(V/V)的二甲基亚硝胺(DMN)稀释溶液,每周前3天对大鼠进行腹腔注射,剂量为10μL DMN/kg体重,共4周,空白对照组腹腔注射生理盐水。从造模第2周开始,模型+秋水仙碱组按照秋水仙碱0.1mg/kg体重灌胃,1次/日×3周;模型+中药复方大、小剂量组分别以20g/kg、5g/kg体重灌胃,1次/日×3周。模型组及空白对照组均灌胃生理盐水10ml/kg,1次/日×3周。从造模第3周开始,模型+去铁铵组以去铁铵100mg/kg腹腔注射,3次/周×2周。从开始造模到试验结束观察记录动物死亡情况。实验结束时摘眼球取血收集血清,并取左前叶肝脏,部分置于Bouin's固定液中固定,另取50mg左右肝组织洗净后置于Trizol中并冻存于—70℃待检,剩余部分—70℃冷冻保存。取肝组织左叶分别行HE染色、Masson染色、普鲁士蓝染色、普鲁士蓝和α-SMA双染色作组织病理学观察;电镜观察肝组织超微结构;ELISA法检测大鼠血清铁蛋白、转铁蛋白;采用火焰原子吸收光谱法测定大鼠肝组织肝铁浓度;采用AU400型奥林巴斯全自动生化分析仪测定肝功能、血清铁和血脂;定量PCR法检测TGF-β_1 mRNA的表达。
三、结果
(一)细胞实验:铁沉积于HSC细胞对肝纤维化的影响及复方肝毒清的干预作用
1.建立铁沉积HSC细胞模型
经普鲁士蓝染色后,镜下HSC细胞核呈红色,铁沉积模型组细胞可见蓝色铁颗粒沉积于细胞质中,铁沉积模型+去铁铵组细胞质中亦见不同程度蓝色铁颗粒沉积,但较铁沉积模型组明显减少。正常对照组细胞未见铁颗粒沉积。
2.铁沉积于HSC细胞对其活化和转归的影响及去铁铵的干预作用
免疫组化结果显示,镜下可见正常对照组HSC细胞明显活化,α-SMA大量表达。铁沉积模型组HSC细胞α-SMA亦大量表达。而去铁铵组HSC细胞α-SMA表达较正常对照组和铁沉积模型组明显减少,说明细胞活化受到不同程度抑制。
HSC细胞Ⅰ型胶原、TGF-β_1 mRNA表达结果显示,与对照组比较,铁沉积模型组Ⅰ型胶原mRNA表达显著增强(P＜0.01),而TGF-β_1 mRNA表达无显著差异(P＞0.05)。50μM去铁铵和25μM去铁铵均能下调Ⅰ型胶原、TGF-β_1 mRNA的表达(P＜0.01),且50μM去铁铵组优于25μM去铁铵组(P＜0.01)。提示去铁治疗能在转录水平抑制Ⅰ型胶原和TGF-β_1的分泌。
HSC细胞凋亡的病理结果显示,正常对照组、铁沉积模型组HSC细胞未见或仅见有较少细胞发生凋亡。而去铁铵组HSC细胞则见明显细胞凋亡,凋亡细胞的细胞核呈棕褐色,并可见凋亡小体。
电镜结果显示,正常对照组HSC细胞内细胞器结构如线粒体、粗面内质网形态正常。铁沉积模型组出现大批双核细胞,细胞质中线粒体、内质网等细胞器明显减少。去铁铵组HSC细胞发生凋亡,其特点是凋亡细胞形态变小,核染色体堆积,线粒体、内质网等细胞器明显减少,代之以大量空泡。
3.复方肝毒清对铁沉积诱导HSC细胞活化和转归的干预作用
免疫组化结果显示,铁沉积模型+中药组HSC细胞α-SMA表达较正常对照组和铁沉积模型组明显减少,说明细胞活化受到不同程度抑制。
HSC细胞TGF-β_1 mRNA表达结果显示,铁沉积模型+中药组TGF-β_1 mRNA的表达不同程度下调(P＜0.05),提示中药复方能在转录水平抑制TGF-β_1的分泌。
HSC细胞凋亡的病理结果显示,铁沉积模型+中药组HSC细胞可见明显细胞凋亡,凋亡细胞的细胞核呈棕褐色,并可见凋亡小体。
电镜结果显示,铁沉积模型+中药组HSC细胞发生凋亡,其特点是凋亡细胞形态变小,核染色体堆积,线粒体、内质网等细胞器明显减少,代之以大量空泡。
(二)动物实验:铁沉积于HSC细胞在DMN诱导大鼠肝纤维化中的作用及复方肝毒清的干预机理
1.DMN诱导的肝纤维化大鼠血清铁蛋白、转铁蛋白的变化:对大鼠血清铁蛋白、转铁蛋白的检测发现,与对照组比较,模型组血清铁蛋白明显增加(P＜0.05),模型+去铁铵组血清铁蛋白有所减少,但无统计学意义(P＞0.05);而与模型组比较,模型+去铁铵组血清铁蛋白明显减少,有统计学意义(P＜0.01)。就转铁蛋白而言,与对照组比较,模型组血清转铁蛋白明显减少(P＜0.01),模型+去铁铵组血清铁蛋白虽有所减少,但无统计学意义(P＞0.05):与模型组比较,模型+去铁铵组血清转铁蛋白明显增加,有统计学意义(P=0.01)。结果表明,在铁沉积大鼠模型中,铁含量增加伴随着血清转铁蛋白水平减少。
2.DMN诱导的肝纤维化大鼠肝组织肝铁浓度的变化:对大鼠肝组织肝铁浓度的检测发现,与对照组比较,模型组和模型+去铁铵组肝铁浓度明显增加(P＜0.01或P＜0.05),而与模型组比较,模型+去铁铵组肝铁浓度明显减少(P＜0.05)。
3.DMN诱导的肝纤维化大鼠中铁沉积于HSC细胞的组织病理学观察:普鲁士蓝染色结果显示,在模型组,深蓝色铁颗粒主要沿小叶间隔分布,且大部分铁沉积在细胞外;模型+去铁铵组铁颗粒分布较模型组有所减少;正常组肝细胞排列整齐,无铁负载。
普鲁士蓝和α-SMA双染色结果显示,与深蓝色铁颗粒分布一致,在坏死区周围,可见大量α-SMA表达阳性的HSC细胞,并且铁颗粒沉积于HSC细胞中。
4.去铁治疗和中药复方对DMN诱导的肝纤维化大鼠肝功能、血清铁和血脂的影响:大鼠肝功能、血清铁和血脂的检测结果发现,模型组ALT明显升高(P＜0.01),同时ALB、G水平显著降低(P＜0.01)。去铁铵、秋水仙碱、中药大小剂量均能降低ALT水平,其中,去铁铵、中药大小剂量组优于秋水仙碱组(P＜0.01或P＜0.05)。就降低AST水平而言,去铁铵和中药小剂量组无显著差异(P＞0.05),但均优于秋水仙碱组(P＜0.01或P＜0.05)。对于提升ALB水平,中药大剂量组优于去铁铵、秋水仙碱和中药小剂量组(P＜0.01或P＜0.05)。对于G而言,去铁铵、秋水仙碱、中药小剂量均能降低G水平(P＜0.01或P＜0.05)。另外,去铁铵能显著降低血清铁和TG水平(P＜0.01或P＜0.05),而秋水仙碱和中药大小剂组均不能降低血清铁、TG和CHOL(P＞0.05)。
5.去铁治疗和中药复方对DMN诱导的肝纤维化大鼠肝组织病理学的影响:HE染色结果显示,正常对照组肝脏肝板以中央静脉为中心呈条索状,向四周放射样排列,板间有不规则肝窦,分布规律,无胶原纤维存在。模型组肝细胞变性坏死,肝小叶结构破坏,纤维结缔组织增生,假小叶形成。在纤维化组织周围可见出血性坏死,并可见棕褐色颗粒样物质(铁)沉积在肝组织中。模型+去铁铵组、模型+秋水仙碱组、模型+中药大剂量组、模型+中药小剂量组肝细胞变性坏死有所减轻,纤维结缔组织、假小叶形成均不同程度减少。
Masson染色结果显示,正常组仅在汇管区和中央静脉壁见少量胶原纤维。模型组大鼠肝组织胶原增生明显,大量较厚的纤维间隔,向肝小叶内伸展,分割包绕肝组织,形成假小叶。模型+去铁铵组、模型+秋水仙碱组、模型+中药大剂量组、模型+中药小剂量组大鼠纤维组织增生程度明显减轻,纤维间隔变窄,着色浅,假小叶较少。
电镜下观察,正常对照组大鼠肝细胞呈多面体,肝细胞内细胞器结构如线粒体、粗面内质网形态正常,肝细胞核为圆形或椭圆形,双层膜完整,核孔清楚。铁沉积模型组肝细胞内可见大量脂滴,Disse腔充满大量红细胞,其它肝细胞结构变化包括线粒体空泡化,内质网扩张,呈大囊泡。模型+去铁铵组肝细胞可见脂滴与红细胞明显减少,核染色体堆积,细胞形态变小,线粒体、内质网等细胞器明显减少。模型+秋水仙碱组、模型+中药大剂量组、模型+中药小剂量组脂肪变性和出血较铁沉积模型组不同程度减轻。
6.去铁治疗和中药复方对DMN诱导的肝纤维化大鼠肝组织胶原的影响:对铁沉积大鼠肝组织胶原的Ridit分析显示,6组总体平均Ridit值不等或不全相等(χ~2=44.2236,v=5,P＜0.01)。进一步进行多样本两两比较的秩和检验(采用Nemenyi法),结果显示,与对照组比较,模型组、模型+秋水仙碱组、模型+中药小剂量组胶原沉积显著增强(P＜0.05);但与模型组比较,模型+去铁铵组、模型+中药小剂量组胶原沉积明显减少(P＜0.05);与模型+去铁铵组比较,模型+秋水仙碱组、模型+中药大剂量组、模型+中药小剂量组胶原沉积无显著差异(P＞0.05)。
7.去铁治疗和中药复方对DMN诱导的肝纤维化大鼠肝组织TGF-β_1 mRNA表达的影响:大鼠肝组织TGF-β_1 mRNA的表达结果显示,与对照组比较,模型组及其它各处理组TGF-β_1 mRNA表达显著增强(P＜0.01);但与模型组比较,模型+去铁铵组、模型+秋水仙碱组、模型+中药小剂量组、模型+中药大剂量组TGF-β_1mRNA表达明显下降(P＜0.01);对于下调TGF-β_1 mRNA的表达,去铁铵、中药大、小剂量明显优于秋水仙碱(P＜0.01);其中,模型+中药大剂量组与模型+中药小剂量组相比较,无显著差异(P＞0.05)。
四、结论
1.细胞实验:外源性铁能够沉积于HSC细胞,诱导HSC细胞活化并促进肝纤维化的持续进展。去铁铵能使HSC细胞活化受到不同程度抑制,甚至诱导其转为静止或发生细胞凋亡。复方肝毒清能在体外发挥抗肝纤维化作用,其机理与其通过抑制TGFβ_1的分泌,抑制HSC细胞活化;诱导HSC细胞转为静止或发生细胞凋亡有关。
2.动物实验:在DMN诱导的肝纤维化大鼠中,伴随着胶原的沉积和肝细胞变性坏死,铁不仅沉积于肝组织,而且铁沉积于HSC细胞;用去铁铵治疗后,肝组织铁沉积明显减少,肝组织胶原染色程度和肝细胞变性坏死明显减轻,TGF-β_1 mRNA表达下调,提示肝组织铁沉积是DMN诱导的大鼠肝纤维化形成的促进因素之一,其机制与铁沉积于HSC细胞并促进HSC细胞活化有关。复方肝毒清能保护肝细胞,改善肝功能;在转录水平抑制TGFβ_1的分泌,抑制HSC细胞活化;抑制胶原纤维的增生,从而起到抗肝纤维化作用。
Objectives
Iron deposition and liver fibrosis are closely related.Iron overload has been proved to play an important role in liver fibrosis during its occurrence and development.In the present study,we took hepatic stellate cell(HSC),which is the key in liver fibrogenesis, as a breakthrough point,and established HSC model with iron deposition,to study the influence of iron deposition in HSC onα-SMA protein expression,collagen typeⅠand TGF-β_1 mRNA expression,as well as HSC apoptosis.Rat liver fibrosis models were established with dimethylnitrosamine(DMN),Rat serum ferritin,transferrin and hepatic iron concentration of liver tissue were used to evaluate iron load of rats.Through prussian blue andα-SMA double staining,iron loaded HSC was located,which made clear the connection between iron deposition and liver fibrosis.Furthermore,by studying the mRNA expression of HSC activation related factor TGFβ1,the effect of iron deposition in HSC on the effect of liver fibrosis was investigated.The reseach of correlation between iron deposition in HSC and liver fibrosis was carried out at histopathological,cellular and molecular levels,in order to illustrate the mechanism of iron deposition in HSC promoting liver fibrogenesis.For in vivo and in vitro experiments,herbal decoction Compound Gan Du Qing was adopted to conduct interference on DMN induced rat liver fibrosis model and HSC model of iron deposition,in the attempt to explain the influence of Compound Gan Du Qing on iron deposition promoting liver fibrogenesis based on the therapy of strengthening healthy qi combined with draining dampness and relieving toxin.
Methods
(一) Cellular experiment:Influence of iron deposition in HSC on liver fibrosis and the effect of Compound Gan Du Qing
1.Establishment of iron deposited HSC model
HSC-T6 was taken as the model cell line and cultured in the 6-well plate,with an 1.8cm square cover glass(sterile) put into each well,and cells were divided into the following 3 groups:iron deposited model group,iron deposited model + desferrioxamine group and normal control group,each group repeated for 2 wells.According to the cytotoxicity experiment,25μM concentration of ferric ammonium citrate(FAC) was chosen as the experimental concentration.After cells grew full adherent the wall,the iron deposited model group was added with FAC,the iron deposited model + desferrioxamine group was supplemented with FAC and desferrioxamine at 1:1 mole ratio.The normal control group was normally cultured HSC-T6.After additional incubation for 24 h,prussian blue staining was performed.
2.Iron deposition in HSC on the activation and reversion of HSC and the effect of desferrioxamine
HSC-T6 cells were divided into normal control group,iron deposited model group, 50μM and 25μM desferrioxamine group.The normal control group was normally cultured HSC-T6,iron deposited model group was the HSC-T6 cells treated with 25μM FAC which was confirmed to be iron deposited,50μM and 25μM desferrioxamine groups were the HSC-T6 cells treated with 50μM and 25μM desferrioxamine respectively.After additional incubation for 24 h,immunohistochemistry was conducted to detect the expression ofα-SMA,and quantitative PCR was performed to examine the mRNA expression of collagen typeⅠand TGF-β_1,TUNEL assay was used to detect HSC-T6 apoptosis,and electronic microscope was adopted to observe the ultrastructure of HSC-T6 cells.
3.Effect of Compound Gan Du Qing on iron deposition induced HSC activation and reversion
HSC-T6 cells were divided into normal control group,iron deposited model group, and iron deposited model + traditional chinese medicine group.The normal control group was normally cultured HSC-T6 cells,iron deposited model group was the HSC-T6 cells treated with 25μM FAC which was confirmed to be iron overloaded,and the iron deposited model + traditional chinese medicine group was the HSC-T6 cells of iron overload model group treated with Compound Gan Du Qing(using the maximum concentration experimentally confirmed to be nontoxic).After incubation for 24 h,immunohistochemistry was conducted to detect the expression ofα-SMA,and quantitative PCR was performed to examine TGF-β_1 mRNA expression,TUNEL assay was used to detect HSC-T6 apoptosis, and electronic microscope was adopted to observe the ultrastructure of HSC-T6 cells.
(二) Animal experiments:Liver fibrogenesis of iron deposition in HSC in DMN induced rat liver fibrosis and the effect of Compound Gan Du Qing
75 of SD rats were randomly divided into 6 groups:blank control group(12),model group(15),model + desferrioxamine group(12),model + colchicine group(12),model + Compound Gan Du Qing large dosage group(12),model + Compound Gan Du Qing small dosage group(12).The method of making rat liver fibrosis model was as follows:prepare 1: 100(V/V) dimethylnitrosamine(DMN) using normal saline as the dilution solution,and perform intraperitoneal injection on rats during the first 3 days each week,at the dosage of 10μL DM-N/kg body weight,for 4 weeks altogether,with the blank control group injected with normal saline.From the second week of making rat models,rats of the model + colchicine group were subjected to colchicine gastric perfusion at a dosage of 0.1mg/kg body weight once daily for 3 weeks;the model + Compound Gan Du Qing large and small dosage groups were gastrically perfused at the dosage of 20g/kg and 5g/kg body weight, respectively,once daily for 3 weeks.The model group and blank control group were both perfused gastrically with normal saline at 10ml/kg once daily for 3 weeks.From the third week,rats of the model + desferrioxamine group were injected intraperitoneally with desferrioxamine at a dosage of 100mg/kg body weight three times a week for 2 weeks. Animal death was recorded from the beginning of making model to the end of the experiment.At the end of the experiment,the eyeballs were extracted and blood was drawn, and the left anterior lobe of the liver was resected,with one portion fixed in Bouin's, another about 50mg liver tissue cleaned and preserved in Trizol at-70℃for further examination.The left liver tissue was preserved at - 70℃.The left lobe of liver tissue was removed and histopathology investigation was conducted by HE,Masson,prussian blue staining,as well as prussian blue andα-SMA double staining.The ultrastructure of liver tissues was observed with electronic microscope.And ELISA assay was carried to examine the concentrations of rat serum ferritin and transferrin.Hepatic iron concentration of rat liver tissue was evaluated by Flame Atomic Absorption Spectrophotometry(FAAS).AU400 Olympus Automatic Biochemistry Analyzer was adopted to detect hepatic function,serum iron and serum lipid.And quantitative PCR was performed to examine the expression of TGF-β_1 mRNA.
Results
(二) Cellular experiment:Influence of iron deposition in HSC on liver fibrosis and the effect of Compound Gan Du Qing
1.Establishment of iron deposited HSC model
After Prussian blue staining,HSC-T6 cells nucleus turned red,and blue iron pellet could be observed in the cytoplasm of HSC-T6 cells in iron deposited model group,which could also be detected in the iron deposited model + desferrioxamine group,with significantly decrease of iron deposition.There was no iron pellet deposition in the normal control group.
2.Iron deposition in HSC on the activation and reversion of HSC and the effect of desferrioxamine
Immunohistochemistry results showed that obvious activation occurred in HSC-T6 cells of the normal control group,with highα-SMA expression.In iron deposited model group,α-SMA was also highly expressed.And in desferrioxamine group,α-SMA expression of HSC-T6 cells was significantly decreased compared with the normal control group and iron deposited model group,implying that activation of cells was inhibited to certain extent.
Collagen typeⅠand TGF-β_1mRNA expression of HSC-T6 cells revealed that collagen typeⅠmRNA expression was significantly enhanced(P＜0.01) in iron deposited model group compared with the control group,whereas there was no significant difference in TGF-β_1 mRNA expression(P＞0.05) between these two groups.Both 50μM and 25μM desferrioxamine could down regulate collagen typeⅠand TGF-β_1 mRNA expression(P＜0.01),and 50μM desferrioxamine was superior to 25μM desferrioxamine(P＜0.01).These results suggested that iron removal therapy could reduce collagen typeⅠand TGF-β_1 expression at transcription level.
Pathology of HSC-T6 cells apoptosis showed that none or only a few cells undewent apoptosis in the control group and iron deposited model group,whereas in desferrioxamine group,HSC-T6 cells undewent apoptosis,whose nucleus appeared to be dark brown,and apoptotic bodies could be observed.
Observation by electronic microscope demonstrated that in the control group, organelles such as mitochondria and rough endoplasmic reticulum in HSC-T6 cells exhibited normal morphology.However,in iron deposited model group,there appeared a large number of binucleated cells,in which organelles such as mitochondria and endoplasmic reticulum decreased dramatically.And in the desferrioxamine group,HSC-T6 cells underwent cell apoptosis,with typical features of a reduction in cell volume,nuclear chromatin condensation,obvious reduction of organelles such as mitochondria and endoplasmic reticulum,which were substituted by a large amount of vacuoles
3.Effect of Compound Gan Du Qing on iron deposition induced HSC activation and reversion
Immunohistochemistry results demonstrated that the expression ofα-SMA decreased significantly in HSC-T6 cells of the iron deposited model+traditional Chinese medicine group compared with the normal control group,implying that activation of cells was inhibited to certain extent.
TGF-β_1 mRNA expression of HSC-T6 cells exhibited that TGF-β_1 mRNA expression was down regulated at different degrees in the iron deposited model + traditional chinese medicine group(P＜0.05).Results implied that Compound Gan Du Qing could inhibit TGF-β_1 expression at transcription level.
Pathology of HSC-T6 cells apoptosis showed that apoptosis occurred in the HSC-T6 cells of the iron deposited model + traditional Chinese medicine group,with the nucleus densely stained dark brown and apoptotic bodies could be detected.
Observation by electronic microscope demonstrated that apoptosis occurred in the HSC-T6 cells of the iron deposited model + traditional Chinese medicine group,with typical apoptosis characteristics of reduction in cell volume,nuclear chromatin condensation,obvious reduction of organelles such as mitochondria and endoplasmic reticulum,which were substituted by a large amount of vacuoles.
(二) Animal experiments:Liver fibrogenesis of iron deposition in HSC in DMN induced rat liver fibrosis and the effect of Compound Gan Du Qing
1.Changes of rat serum ferritin and transferrin in DMN induced rat liver fibrosis:As for the concentration of rat serum ferritin and transferrin,we can see that the concentration of rat serum ferritin in the model group has an significant increase(P＜0.05) than in the control group,and for model + desferrioxamine group,the serum ferritin concentration has a decrease,but there is no statistical significance(P＞0.05),whereas when compared with the model group,the serum ferritin concentration of the model + desfen-ioxamine group decrease dramatically with a statistical significance(P＜0.01).As for transferrin,the concentration in the model group has a significant decrease(P＜0.01),and though transferrin concentration could be detected in the model + desferrioxamine group,there was no statistical significance(P＞0.05).However,when compared with the model group,the concentration of transferrin increase dramatically with a statistical significance(P=0.01). Results revealed that in iron overloaded rat models,the increase of iron concentration is followed by the reduction of transfenin level.
2.Changes of hepatic iron concentration of liver tissue in DMN induced rat liver fibrosis:Investigation of hepatic iron concentration demonstrated that the hepatic iron concentration increased in both the model group and the model + desferrioxamine group(P＜0.01 or P＜0.05) compared with the control group,and when compared with the model group,hepatic iron concentration has a significant decrease in the model + desferrioxamine group(P＜0.05).
3.Histopathology of iron deposition in HSC in rats of DMN induced liver fibrosis: Prussian blue staining demonstrated that,in the model group,the dark blue iron pellet mainly distributed along the space between lobules,and most iron deposit outside cells.For the model + desferrioxamine group,the distribution of iron pellet decreased compared with the model group.And in normal liver tissues,cells are arranged uniformly,with no iron overload.Prussian blue andα-SMA double staining showed that consistent with the dark blue iron pellet distribution,and around the necrosis area,large number ofα-SMA positive HSC cells could be detected,with iron pellet distributed in HSC.
4.Effect of desferrioxamine therapy and Compound Gan Du Qing on hepatic function, serum iron and serum lipid in rats of DMN induced liver fibrosis:Examination of hepatic function,serum iron and serum lipid revealed that,ALT in model group increased significantly(P＜0.01),and at meantime,ALB and G levels decreased significantly(P＜0.01).Desferrioxamine,colchicines,large and small dosage of Compound Gan Du Qing could all decrease ALT level,with desferrioxamine,large and small dosage of Compound Gan Du Qing superior to colchicine(P＜0.01 or P＜0.05).As for the decreasing of AST levels,there was no significant difference between desferrioxamine and small dosage of Compound Gan Du Qing,but both are superior to colchicine(P＜0.01 or P＜0.05).For ALB level increasing,Compound Gan Du Qing large dosage was superior to desferrioxamine,colchicine and Compound Gan Du Qing small dosage(P＜0.01 or P＜0.05).And for G levels,desferrioxamine,colchicine and Compound Gan Du Qing small dosage could all decrease G level(P＜0.01 or P＜0.05). Moreover,desferrioxamine could significantly decrease the levels of serum iron and TG(P＜0.01 or P＜0.05),whereas neither colchicine nor large or small dosage of Compound Gan Du Qing could decrease the levels of serum iron,TG or CHOL(P＞0.05).
5.Effect of desferrioxamine therapy and Compound Gan Du Qing on histopathology in rats of DMN induced liver fibrosis:By HE staining we can see that the hepatocellular plates in normal control group was in streaky-shape with the central vein as center and distributed radially outward.There was hepatic sinusoid with regular distribution,but no collagen fiber could be seen.In the model group,hepatocytes got denatured and necrotic, and the hepatic lobule structure was destroyed,fibrous tissues proliferated and pseudolobules formed,and hemorrhagic necrosis can be detected around the fibrotic tissues, with dark brown particles(iron) deposited in liver tissues.And for model + desferrioxamine group,model + colchicine group,model + Compound Gan Du Qing large dosage group and model + Compound Gan Du Qing small dosage group,there was decrease at various degrees in necrotic hepatocytes,fibrous tissues and pseudolobules.
Masson staining exhibited that only a small amount of collagen fibers could be observed in portal areas and the wall of central vein in normal control group.And in the model group,there was obvious collagen proliferation in rat hepatic tissues,and a large number of thick fibrous septa extended inward the hepatic lobules,dividing and enwraping the liver tissue,leading to pseudolobule formation.And in model + desferrioxamine group, model + colchicine group,model +Compound Gan Du Qing large dosage group and model + Compound Gan Du Qing small dosage group,there was obvious decrease in fibrous tissue proliferation,and fibrous septa turned narrow with faint coloring,and pseudolobules decreased as well.
By electronic microscope we can detect that in control group,hepatocytes appeared to be polyhedron,with organelles such as mitochondria and rough endoplasmic reticulum exhibiting normal morphology,the nucleus of hepatocytes appeared to be circular or elliptical,the bilayer film was intact,and the nuclear pore was clear.In iron overload model group,large amount of lipid droplet could be observed in hepatocytes,Disse space was filled with large number of red cells,and other changes in hepatocytes structure include mitochondrial vacuolation and endoplasmic reticulum dilatation,appearing to be giant vesicles.In the model + desferrioxamine group,the amount of lipid droplet and red blood cells in hepatocytes reduced greatly,and the nuclear chromatin condensed,the cell volume got reduced,organelles such as mitochondria and endoplasmic reticulum reduced greatly as well.In the model + colchicine group,model + Compound Gan Du Qing large dosage group and model + Compound Gan Du Qing small dosage group,various degrees of reduction of lipid denaturation and bleeding in hepatocytes can all be detected compared with iron overloaded model group.
6.Effect of desferrioxamine therapy and Compound Gan Du Qing on liver tissue collagen in rats of DMN induced liver fibrosis:Ridit analysis of iron overloaded rat liver tissue collagen indicated that the average Ridit values of the six groups were unequal or not completely equal(χ~2=44.2236,ν=5,P＜0.01).Further rank-sum test by multi sample and multiple comparison(Nemenyi method) showed that compared with the control group, collagen deposition was greatly enhanced in model group,model+colchicine group and model +Compound Gala Du Qing small dosage group(P＜0.05).However,compared with the model group,collagen deposition was significantly decreased in the model + desferrioxamine group and model + Compound Gan Du Qing small dosage group.And compared with the model + desferrioxamine group,there was no significant differences(P＞0.05) in collagen deposition for the model + colchicine group,model + Compound Gan Du Qing large dosage group,and model + Compound Gan Du Qing small dosage group.
7.Effect of desferrioxamine therapy and Compound Gan Du Qing on TGF-β_1 mRNA expression in rats of DMN induced liver fibrosis:Expression of TGF-β_1 mRNA in rat liver tissues indicated that TGF-β_1 lnRNA expression was greatly enhanced(P＜0.01) in the model group and the other groups compared with the control group,whereas compared with the model group,TGF-β_1mRNA expression was significantly down regulated(P＜0.01) in the model + desferrioxamine group,model + colchicine group,model + Compound Gan Du Qing small dosage group,and model + Compound Gan Du Qing large dosage group,with desferrioxamine,small and large dosage of Compound Gan Du Qing significantly superior to colchicine(P＜0.01).Between model + Compound Gan Du Qing small dosage group and model + Compound Gan Du Qing large dosage group,there was no significant difference(P＞0.05) in down regulation of TGF-β_1mRNA expression.
Conclusions
1.Cellular experiment:Confirmed that exogenous iron could deposit in HSC,thus induce HSC activation,and further lead to liver fibrogenesis.By desferrioxamine therapy,HSC activation was suppressed at various degrees,some of which became quiescent or even undewent apoptosis.Compound Gan Du Qing could play the anti-fibrosis role in vitro, whose mechanism might be its down regulation of TGFβ1 expression,inhibition of HSC activation,and inducing HSC to become quiescent or even undewent apoptosis.
2.Animal experiment:In rats of DMN induced liver fibrosis,accompanied with collagen deposition and hepatocytes denaturation and necrocytosis,iron not only deposited in liver tissue,but also in HSC.By desferrioxamine therapy,iron deposition in liver tissue had decreased,and the degree of collagen staining and hepatocytes denaturation and necrocytosis was improved significanttly,which showed that iron deposition in liver tissue was one of the stimulatives in DMN induced rat liver fibrosis.The mechanism related to iron deposition in HSC promoting the activation of HSC.Compound Gan Du Qing could protect hepatocytes and improve liver function.It could inhibit the secretion of TGFβ1 at the transcription level,thus suppress HSC activation.It could also prevent the proliferation of collagen fibers in liver.Therefore,Compound Gan Du Qing can play the anti- fibrosis role in vivo.
引文
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