大黄蟅虫超微粉剂干预大鼠肝、肾纤维化的作用机制研究
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摘要
背景
纤维化是组织、器官炎症损伤后的常见病理转归,可见于多种脏器和组织,是一个涉及细胞、细胞因子、细胞外基质及其降解酶类等多个复杂因素的病理生理过程。目前关于纤维化的形成机制尚不完全清楚,临床缺乏有效的早期诊断方法和治疗方法。
肝肾等不同器官的纤维化,虽然发病病因与临床表现不同,但其基本病理特点与形成机制相同。而临床上慢性肝病也常可导致慢性肾脏病变,如乙肝相关性肾病,慢性乙型肝炎肝纤维化病变可形成免疫复合物导致肾小球基膜增厚相关膜性肾炎,最终可导致肾纤维化、慢性肾衰等病变。在器官纤维化中研究对肝肾纤维化同时有效的药物及治疗方法有其特殊意义。
大黄蟅虫丸出自《金匮要略》,功能活血祛瘀、扶正补虚,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。大黄蟅虫丸临床应用广泛,其抗组织器官纤维化的作用受到关注。大黄蟅虫超微粉剂是对传统丸剂的剂型改进,能减少药量的同时提高药效。
中医学认为,肝肾纤维化均存在“正虚”与“血瘀”的病机。大黄蟅虫丸能扶正祛瘀,因此,研究同一方剂对不同脏器纤维化的作用,对于防治慢性肝病与慢性肾病,探讨异病同治的内涵有重要意义。
目的
研究大黄蟅虫超微粉剂对实验性大鼠肝纤维化、肾纤维化的干预作用,用荧光差异凝胶电泳技术和质谱分析技术研究其对肝、肾纤维化大鼠肝、肾组织蛋白表达的影响,用Western免疫印迹方法及免疫组化方法对差异蛋白进行验证,探讨大黄蟅虫超微粉剂干预肝、肾纤维化的蛋白质水平的作用机制。
方法
1.大黄蟅虫超微粉剂对实验性大鼠肝纤维化的干预作用
24只SD大鼠(雌雄各半,体重100-120g)随机分成3组：正常组、模型组、给药组。正常组腹腔注射等渗生理盐水0.5 mL,其余组腹腔注射猪血清0.5 mL,每周2次,连续12周。药物组(0.27g／kg／d)于造模之日起灌胃给药,正常组与模型组灌服等容积生理盐水,每天1次,连续12周。第12周末处死大鼠,取其血与肝组织。
指标检测。肝组织病理学检查：HE染色与Masson染色观察其肝纤维化程度。血清纤维化指标：透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(Ⅳ-C)含量。肝功能指标：血清谷丙转氨酶(ALT)、谷草转氨酶(AST)含量。血液流变学指标：反映血液粘度的4个全血粘度值(切变率150／s、30／s、5／s、1／s)、全血高切还原粘度、全血低切还原粘度,与反映红细胞流变性的红细胞压积、红细胞聚集指数。
2.大黄蟅虫超微粉剂对实验性大鼠肾间质纤维化的干预作用
24只SD大鼠(雌雄各半,体重180-200g)随机分成3组：假手术组、模型组、给药组。假手术组：暴露肾脏后游离左侧输尿管,然后逐层缝合；其他组：暴露肾脏后游离左侧输尿管,在肾盏处和肾下极处分别结扎输尿管,然后逐层缝合。给药组于造模之日起灌胃给药,假手术组与模型组灌服等容积生理盐水,每天1次,连续5周。第5周末处死大鼠,取其血与梗阻侧肾组织。
指标检测。肾组织病理学检查：HE染色与Masson染色观察其肝纤维化程度。血清纤维化指标：透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(Ⅳ-C)含量。肾功能指标：血清肌酐(Cr)、尿素氮(Urea)含量。血液流变学指标：反映血液粘度的4个全血粘度值(切变率150／s、30／s、5／s、1／s)、全血高切还原粘度、全血低切还原粘度,与反映红细胞流变性的红细胞压积、红细胞聚集指数。
3.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织蛋白表达的影响
采用Ettan DIGETM蛋白质组学技术平台比较肝纤维化大鼠与给予中药治疗的肝纤维化大鼠的肝组织的蛋白表达差异。蛋白样品处理：肝组织研粉,加入DIGE用裂解液,制备肝组织全蛋白裂解液→蛋白定量：采用EttanTM 2-D QuantKit定量蛋白。标记样品：采用CyDye DIGE最小标记法,用不同的CyDye荧光染料标记不同组样品(Cy2、Cy3、Cy5)。双向凝胶电泳：固相pH梯度胶条被动重水化14小时后用GE公司电泳仪等电聚焦电泳。等电聚焦完毕,将固相pH梯度胶条立即在平衡液中平衡,之后进行垂直板SDS-聚丙烯酰胺凝胶(SDS-PAGE)电泳,得到凝胶。图像扫描与分析：用Typhoon扫描仪在不同波长分别对Cy2,Cy3,Cy5荧光染料标记的图像进行扫描,用DeCyder Image QuantTM图像分析软件对DIGE图像进行凝胶内差异分析,凝胶间生物学误差分析,确定差异蛋白质,筛选表达量差异2倍以上的蛋白质点。制备胶与染色、酶解：制作一块制备胶,硝酸银染色后切下感兴趣的蛋白质点进行酶解。质谱鉴定：初步鉴定12个表达量差异2倍以上的蛋白点,进行MALDI-TOF MS/MS分析,获得肽质量指纹图谱,采用Mascot软件在NCBInr数据库中进行检索,鉴定蛋白。蛋白功能分析和作用网络分析：对于质谱鉴定出的差异蛋白质,用蛋白质序列数据库Swiss Prot + TrEMBL查询相关信息,用String数据库进行蛋白质作用网络分析。
4.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织与肾间质纤维化大鼠肾组织中蛋白Regucalcin、ERp57、CAⅢ表达水平的影响
采用免疫印迹(Western blot)与免疫组化(Immunohistochemistry)方法对前部分实验中得到的差异蛋白质Regucalcin、ERp57、CAⅢ在肝组织、肾组织中的表达水平进行定量分析。免疫印迹方法：组织蛋白样品提取,SDS-PAGE电泳,转膜,免疫检测及ECL化学发光,软件进行图像分析及数据处理。免疫组化方法：制备组织切片,二甲苯脱蜡,乙醇水化,3%H2O2孵育,PBS液洗,加一抗,PBS液洗,加二抗,PBS液,DAB显色,苏木素复染,梯度酒精脱水,中性树胶封片,软件进行图像分析及数据处理。
5.统计学方法
用SPSS 13.0统计软件进行数据分析。数据满足方差齐性检验,则用One-way ANOVA与LSD法进行组间两两比较。数据以“x±s”表示,P<0.05为差异有统计学意义。
结果
1.大黄蟅虫超微粉剂对实验性大鼠肝纤维化的干预作用
模型组肝组织假小叶形成,肝组织胶原纤维含量与血清HA. LN. PCⅢ. IV-C、ALT、AST及血液流变学指标显著高于正常组(P<0.01)。给药组肝脏无假小叶形成,肝组织胶原纤维含量与血清HA、LN、PCⅢ、IV-C、ALT、AST及血液流变学指标显著低于模型组(P<0.01)。
2.大黄蟅虫超微粉剂对实验性大鼠肾间质纤维化的干预作用
模型组肾组织弥漫性间质纤维化形成,肾组织胶原纤维含量与血清HA、LN、PCⅢ、Ⅳ-C、Cr、Urea及血液流变学指标显著高于假手术组(P<0.01)。给药组肾组织轻度间质纤维化形成,肾组织胶原纤维含量与血清HA、LN、PCⅢ、Ⅳ-C、Cr、Urea及血液流变学指标显著低于模型组(P<0.01)。
3.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织蛋白表达的影响
利用荧光差异凝胶电泳(2D DIGE)技术比较了肝纤维化大鼠与给予中药治疗的肝纤维化大鼠肝组织蛋白质组学差异,在58个表达量差异大于2倍的蛋白质点中,利用MALDI-TOF MS/MS质谱分析技术初步鉴定了12个。其中Regucalcin、ERp57、CAⅢ蛋白在慢性肝、肾疾病中报道颇多,故对三者进行进一步研究。
4.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织与肾间质纤维化大鼠肾组织中蛋白Regucalcin、ERp57、CAⅢ表达水平的影响
免疫印迹与免疫组化结果显示：Regucalcin、ERp57、CAⅢ三个蛋白在正常肝组织、肾组织都有一定表达；肝纤维化模型组与肾间质纤维化模型组大鼠组织中Regucalcin、ERp57的表达量显著高于正常组／假手术组、CAⅢ的表达量显著低于正常组／假手术组；给药组大鼠组织中Regucalcin、ERp57的表达量均显著低于模型组、CAⅢ表达量均显著高于模型组；这与2D DIGE结果一致。
结论
1.大黄蟅虫超微粉剂对猪血清诱导的大鼠肝纤维化有一定的干预作用：能减轻肝纤维化大鼠的肝组织损害、保护肝细胞,降低肝组织纤维化程度与血清纤维化指标(HA、LN、PCⅢ、Ⅳ-C),改善血清肝功能指标(ALT、AST),改善血液流变学性能与微循环。
2.大黄蟅虫超微粉剂对单侧输尿管结扎诱导的大鼠肾间质纤维化有一定的干预作用：能减轻肾间质纤维化大鼠的肾组织损害、保护肾小管上皮细胞,降低肾组织纤维化程度与血清纤维化指标(HA、LN、PCⅢ、Ⅳ-C),改善血清肾功能指标(Cr、Urea),改善血液流变学性能与微循环。
3.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织的蛋白质表达水平有影响。利用2D DIGE技术和MALDI-TOF MS/MS技术比较了肝纤维化大鼠与给予药物治疗的肝纤维化大鼠的肝组织蛋白质组学差异,在58个差异表达蛋白质点(表达量差异大于2倍)中,初步鉴定了12个。这12个蛋白参与机体的损伤修复、氧化应激、炎症反应、免疫反应、凋亡、细胞因子与炎症介质的释放及信号传导通路等机制。这12个蛋白是否参与大黄蟅虫超微粉剂干预实验性肝、肾纤维化的作用机制,需要进一步研究。
4.大黄蟅虫超微粉剂干预实验性肝纤维化、肾间质纤维化的部分作用机制与Regucalcin、ERp57、CAⅢ三个蛋白相关。大黄蛰虫超微粉剂能够调节这三个蛋白在肝纤维化大鼠肝组织及肾纤维化大鼠肾组织的表达量,而这三个蛋白通过细胞钙稳态失调(参与细胞损伤、增殖、凋亡)、过氧化应激、炎症反应、免疫反应、纤维组织癌变等机制参与器官纤维化的病理过程。
5、本研究建立的2D DIGE到MALDI-TOF MS/MS的技术平台相对稳定,在此基础上开展药物蛋白质组学研究,探讨中药复方干预器官纤维化的作用机制是切实可行的。
Background
Organic fibrosis are the result of abnormally depositing of extracellular matrix, the mechanism of organic fibrosis is unknown. The study of the forming mechanism and the methods of diagnosis in early period and effective blocking ways of organic fibrosis are still the urgently probles of basic and clinical studies.
Dahuang Zhechong recipe (DHZC), come from Golden Chamber, is a traditional Chinese complex prescription, which can promot blood circulation, strengthening healthy Qi and dispelling pathogenic factors. It is the Chinese patent medicine for preventing and curing chronic liver disease including fibrosis and cirrhosis recommended by Chinese association of integrative medicine. The antifibrotic effects of DHZC continues to remain a major concern in clinical treatment.
According to the theories of Traditonal Chinese Medicine, there are same mechanisms between hepatic fibrosis and renal fibrosis. healthy Qi-deficiency and Blood-stagnation are the main pathogenesis of organic fibrosis. The key principles for the treatment are strengthening healthy Qi and dispelling pathogenic factors. On the one hand, to reinforce deficiency and strengthening body resistance was used for retrieving function of organ and elevating resistant-illness power. On the other hand, to activate the blood circulation and removing the blood stasis. Therefore, DHZC maybe have the protective and therapeutic effect of organic fibrosis.
Objective
To examine the protective and therapeutic effect of ultramicro-powder of Dahuang Zhechong recipe (DHZC) on liver fibrosis and renal interstitial fibrosis of rats. To investigate its possible mechanisms of anti-hepatic fibrosis effect by 2D DIGE based proteomics analysis and MALDI-TOF MS/MS. Three proteins representing significant changes of expression were confirmed by Western blot and immunohistochemistry. To find some of drug targets of traditional Chinese formulations by Pharmacoproteomics Technology.
Methods
1. Effect of ultramicro-powder of DHZC on hepatic fibrosis in rats.
Wistar rats (n=24,12 males,12 females, SPF) weighing 100g-120g were housed in a pathogen-free environment at room temperature(22-25℃) and maintained on rat chow. All animals received humane care in compliance with the institution's guidelines. They were provided with commercial rodent diet and tap water adlibitum throughout the experiments.
All rats were divided randomly into three groups:normal group, model group, DHZC-treated group. Eight rats were treated in each group. Except in normol group, liver fibrosis in rat was produced in each group. The rats were injected intraperitoneally with 0.5 ml sterile porcine serum twice a week for 12 weeks. In addition, rats of normal group treated with physiological saline in the same way. Ultramicro powders of DHZC were processed by ultramicro pulverization technology. At the same time, DHZC was given to the DHZC-treated group (0.27g/kg/d, i.g.) for 12 weeks. The model group and the normal group were given equal volume of physiological saline. The rats were killed in fasted status after 12 weeks of porcine serum treatment. Animals were anesthetized with isoflurane according to their weight and killed by cervical dislocation. Blood samples of all groups were drawn from the abdominal aorta. Livers were rapidly excised, cut into parts.
liver samples for histopathological evaluation were fixed in 4% neutral buffered formalin, dehydrated with 50%-100% ethanol, and embedded in paraffin. Paraffin sections were cut and stained with HE and Masson to obeserve the histopathological changs. The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by standard spectrophotomeric method using commercial test reagents (BioSino Bio-technology and Science Inc, China). Serum content of hyaluronic acid (HA), laminin (LN), procollagen typeⅢ(PC-Ⅲ) and collagen typeⅣ(Ⅳ-C) were assessed by radio-immune method using commercial kits (BioSino Bio-technology and Science Inc, China). And indexes of blood rheology (four index of whole blood viscosity, two index of whole blood reduction viscosity of high shear rate, hematocrit, erythrocyte aggregation index)were measured.
2. Effect of ultramicro-powder of DHZC on kidney interstitial fibrosis in rats.
Wistar rats (n=24,12 males,12 females, SPF) weighing 100g-120g were housed in a pathogen-free environment at room temperature(22-25℃) and maintained on rat chow. All animals received humane care in compliance with the institution's guidelines. They were provided with commercial rodent diet and tap water adlibitum throughout the experiments.
All rats were divided randomly into three groups:sham-operated group, model group, DHZC-treated group. Eight rats were treated in each group. Except in sham-operated group, liver fibrosis in rat was produced in each group. The renal interstitial fibrosis was induced by unilateral ureter obstruction before the start of experiment. In addition, rats of sham-operated group were just separated left ureter. Ultramicro powders of DHZC were processed by ultramicro pulverization technology. At the same time, DHZC was given to the DHZC-treated group (0.27g/kg/d, i.g.). The model group and the sham-operated group were given equal volume of physiological saline. The rats were killed in fasted status after 5 weeks. Animals were anesthetized with isoflurane according to their weight and killed by cervical dislocation. Blood samples of all groups were drawn from the abdominal aorta, kidneys were rapidly excised, cut into parts.
Renal samples for histopathological evaluation were fixed in 4% neutral buffered formalin, dehydrated with 50%-100% ethanol, and embedded in paraffin. Paraffin sections were cut and stained with HE and Masson to obeserve the histopathological changs. The activity of serum creatinine (Cr) and urea nitrogen (Urea) were determined using commercial test reagents (BioSino Bio-technology and Science Inc, China). Serum content of hyaluronic acid (HA), laminin (LN), procollagen typeⅢ(PC-Ⅲ) and collagen typeⅣ(Ⅳ-C) were assessed by radio-immune method using commercial kits (BioSino Bio-technology and Science Inc, China). And indexes of blood rheology (four index of whole blood viscosity, two index of whole blood reduction viscosity of high shear rate, hematocrit, erythrocyte aggregation index) were measured.
3. Effect of ultramicro-powder of DHZC on expression of proteins in liver tissues.
Using 2D-DIGE to find differentially expressed protein spots between model group (rats were treated with porcine serum) and DHZC-treated group (rats were treated with porcine serum and DAZC).
Preparation of liver lysates. Liver samples of model group and DHZC-treated group for proteomic analysis were immersed in ice-cold PBS buffer, and washed three times. After washing, the livers were cut into large pieces, weighed, and minced into 1 mm3-2 mm3. Then, liver tissue of model group and DHZC-treated group were pulverized in liquid nitrogen. Twenty milligrams of liver tissue were dissolved in 1 mL of lysis buffer (7 M urea,2 M thiourea,4%CHAPS,30 mM Tris-HCL, pH8.5). the solution was sonicated on ice for 15 min (with 5 s pulse-on and 3 s pulse-off to prevent overheating).The sample was incubated for 60 min at room temperature. After centrifugation (45 min,12000 rpm,4℃) the protein concentration of the supernatant was determined by EttanTM2-D Quant Kit (GE healthcare, USA). Samples were stored at-80℃until use.
2D-DIGE. Protein labelling with cyanine dyes (Cy2, Cy3 and Cy5) according to the instructions of the manufacturer (GE healthcare, USA). Sample from DHZC-treated group were labelled with Cy5, sample from model group with labelled with Cy3, and the internal standards with Cy2. Thus, qualitative and quantitative analysis of protein expression was carried out. IEF was carried out by the IPGphor system (GE Healthcare) utilizing IPG strips (Immobiline DryStrip, pH4-7,24cm, GE Healthcare). The labelled mixture was added to the IPG-strips and rehydrated with 30V for 9h. The first dimension was performed with a total of 49.5kV-h of isoelectric focussing at 20℃. The second dimension was carried out with 12.5% SDS/PAGE gels. Gel images were scanned using the Typhoon 9410 imager (GE Healthcare). Images were analysed using DeCyder software (GE Healthcare). The quantitative statistical analysis was performed using the BVA (Biological Variation Analysis) module from the DeCyder Software setting 2.0-fold protein expression change as the differential threshold. Spots with a Student's t-test p value less than 0.05 were included for further investigations.
Protein identification by MALDI-TOF MS/MS. Gels were fixed and then under went silver staining in order to visualize proteins. Protein spots of interest, as defined by the 2D-DIGE/DeCyder analysis, were excised from the gels. Proteins were digested with trypsin, and peptide mass mapping was per-formed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) using an ABI Voyager DE-STR mass spectrometer. Each sample is first analyzed with MALDI-TOF MS to generate the PMF data. Then the five most abundant peptides (precursors) are selected for further fragmentation (MALDI-TOF/TOF) analysis to generate the full scan mass spectrum. To identify the original protein, the masses of the tryptic peptides were entered into MASCOT Database. The database search was restricted to rat proteins, with no constraints on either the molecular weight or the isoelectric point of the protein.
4. Effect of ultramicro-powder of DHZC on expression of Regucalcin, ERp57 and CAⅢin liver tissues and kidney tissues.
Verification of DIGE results by Western blotting and immunostaining were performed for proteins of Regucalcin、ERp57 and CAⅢ. Differences of Regucalcin, ERp57 and CAⅢexpression in liver tissues and kidney tissues between two model groups and two DHZC-treated groups were observed.
Western blot analysis experiment. Selected proteins based on the 2-D MALDI experiments were analyzed further using commercially available antibodies. Liver and renal cell lysates from six groups were prepared on ice in RIPA buffer [1×PBS, 1% NP40,0.1% sodium dodecyl sulfate (SDS),5 mM EDTA,0.5% sodium deoxycholate, and 1 mM sodium orthovanadate] with protease inhibitors. Protein concentration was determined by the modified Bradford method[9]. Equal amounts of proteins were separated electrophoretically on 12% SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes(PVDF) (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was probed with primary antibodies, which were anti-Regucalcin antibody (diluted 1:10, abcam, UK), anti-EREp57 antibody (diluted 1:10, abcam, UK) and anti-CAⅢantibody (diluted 1:10, abcam, UK). Expression was determined with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL) (Pierce, Rockford, IL). The immunoreactive bands were visualized on Kodak 2000M camera system (Eastman Kodak, Rochester, NY) and analysed by HMIAS-2000 software according to the instructions of the manufacturer.β-actine was used to confirm equal loading. The experiments were repeated three times.
Immunohistochemistry experiment. Regucalcin, ERp57 and CAⅢexpression were studied by immunohistochemical analysis conducted on 5μm thick paraffin-embedded liver and renal sections from rats of six groups (n=8, each) Briefly, deparaffinized unstained tissue sections were treated with 3% solution of H2O2 in order to quench the endogenous peroxidase activity. ChemMateTM EnVisionTM Detection Kit, Peroxidase/DAB is employed in a two-step procedure where the first step is incubation of the tissue with primary antibodies, anti-Regucalcin antibody (diluted 1:10, abcam, UK), anti-EREp57 antibody (diluted 1:10, abcam, UK) and anti-CAⅢantibody (diluted 1:10, abcam, UK), and the second step is incubation with the ChemMateTM EnVisionTM/HRP, Rabbit/Mouse reagent. The reaction is visualized by ChemMateTM DAB+ Chromogen. Expression was identified by the brown-colored liver and renal tissue staining. For immunostaining, all samples were treated identically and digital artwork was performed in the same manner for all micrographs.
5. Statistical analysis.
All statistical analyses were carried out using the SPSS 13.0 statistical software package. Quantitative data were expressed as mean±SD and compared using the One-way ANOVA and LSD. Statistical significance was set at p≤0.05.
Results
1.Effect of ultramicro-powder of DHZC on hepatic fibrosis in rats.
Rats of model group with hepatic fibrosis showed significantly increased serum ALT, AST, HA, LN, PCⅢ,Ⅳ-C and indexes of blood rheology levels compared with rats of normal group (P<0.01). Concurrent administration of ultramicro-powder of DHZC group significantly reduced the increase in ALT, AST, HA, LN, PCⅢ,Ⅳ-C and indexes of blood rheology levels, compared with rats of model group (P<0.01). The histopathological analysis suggested that the severe liver fibrosis happened in porcine serum alone treatment group compared with control group. Administration with ultramicro-powder of DHZC obviously alleviated the degree of liver fibrosis based on HE stained and Masson stain tissue sections
2. Effect of ultramicro-powder of DHZC on kidney interstitial fibrosis in rats.
Rats of model group with renal interstitial fibrosis showed significantly increased serum ALT, AST, HA, LN, PCⅢ,Ⅳ-C and indexes of blood rheology levels compared with rats of sham-operated group (P<0.01). Concurrent administration of ultramicro-powder of DHZC group significantly reduced the increase in ALT, AST, HA, LN, PCⅢ,Ⅳ-C and indexes of blood rheology levels, compared with rats of model group (P<0.01). The histopathological analysis suggested that the severe renal interstitial fibrosis happened in alone treatment of unilateral ureter obstruction group compared with control group. Administration with ultramicro-powder of DHZC obviously alleviated the degree of renal interstitial fibrosis based on HE stained and Masson stain tissue sections
3. Effect of ultramicro-powder of DHZC on expression of proteins in liver tissues.
Differentially expressed protein spots were identified by computer analysis using the DeCyder Software.58 spots with greater than 2.0-fold differences in abundance representing significant changes in protein expression were identified. Thirty of these proteins were down-regulated while 28 were up-regulated compared to fibrotic liver. Preliminary choosed twelve protein spots to identificat by MALDI-TOF MS/MS. The regulation on the expressions of these proteins may be one of the pathways for ultramicro-powder of DHZC to exert its anti-organizational fibrosis effect.
4. Effect of ultramicro-powder of DHZC on expression of Regucalcin, ERp57 and CAⅢin liver tissues and kidney tissues.
Differences of proteins of Regucalcin, ERp57 and CAⅢexpression in liver tissues and kidney tissues were observed via immunostaining and Western blotting.
The specificity of the antibodies against Regucalcin, ERp57, CAⅢhad been testified by Western blot. The up-regulation of Regucalcin and ERp57 and the down-regulation of CAⅢwere observed in model group with hepatic fibrosis compared with normal group. These results were consistent with the DIGE results. Furthermore, the up-regulation of Regucalcin and ERp57 and the down-regulation of CAⅢwere observed in model group with renal interstitial fibrosis compared with sham-operated group.
Expression of Regucalcin, ERp57 and CAⅢin hepatocyte cytoplasm and renal tubular epithelial cells were observed in liver tissues from normal group and kidney tissues from sham-operated group. Immunostaining of liver tissues and kidney tissues verified markedly increased levels of Regucalcin and ERp57 in samples from two model groups and decreased levels in samples from two DHZC-treated groups. It also verified markedly decreased levels of CAIII in samples from two model groups and increased levels in samples from two DHZC-treated groups.
Conclusions
1. The ultramicro-powder of Dahuang Zhechong recipe significantly inhibited the progression of hepatic fibrosis induced by porcine serum. The inhibitory effect of DHZC on hepatic fibrosis might be associated with its ability to significantly improve the liver function, markedly reduced the levels of serum aminotransferase and serum fibrosis makers, greatly improve blood rheology and microcirculation, protect the liver cells, repaire damage of liver tissue.
2. The ultramicro-powder of Dahuang Zhechong recipe significantly inhibited the progression of renal interstitial fibrosis induced by unilateral ureter obstruction. The inhibitory effect of DHZC on renal interstitial fibrosis might be associated with its ability to significantly improve the renal function, markedly reduced the levels of serum fibrosis makers, greatly improve blood rheology and microcirculation, protect the renal tubular epithelial cells, repaire damage of renal tissue.
3. According to 2-D DIGE and MALDI-TOF MS/MS, considerable differences in protein regulation during fibrogenesis exist. DHZC's mechanism of action is related to regulation of these differentially expressed proteins. Preliminary choosed twelve protein spots to identificat by MALDI-TOF MS/MS. these proteins are relevant to mechanism of cell damage c, oxidative stress, inflammatory reaction, immune reaction, the process of apoptosis, release of various cytokines and inflammatory mediators, signal transduction and molecular regulation. The regulation on the expressions of these proteins may be one of the pathways for ultramicro-powder of DHZC to exert its anti-fibrosis effect.
4. Verification of DIGE results by Western blotting and immunostaining were performed for proteins of Regucalcin、ERp57 and CAⅢ. The inhibitory effect of ultramicro-powder of DHZC on hepatic fibrosis and renal interstitial fibrosis might be associated with the ability of Regucalcin、ERp57、CAⅢto rehabilitate calcium ions homeostasis, restrain early malignant transformation of tissues, interpose the immune-regulating mechanism and inflammation reaction, protect the cells function of liver and kidney, inhibit oxidative stress.
5. Verification of protein expression confirmed that the technology platform of 2D DIGE and MALDI-TOF MS/MS was relatively stable. Our study shows the great development potential of finding drug target of traditional Chinese formulations by Pharmacoproteomics Technology.
纤维化是组织、器官炎症损伤后的常见病理转归,可见于多种脏器和组织,是一个涉及细胞、细胞因子、细胞外基质及其降解酶类等多个复杂因素的病理生理过程。目前关于纤维化的形成机制尚不完全清楚,临床缺乏有效的早期诊断方法和治疗方法。
肝肾等不同器官的纤维化,虽然发病病因与临床表现不同,但其基本病理特点与形成机制相同。而临床上慢性肝病也常可导致慢性肾脏病变,如乙肝相关性肾病,慢性乙型肝炎肝纤维化病变可形成免疫复合物导致肾小球基膜增厚相关膜性肾炎,最终可导致肾纤维化、慢性肾衰等病变。在器官纤维化中研究对肝肾纤维化同时有效的药物及治疗方法有其特殊意义。
大黄蟅虫丸出自《金匮要略》,功能活血祛瘀、扶正补虚,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。大黄蟅虫丸临床应用广泛,其抗组织器官纤维化的作用受到关注。大黄蟅虫超微粉剂是对传统丸剂的剂型改进,能减少药量的同时提高药效。
中医学认为,肝肾纤维化均存在“正虚”与“血瘀”的病机。大黄蟅虫丸能扶正祛瘀,因此,研究同一方剂对不同脏器纤维化的作用,对于防治慢性肝病与慢性肾病,探讨异病同治的内涵有重要意义。
目的
研究大黄蟅虫超微粉剂对实验性大鼠肝纤维化、肾纤维化的干预作用,用荧光差异凝胶电泳技术和质谱分析技术研究其对肝、肾纤维化大鼠肝、肾组织蛋白表达的影响,用Western免疫印迹方法及免疫组化方法对差异蛋白进行验证,探讨大黄蟅虫超微粉剂干预肝、肾纤维化的蛋白质水平的作用机制。
方法
1.大黄蟅虫超微粉剂对实验性大鼠肝纤维化的干预作用
24只SD大鼠(雌雄各半,体重100-120g)随机分成3组：正常组、模型组、给药组。正常组腹腔注射等渗生理盐水0.5 mL,其余组腹腔注射猪血清0.5 mL,每周2次,连续12周。药物组(0.27g／kg／d)于造模之日起灌胃给药,正常组与模型组灌服等容积生理盐水,每天1次,连续12周。第12周末处死大鼠,取其血与肝组织。
指标检测。肝组织病理学检查：HE染色与Masson染色观察其肝纤维化程度。血清纤维化指标：透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(Ⅳ-C)含量。肝功能指标：血清谷丙转氨酶(ALT)、谷草转氨酶(AST)含量。血液流变学指标：反映血液粘度的4个全血粘度值(切变率150／s、30／s、5／s、1／s)、全血高切还原粘度、全血低切还原粘度,与反映红细胞流变性的红细胞压积、红细胞聚集指数。
2.大黄蟅虫超微粉剂对实验性大鼠肾间质纤维化的干预作用
24只SD大鼠(雌雄各半,体重180-200g)随机分成3组：假手术组、模型组、给药组。假手术组：暴露肾脏后游离左侧输尿管,然后逐层缝合；其他组：暴露肾脏后游离左侧输尿管,在肾盏处和肾下极处分别结扎输尿管,然后逐层缝合。给药组于造模之日起灌胃给药,假手术组与模型组灌服等容积生理盐水,每天1次,连续5周。第5周末处死大鼠,取其血与梗阻侧肾组织。
指标检测。肾组织病理学检查：HE染色与Masson染色观察其肝纤维化程度。血清纤维化指标：透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(Ⅳ-C)含量。肾功能指标：血清肌酐(Cr)、尿素氮(Urea)含量。血液流变学指标：反映血液粘度的4个全血粘度值(切变率150／s、30／s、5／s、1／s)、全血高切还原粘度、全血低切还原粘度,与反映红细胞流变性的红细胞压积、红细胞聚集指数。
3.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织蛋白表达的影响
采用Ettan DIGETM蛋白质组学技术平台比较肝纤维化大鼠与给予中药治疗的肝纤维化大鼠的肝组织的蛋白表达差异。蛋白样品处理：肝组织研粉,加入DIGE用裂解液,制备肝组织全蛋白裂解液→蛋白定量：采用EttanTM 2-D QuantKit定量蛋白。标记样品：采用CyDye DIGE最小标记法,用不同的CyDye荧光染料标记不同组样品(Cy2、Cy3、Cy5)。双向凝胶电泳：固相pH梯度胶条被动重水化14小时后用GE公司电泳仪等电聚焦电泳。等电聚焦完毕,将固相pH梯度胶条立即在平衡液中平衡,之后进行垂直板SDS-聚丙烯酰胺凝胶(SDS-PAGE)电泳,得到凝胶。图像扫描与分析：用Typhoon扫描仪在不同波长分别对Cy2,Cy3,Cy5荧光染料标记的图像进行扫描,用DeCyder Image QuantTM图像分析软件对DIGE图像进行凝胶内差异分析,凝胶间生物学误差分析,确定差异蛋白质,筛选表达量差异2倍以上的蛋白质点。制备胶与染色、酶解：制作一块制备胶,硝酸银染色后切下感兴趣的蛋白质点进行酶解。质谱鉴定：初步鉴定12个表达量差异2倍以上的蛋白点,进行MALDI-TOF MS/MS分析,获得肽质量指纹图谱,采用Mascot软件在NCBInr数据库中进行检索,鉴定蛋白。蛋白功能分析和作用网络分析：对于质谱鉴定出的差异蛋白质,用蛋白质序列数据库Swiss Prot + TrEMBL查询相关信息,用String数据库进行蛋白质作用网络分析。
4.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织与肾间质纤维化大鼠肾组织中蛋白Regucalcin、ERp57、CAⅢ表达水平的影响
采用免疫印迹(Western blot)与免疫组化(Immunohistochemistry)方法对前部分实验中得到的差异蛋白质Regucalcin、ERp57、CAⅢ在肝组织、肾组织中的表达水平进行定量分析。免疫印迹方法：组织蛋白样品提取,SDS-PAGE电泳,转膜,免疫检测及ECL化学发光,软件进行图像分析及数据处理。免疫组化方法：制备组织切片,二甲苯脱蜡,乙醇水化,3%H2O2孵育,PBS液洗,加一抗,PBS液洗,加二抗,PBS液,DAB显色,苏木素复染,梯度酒精脱水,中性树胶封片,软件进行图像分析及数据处理。
5.统计学方法
用SPSS 13.0统计软件进行数据分析。数据满足方差齐性检验,则用One-way ANOVA与LSD法进行组间两两比较。数据以“x±s”表示,P<0.05为差异有统计学意义。
结果
1.大黄蟅虫超微粉剂对实验性大鼠肝纤维化的干预作用
模型组肝组织假小叶形成,肝组织胶原纤维含量与血清HA. LN. PCⅢ. IV-C、ALT、AST及血液流变学指标显著高于正常组(P<0.01)。给药组肝脏无假小叶形成,肝组织胶原纤维含量与血清HA、LN、PCⅢ、IV-C、ALT、AST及血液流变学指标显著低于模型组(P<0.01)。
2.大黄蟅虫超微粉剂对实验性大鼠肾间质纤维化的干预作用
模型组肾组织弥漫性间质纤维化形成,肾组织胶原纤维含量与血清HA、LN、PCⅢ、Ⅳ-C、Cr、Urea及血液流变学指标显著高于假手术组(P<0.01)。给药组肾组织轻度间质纤维化形成,肾组织胶原纤维含量与血清HA、LN、PCⅢ、Ⅳ-C、Cr、Urea及血液流变学指标显著低于模型组(P<0.01)。
3.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织蛋白表达的影响
利用荧光差异凝胶电泳(2D DIGE)技术比较了肝纤维化大鼠与给予中药治疗的肝纤维化大鼠肝组织蛋白质组学差异,在58个表达量差异大于2倍的蛋白质点中,利用MALDI-TOF MS/MS质谱分析技术初步鉴定了12个。其中Regucalcin、ERp57、CAⅢ蛋白在慢性肝、肾疾病中报道颇多,故对三者进行进一步研究。
4.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织与肾间质纤维化大鼠肾组织中蛋白Regucalcin、ERp57、CAⅢ表达水平的影响
免疫印迹与免疫组化结果显示：Regucalcin、ERp57、CAⅢ三个蛋白在正常肝组织、肾组织都有一定表达；肝纤维化模型组与肾间质纤维化模型组大鼠组织中Regucalcin、ERp57的表达量显著高于正常组／假手术组、CAⅢ的表达量显著低于正常组／假手术组；给药组大鼠组织中Regucalcin、ERp57的表达量均显著低于模型组、CAⅢ表达量均显著高于模型组；这与2D DIGE结果一致。
结论
1.大黄蟅虫超微粉剂对猪血清诱导的大鼠肝纤维化有一定的干预作用：能减轻肝纤维化大鼠的肝组织损害、保护肝细胞,降低肝组织纤维化程度与血清纤维化指标(HA、LN、PCⅢ、Ⅳ-C),改善血清肝功能指标(ALT、AST),改善血液流变学性能与微循环。
2.大黄蟅虫超微粉剂对单侧输尿管结扎诱导的大鼠肾间质纤维化有一定的干预作用：能减轻肾间质纤维化大鼠的肾组织损害、保护肾小管上皮细胞,降低肾组织纤维化程度与血清纤维化指标(HA、LN、PCⅢ、Ⅳ-C),改善血清肾功能指标(Cr、Urea),改善血液流变学性能与微循环。
3.大黄蟅虫超微粉剂对肝纤维化大鼠肝组织的蛋白质表达水平有影响。利用2D DIGE技术和MALDI-TOF MS/MS技术比较了肝纤维化大鼠与给予药物治疗的肝纤维化大鼠的肝组织蛋白质组学差异,在58个差异表达蛋白质点(表达量差异大于2倍)中,初步鉴定了12个。这12个蛋白参与机体的损伤修复、氧化应激、炎症反应、免疫反应、凋亡、细胞因子与炎症介质的释放及信号传导通路等机制。这12个蛋白是否参与大黄蟅虫超微粉剂干预实验性肝、肾纤维化的作用机制,需要进一步研究。
4.大黄蟅虫超微粉剂干预实验性肝纤维化、肾间质纤维化的部分作用机制与Regucalcin、ERp57、CAⅢ三个蛋白相关。大黄蛰虫超微粉剂能够调节这三个蛋白在肝纤维化大鼠肝组织及肾纤维化大鼠肾组织的表达量,而这三个蛋白通过细胞钙稳态失调(参与细胞损伤、增殖、凋亡)、过氧化应激、炎症反应、免疫反应、纤维组织癌变等机制参与器官纤维化的病理过程。
5、本研究建立的2D DIGE到MALDI-TOF MS/MS的技术平台相对稳定,在此基础上开展药物蛋白质组学研究,探讨中药复方干预器官纤维化的作用机制是切实可行的。
Background
Organic fibrosis are the result of abnormally depositing of extracellular matrix, the mechanism of organic fibrosis is unknown. The study of the forming mechanism and the methods of diagnosis in early period and effective blocking ways of organic fibrosis are still the urgently probles of basic and clinical studies.
Dahuang Zhechong recipe (DHZC), come from Golden Chamber, is a traditional Chinese complex prescription, which can promot blood circulation, strengthening healthy Qi and dispelling pathogenic factors. It is the Chinese patent medicine for preventing and curing chronic liver disease including fibrosis and cirrhosis recommended by Chinese association of integrative medicine. The antifibrotic effects of DHZC continues to remain a major concern in clinical treatment.
According to the theories of Traditonal Chinese Medicine, there are same mechanisms between hepatic fibrosis and renal fibrosis. healthy Qi-deficiency and Blood-stagnation are the main pathogenesis of organic fibrosis. The key principles for the treatment are strengthening healthy Qi and dispelling pathogenic factors. On the one hand, to reinforce deficiency and strengthening body resistance was used for retrieving function of organ and elevating resistant-illness power. On the other hand, to activate the blood circulation and removing the blood stasis. Therefore, DHZC maybe have the protective and therapeutic effect of organic fibrosis.
Objective
To examine the protective and therapeutic effect of ultramicro-powder of Dahuang Zhechong recipe (DHZC) on liver fibrosis and renal interstitial fibrosis of rats. To investigate its possible mechanisms of anti-hepatic fibrosis effect by 2D DIGE based proteomics analysis and MALDI-TOF MS/MS. Three proteins representing significant changes of expression were confirmed by Western blot and immunohistochemistry. To find some of drug targets of traditional Chinese formulations by Pharmacoproteomics Technology.
Methods
1. Effect of ultramicro-powder of DHZC on hepatic fibrosis in rats.
Wistar rats (n=24,12 males,12 females, SPF) weighing 100g-120g were housed in a pathogen-free environment at room temperature(22-25℃) and maintained on rat chow. All animals received humane care in compliance with the institution's guidelines. They were provided with commercial rodent diet and tap water adlibitum throughout the experiments.
All rats were divided randomly into three groups:normal group, model group, DHZC-treated group. Eight rats were treated in each group. Except in normol group, liver fibrosis in rat was produced in each group. The rats were injected intraperitoneally with 0.5 ml sterile porcine serum twice a week for 12 weeks. In addition, rats of normal group treated with physiological saline in the same way. Ultramicro powders of DHZC were processed by ultramicro pulverization technology. At the same time, DHZC was given to the DHZC-treated group (0.27g/kg/d, i.g.) for 12 weeks. The model group and the normal group were given equal volume of physiological saline. The rats were killed in fasted status after 12 weeks of porcine serum treatment. Animals were anesthetized with isoflurane according to their weight and killed by cervical dislocation. Blood samples of all groups were drawn from the abdominal aorta. Livers were rapidly excised, cut into parts.
liver samples for histopathological evaluation were fixed in 4% neutral buffered formalin, dehydrated with 50%-100% ethanol, and embedded in paraffin. Paraffin sections were cut and stained with HE and Masson to obeserve the histopathological changs. The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by standard spectrophotomeric method using commercial test reagents (BioSino Bio-technology and Science Inc, China). Serum content of hyaluronic acid (HA), laminin (LN), procollagen typeⅢ(PC-Ⅲ) and collagen typeⅣ(Ⅳ-C) were assessed by radio-immune method using commercial kits (BioSino Bio-technology and Science Inc, China). And indexes of blood rheology (four index of whole blood viscosity, two index of whole blood reduction viscosity of high shear rate, hematocrit, erythrocyte aggregation index)were measured.
2. Effect of ultramicro-powder of DHZC on kidney interstitial fibrosis in rats.
Wistar rats (n=24,12 males,12 females, SPF) weighing 100g-120g were housed in a pathogen-free environment at room temperature(22-25℃) and maintained on rat chow. All animals received humane care in compliance with the institution's guidelines. They were provided with commercial rodent diet and tap water adlibitum throughout the experiments.
All rats were divided randomly into three groups:sham-operated group, model group, DHZC-treated group. Eight rats were treated in each group. Except in sham-operated group, liver fibrosis in rat was produced in each group. The renal interstitial fibrosis was induced by unilateral ureter obstruction before the start of experiment. In addition, rats of sham-operated group were just separated left ureter. Ultramicro powders of DHZC were processed by ultramicro pulverization technology. At the same time, DHZC was given to the DHZC-treated group (0.27g/kg/d, i.g.). The model group and the sham-operated group were given equal volume of physiological saline. The rats were killed in fasted status after 5 weeks. Animals were anesthetized with isoflurane according to their weight and killed by cervical dislocation. Blood samples of all groups were drawn from the abdominal aorta, kidneys were rapidly excised, cut into parts.
Renal samples for histopathological evaluation were fixed in 4% neutral buffered formalin, dehydrated with 50%-100% ethanol, and embedded in paraffin. Paraffin sections were cut and stained with HE and Masson to obeserve the histopathological changs. The activity of serum creatinine (Cr) and urea nitrogen (Urea) were determined using commercial test reagents (BioSino Bio-technology and Science Inc, China). Serum content of hyaluronic acid (HA), laminin (LN), procollagen typeⅢ(PC-Ⅲ) and collagen typeⅣ(Ⅳ-C) were assessed by radio-immune method using commercial kits (BioSino Bio-technology and Science Inc, China). And indexes of blood rheology (four index of whole blood viscosity, two index of whole blood reduction viscosity of high shear rate, hematocrit, erythrocyte aggregation index) were measured.
3. Effect of ultramicro-powder of DHZC on expression of proteins in liver tissues.
Using 2D-DIGE to find differentially expressed protein spots between model group (rats were treated with porcine serum) and DHZC-treated group (rats were treated with porcine serum and DAZC).
Preparation of liver lysates. Liver samples of model group and DHZC-treated group for proteomic analysis were immersed in ice-cold PBS buffer, and washed three times. After washing, the livers were cut into large pieces, weighed, and minced into 1 mm3-2 mm3. Then, liver tissue of model group and DHZC-treated group were pulverized in liquid nitrogen. Twenty milligrams of liver tissue were dissolved in 1 mL of lysis buffer (7 M urea,2 M thiourea,4%CHAPS,30 mM Tris-HCL, pH8.5). the solution was sonicated on ice for 15 min (with 5 s pulse-on and 3 s pulse-off to prevent overheating).The sample was incubated for 60 min at room temperature. After centrifugation (45 min,12000 rpm,4℃) the protein concentration of the supernatant was determined by EttanTM2-D Quant Kit (GE healthcare, USA). Samples were stored at-80℃until use.
2D-DIGE. Protein labelling with cyanine dyes (Cy2, Cy3 and Cy5) according to the instructions of the manufacturer (GE healthcare, USA). Sample from DHZC-treated group were labelled with Cy5, sample from model group with labelled with Cy3, and the internal standards with Cy2. Thus, qualitative and quantitative analysis of protein expression was carried out. IEF was carried out by the IPGphor system (GE Healthcare) utilizing IPG strips (Immobiline DryStrip, pH4-7,24cm, GE Healthcare). The labelled mixture was added to the IPG-strips and rehydrated with 30V for 9h. The first dimension was performed with a total of 49.5kV-h of isoelectric focussing at 20℃. The second dimension was carried out with 12.5% SDS/PAGE gels. Gel images were scanned using the Typhoon 9410 imager (GE Healthcare). Images were analysed using DeCyder software (GE Healthcare). The quantitative statistical analysis was performed using the BVA (Biological Variation Analysis) module from the DeCyder Software setting 2.0-fold protein expression change as the differential threshold. Spots with a Student's t-test p value less than 0.05 were included for further investigations.
Protein identification by MALDI-TOF MS/MS. Gels were fixed and then under went silver staining in order to visualize proteins. Protein spots of interest, as defined by the 2D-DIGE/DeCyder analysis, were excised from the gels. Proteins were digested with trypsin, and peptide mass mapping was per-formed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) using an ABI Voyager DE-STR mass spectrometer. Each sample is first analyzed with MALDI-TOF MS to generate the PMF data. Then the five most abundant peptides (precursors) are selected for further fragmentation (MALDI-TOF/TOF) analysis to generate the full scan mass spectrum. To identify the original protein, the masses of the tryptic peptides were entered into MASCOT Database. The database search was restricted to rat proteins, with no constraints on either the molecular weight or the isoelectric point of the protein.
4. Effect of ultramicro-powder of DHZC on expression of Regucalcin, ERp57 and CAⅢin liver tissues and kidney tissues.
Verification of DIGE results by Western blotting and immunostaining were performed for proteins of Regucalcin、ERp57 and CAⅢ. Differences of Regucalcin, ERp57 and CAⅢexpression in liver tissues and kidney tissues between two model groups and two DHZC-treated groups were observed.
Western blot analysis experiment. Selected proteins based on the 2-D MALDI experiments were analyzed further using commercially available antibodies. Liver and renal cell lysates from six groups were prepared on ice in RIPA buffer [1×PBS, 1% NP40,0.1% sodium dodecyl sulfate (SDS),5 mM EDTA,0.5% sodium deoxycholate, and 1 mM sodium orthovanadate] with protease inhibitors. Protein concentration was determined by the modified Bradford method[9]. Equal amounts of proteins were separated electrophoretically on 12% SDS/polyacrylamide gels and transferred onto polyvinylidene difluoride membranes(PVDF) (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was probed with primary antibodies, which were anti-Regucalcin antibody (diluted 1:10, abcam, UK), anti-EREp57 antibody (diluted 1:10, abcam, UK) and anti-CAⅢantibody (diluted 1:10, abcam, UK). Expression was determined with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL) (Pierce, Rockford, IL). The immunoreactive bands were visualized on Kodak 2000M camera system (Eastman Kodak, Rochester, NY) and analysed by HMIAS-2000 software according to the instructions of the manufacturer.β-actine was used to confirm equal loading. The experiments were repeated three times.
Immunohistochemistry experiment. Regucalcin, ERp57 and CAⅢexpression were studied by immunohistochemical analysis conducted on 5μm thick paraffin-embedded liver and renal sections from rats of six groups (n=8, each) Briefly, deparaffinized unstained tissue sections were treated with 3% solution of H2O2 in order to quench the endogenous peroxidase activity. ChemMateTM EnVisionTM Detection Kit, Peroxidase/DAB is employed in a two-step procedure where the first step is incubation of the tissue with primary antibodies, anti-Regucalcin antibody (diluted 1:10, abcam, UK), anti-EREp57 antibody (diluted 1:10, abcam, UK) and anti-CAⅢantibody (diluted 1:10, abcam, UK), and the second step is incubation with the ChemMateTM EnVisionTM/HRP, Rabbit/Mouse reagent. The reaction is visualized by ChemMateTM DAB+ Chromogen. Expression was identified by the brown-colored liver and renal tissue staining. For immunostaining, all samples were treated identically and digital artwork was performed in the same manner for all micrographs.
5. Statistical analysis.
All statistical analyses were carried out using the SPSS 13.0 statistical software package. Quantitative data were expressed as mean±SD and compared using the One-way ANOVA and LSD. Statistical significance was set at p≤0.05.
Results
1.Effect of ultramicro-powder of DHZC on hepatic fibrosis in rats.
Rats of model group with hepatic fibrosis showed significantly increased serum ALT, AST, HA, LN, PCⅢ,Ⅳ-C and indexes of blood rheology levels compared with rats of normal group (P<0.01). Concurrent administration of ultramicro-powder of DHZC group significantly reduced the increase in ALT, AST, HA, LN, PCⅢ,Ⅳ-C and indexes of blood rheology levels, compared with rats of model group (P<0.01). The histopathological analysis suggested that the severe liver fibrosis happened in porcine serum alone treatment group compared with control group. Administration with ultramicro-powder of DHZC obviously alleviated the degree of liver fibrosis based on HE stained and Masson stain tissue sections
2. Effect of ultramicro-powder of DHZC on kidney interstitial fibrosis in rats.
Rats of model group with renal interstitial fibrosis showed significantly increased serum ALT, AST, HA, LN, PCⅢ,Ⅳ-C and indexes of blood rheology levels compared with rats of sham-operated group (P<0.01). Concurrent administration of ultramicro-powder of DHZC group significantly reduced the increase in ALT, AST, HA, LN, PCⅢ,Ⅳ-C and indexes of blood rheology levels, compared with rats of model group (P<0.01). The histopathological analysis suggested that the severe renal interstitial fibrosis happened in alone treatment of unilateral ureter obstruction group compared with control group. Administration with ultramicro-powder of DHZC obviously alleviated the degree of renal interstitial fibrosis based on HE stained and Masson stain tissue sections
3. Effect of ultramicro-powder of DHZC on expression of proteins in liver tissues.
Differentially expressed protein spots were identified by computer analysis using the DeCyder Software.58 spots with greater than 2.0-fold differences in abundance representing significant changes in protein expression were identified. Thirty of these proteins were down-regulated while 28 were up-regulated compared to fibrotic liver. Preliminary choosed twelve protein spots to identificat by MALDI-TOF MS/MS. The regulation on the expressions of these proteins may be one of the pathways for ultramicro-powder of DHZC to exert its anti-organizational fibrosis effect.
4. Effect of ultramicro-powder of DHZC on expression of Regucalcin, ERp57 and CAⅢin liver tissues and kidney tissues.
Differences of proteins of Regucalcin, ERp57 and CAⅢexpression in liver tissues and kidney tissues were observed via immunostaining and Western blotting.
The specificity of the antibodies against Regucalcin, ERp57, CAⅢhad been testified by Western blot. The up-regulation of Regucalcin and ERp57 and the down-regulation of CAⅢwere observed in model group with hepatic fibrosis compared with normal group. These results were consistent with the DIGE results. Furthermore, the up-regulation of Regucalcin and ERp57 and the down-regulation of CAⅢwere observed in model group with renal interstitial fibrosis compared with sham-operated group.
Expression of Regucalcin, ERp57 and CAⅢin hepatocyte cytoplasm and renal tubular epithelial cells were observed in liver tissues from normal group and kidney tissues from sham-operated group. Immunostaining of liver tissues and kidney tissues verified markedly increased levels of Regucalcin and ERp57 in samples from two model groups and decreased levels in samples from two DHZC-treated groups. It also verified markedly decreased levels of CAIII in samples from two model groups and increased levels in samples from two DHZC-treated groups.
Conclusions
1. The ultramicro-powder of Dahuang Zhechong recipe significantly inhibited the progression of hepatic fibrosis induced by porcine serum. The inhibitory effect of DHZC on hepatic fibrosis might be associated with its ability to significantly improve the liver function, markedly reduced the levels of serum aminotransferase and serum fibrosis makers, greatly improve blood rheology and microcirculation, protect the liver cells, repaire damage of liver tissue.
2. The ultramicro-powder of Dahuang Zhechong recipe significantly inhibited the progression of renal interstitial fibrosis induced by unilateral ureter obstruction. The inhibitory effect of DHZC on renal interstitial fibrosis might be associated with its ability to significantly improve the renal function, markedly reduced the levels of serum fibrosis makers, greatly improve blood rheology and microcirculation, protect the renal tubular epithelial cells, repaire damage of renal tissue.
3. According to 2-D DIGE and MALDI-TOF MS/MS, considerable differences in protein regulation during fibrogenesis exist. DHZC's mechanism of action is related to regulation of these differentially expressed proteins. Preliminary choosed twelve protein spots to identificat by MALDI-TOF MS/MS. these proteins are relevant to mechanism of cell damage c, oxidative stress, inflammatory reaction, immune reaction, the process of apoptosis, release of various cytokines and inflammatory mediators, signal transduction and molecular regulation. The regulation on the expressions of these proteins may be one of the pathways for ultramicro-powder of DHZC to exert its anti-fibrosis effect.
4. Verification of DIGE results by Western blotting and immunostaining were performed for proteins of Regucalcin、ERp57 and CAⅢ. The inhibitory effect of ultramicro-powder of DHZC on hepatic fibrosis and renal interstitial fibrosis might be associated with the ability of Regucalcin、ERp57、CAⅢto rehabilitate calcium ions homeostasis, restrain early malignant transformation of tissues, interpose the immune-regulating mechanism and inflammation reaction, protect the cells function of liver and kidney, inhibit oxidative stress.
5. Verification of protein expression confirmed that the technology platform of 2D DIGE and MALDI-TOF MS/MS was relatively stable. Our study shows the great development potential of finding drug target of traditional Chinese formulations by Pharmacoproteomics Technology.
引文
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