Effects And Mechanisms Of Ultramicro-powder Of Dahuang Zhechong Recipe On Hepatic Fibrosis And Renal Interstitial Fibrosis In Rats

BackgroundOrganic 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.ObjectiveTo 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.Methods1. 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.Results1.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 sections2. 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 sections3. 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.Conclusions1. 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.