ABSTRACT
Salvianolic acid B(Sal B), tanshinone â ¡_A(TSN â ¡_A), and glycyrrhetinic acid(GA) lipid emulsion(GTS-LE) was prepared by the high-speed dispersion method combined with ultrasonic emulsification.The preparation process of the emulsion was optimized by single-factor method and D-optimal method with appearance, centrifugal stability, and particle size of the emulsion as evalua-tion indexes, followed by verification.In vitro release of Sal B, TSN â ¡_A, and GA in GTS-LE was performed by reverse dialysis.In vivo pharmacokinetic evaluation was carried out in mice.The acute liver injury model was induced by acetaminophen.The effect of oral GTS-LE on the acute liver injury was investigated by serum liver function indexes and pathological changes in liver tissues of mice.The results showed that under the optimal preparation process, the average particle size of GTS-LE was(145.4±9.25) nm and the Zeta potential was(-33.6±1.45) mV.The drug-loading efficiencies of Sal B, TSN â ¡_A, and GA in GTS-LE were above 95%, and the drug release in vitro conformed to the Higuchi equation.The pharmacokinetic results showed that the C_(max) of Sal B, TSN â ¡_A, and GA in GTS-LE was 3.128, 2.7, and 2.85 times that of the GTS-S group, and AUC_(0-t) of Sal B, TSN â ¡_A, and GA in GTS-LE was 3.09, 2.23, and 1.9 times that of the GTS-S group.After intragastric administration of GTS-LE, the activities of alanine aminotransferase and aspartate aminotransferase were significantly inhibited, the content of malondialdehyde was reduced, and the structure of hepatocytes recovered to normal.In conclusion, GTS-LE can delay the release of Sal B and promote the release of TSN â ¡_A and GA.The encapsulation of three drug components in the emulsion can improve the oral bioavailability to varying degrees and can effectively prevent the acute liver injury caused by acetaminophen.
Subject(s)
Abietanes , Acetaminophen , Antipyretics , Benzofurans , Chemical and Drug Induced Liver Injury , Depsides , Glycyrrhetinic Acid , Abietanes/therapeutic use , Acetaminophen/adverse effects , Acetaminophen/therapeutic use , Alanine Transaminase/metabolism , Animals , Antipyretics/adverse effects , Antipyretics/therapeutic use , Aspartate Aminotransferases/metabolism , Benzofurans/therapeutic use , Chemical and Drug Induced Liver Injury/prevention & control , Depsides/therapeutic use , Emulsions , Glycyrrhetinic Acid/therapeutic use , Liver/drug effects , Malondialdehyde , MiceABSTRACT
Isoliquiritigenin (ISL) is a ï¬avonoid extracted from licorice root, which is known to serve important antitumor roles in numerous types of cancers; however, its effect on gastric cancer remains to be elucidated. The present study aimed to explore the roles and underlying mechanisms of ISL in MKN28 gastric cancer cells. MKN28 cell proliferation was measured using the Cell Counting Kit8 (CCK8) assay. A Transwell assay was used to determine the effects of ISL on the migration and invasion of MKN28 cells. Apoptosis was assessed by flow cytometry, and the expression levels of apoptosis, autophagy and signaling pathwayrelated proteins were detected by western blot analysis. The results of the CCK8 assay demonstrated that ISL significantly inhibited the proliferation of MKN28 cells (P<0.05). Transwell assays demonstrated that the migration and invasion of MKN28 cells were significantly inhibited following treatment with ISL (P<0.05). Flow cytometric analysis indicated that ISL induced apoptosis of MKN28 cells. In addition, western blot analysis revealed that the ratio of microtubuleassociated proteins 1A/1B light chain 3B (LC3)II/LC3I was upregulated, as was Beclin 1 expression; however, p62 was downregulated following ISL pretreatment, thus suggesting that ISL triggered autophagy in MKN28 cells. In addition, the phosphorylation levels of protein kinase B (AKT) and mammalian target of rapamycin (mTOR) were significantly reduced following ISL treatment. These results indicated that ISL may influence apoptosis and autophagy in MKN28 cells by suppressing the phosphoinositide 3kinase/AKT/mTOR signaling pathway. In conclusion, the findings of the present study suggested that ISL may inhibit MKN28 cell proliferation, migration and invasion by inducing apoptosis and autophagy, implying potential as a therapeutic agent for gastric cancer.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Chalcones/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Animals , Apoptosis/drug effects , Autophagy/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Mice , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathologyABSTRACT
Two zinc-phosphites modified by a rigid amine 3,5-bis(imidazole-1-yl)pyridine (BIP), [Zn2(HPO3)2(BIP)]·H2O (1) and [Zn3(HPO3)3(BIP)(H2O)]·H2O (2), were solvothermally prepared. Compound 1 possesses a zincophosphite layer decorated by BIP moieties via coordinating to intralayer Zn(ii) ions. Compound 2 features a 3D frame with a pillar-layer structure, in which the organic BIP pillars the inorganic zincophosphite layer via coordinating to interlayer Zn(ii) ions. The structural diversity from a 2D layer to a 3D frame was mainly attributable to the different bridging modes of BIP in the process of assembly. Their temperature-dependent photoluminescence properties have also been studied.
ABSTRACT
This study aims to explore the characteristics of crystallization inhibition by cellulose polymers at the supersaturated states of drugs. The study was performed by simulating supersaturated process and preparing supersaturated drug solid, and was carried out by measuring the content of drugs at different time points using dissolution apparatus. The types, amounts, ionic intensity and viscosity of cellulose polymers were examined to assess the crystallization inhibition effect on BCS II class drug indomethacin. HPMC E15 exhibited the strongest crystallization inhibition effect. The more added, more obvious crystallization suppression was observed against indomethacin. The decrease in viscosity and increase in ionic intensity led to an enhanced inhibition. The research provides a scientific guide for the crystallization inhibition of supersaturated drug by cellulose polymers.
Subject(s)
Cellulose/chemistry , Drug Compounding , Indomethacin/chemistry , Polymers/chemistry , Crystallization , Solubility , ViscosityABSTRACT
Aminopeptidase N (APN, also known as CD13) is involved in cellular processes of various types of tumors and a potential anti-cancer therapeutic target. Here, we report the effect of an APN inhibitor 4cc in enhancing sensitivity of hepatocellular carcinoma (HCC) cell lines and xenograft model in response to 5-fluorouracil (5-FU) in vivo and in vitro. The treatment of the combination of 4cc with 5-FU, compared to the combination of bestain with 5-FU, markedly suppressed cell growth and induced apoptosis of HCC cells, accompanying the increase in the level of reactive oxygen species (ROS) and followed by a decrease in the mitochondrial membrane potential (ΔΨM). Furthermore, the combination of 4cc and 5-FU showed a significant inhibitory effect on the growth of HCC xenograft tumors. In addition, following the treatment of 4cc, APN activity and clonogenic formation and the number of CD13-positive cells in PLC/PRF/5 cells were significantly decreased, suggesting that 4cc may also inhibit liver cancer stem cells by CD13 inhibition. These results showed that the APN inhibitor 4cc synergizes antitumor effects of 5-FU on human liver cancer cells via ROS-mediated drug resistance inhibition and concurrent activation of the mitochondrial pathways of apoptosis.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , CD13 Antigens/antagonists & inhibitors , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Animals , Apoptosis/drug effects , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Drug Synergism , Female , Fluorouracil/administration & dosage , Humans , Hydroxamic Acids/administration & dosage , Leucine/administration & dosage , Leucine/analogs & derivatives , Liver Neoplasms/pathology , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Urea/administration & dosage , Urea/analogs & derivatives , Xenograft Model Antitumor AssaysABSTRACT
Mitomycin C (MTC) was incorporated to a micelle system preparing from a polymer named deoxycholic acid chitosan-grafted poly(ethylene glycol) methyl ether (mPEG-CS-DA). mPEG-CS-DA was synthesized and characterized by (1)H nuclear magnetic resonance ((1)H-NMR) and Fourier transform infrared spectroscopy. mPEG-CS-DA formed a core-shell micellar structure with a critical micelle concentration of 6.57 µg/mL. The mPEG-CS-DA micelles were spherical with a hydrodynamic diameter of about 231 nm. After poly(ethylene glycol)ylation of deoxycholic acid chitosan (CS-DA), the encapsulation efficiency and drug loading efficiency increased from 50.62% to 56.42% and from 20.51% to 24.13%, respectively. The mPEG-CS-DA micelles possessed a higher drug release rate than the CS-DA micelles. For pharmacokinetics, the area under the curve (AUC) of the mPEG-CS-DA micelles was 1.5 times higher than that of MTC injection, and these micelles can enhance the bioavailability of MTC. mPEG-CS-DA micelles reduced the distribution of MTC in almost all normal tissues and had the potential to improve the kidney toxicity caused by MTC injection.
Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Drug Carriers/chemistry , Drug Delivery Systems , Mitomycin/administration & dosage , Animals , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacokinetics , Area Under Curve , Biological Availability , Chemistry, Pharmaceutical/methods , Chitosan/chemistry , Deoxycholic Acid/chemistry , Drug Compounding/methods , Magnetic Resonance Spectroscopy , Male , Micelles , Mitomycin/chemistry , Mitomycin/pharmacokinetics , Particle Size , Polyethylene Glycols/chemistry , Rats , Rats, Wistar , Spectroscopy, Fourier Transform Infrared , Tissue DistributionABSTRACT
Aminopeptidase N (APN) is important in tumour processes. The present study detected the antitumour activity of the novel APN inhibitor DH12a, which is an indoline2,3dione derivative. In the present study, Bestatin, a clinical APN inhibitor was used as a positive control. The expression of APN in the ES-2 and 3AO cell lines were assessed using flow cytometry and the drug inhibition constants of DH12a (Ki=13.15 µM) and Bestatin (Ki=16.57 µM) were assessed using a double reciprocal method of competitive inhibition. The in vitro effects of DH12a on cell proliferation were assessed using a 3(4,5dimethylthiazol2yl)2,5diphenyl tetrazolium bromide assay on human cell lines of ES2 (IC50=43.8 µM), A549 (inhibition rate=41.5% at 160 µM DH12a), HL60 (inhibition rate=47.83% at 160 µM DH12a) and 3AO (IC50=70.2 µM). The inhibition rates were consistently higher than those of Bestatin. The effects of DH12a on cell migration (inhibition rates in ES2 cells and 3AO cells were 56.4 and 76.5%, respectively at 15 µM) and invasion (inhibition rates in ES2 cells and 3AO cells were 75.6 and 66.5%, respectively at 15 µM) were assessed using transwell plates. The in vivo effects of DH12a on tumour proliferation and lung tumour metastasis were determined using an H22 xenograft mice model, where DH12a was administered in combination with genotoxic 5fluorouracil. The antitumour activities of DH12a in vivo were also greater than those of Bestatin. In conclusion, the in vitro effects of DH12a on tumour proliferation, migration and invasion were consistent with the in vivo effects. In addition, DH12a exhibited greater antitumour properties compared with Bestatin.
Subject(s)
Antineoplastic Agents/pharmacology , Hydroxamic Acids/pharmacology , Indoles/pharmacology , Lung Neoplasms/drug therapy , Animals , CD13 Antigens/antagonists & inhibitors , CD13 Antigens/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Dose-Response Relationship, Drug , Drug Synergism , Female , Fluorouracil/pharmacology , Inhibitory Concentration 50 , Leucine/analogs & derivatives , Leucine/pharmacology , Lung Neoplasms/secondary , Mice , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Multi-target drug design, in which drugs are designed as single molecules to simultaneously modulate multiple physiological targets, is an important strategy in the field of drug discovery. QT-011, a tamibarotene-furoxan derivative, was here prepared and proposed to exert synergistic effects on antileukemia by releasing nitric oxide and tamibarotene. Compared with tamibarotene itself, QT-011 displayed stronger antiproliferative effects on U937 and HL-60 cells and was more effective evaluated in a nude mice U937 xenograft model in vivo. In addition, QT-011 could release nitric oxide which might contribute to the antiproliferative activity. Autodocking assays showed that QT-011 fits well with the hydrophobic pocket of retinoic acid receptors. Taken together, these results suggest that QT-011 might be a highly effective derivative of tamibarotene and a potential candidate compound as antileukemia agent.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Benzoates/chemistry , Cell Proliferation/drug effects , Leukemia/drug therapy , Ovarian Neoplasms/drug therapy , Oxadiazoles/chemistry , Oxadiazoles/chemical synthesis , Oxadiazoles/pharmacology , Tetrahydronaphthalenes/chemistry , Tetrahydronaphthalenes/chemical synthesis , Tetrahydronaphthalenes/pharmacology , Animals , Benzoates/pharmacology , Blotting, Western , Female , Humans , Leukemia/metabolism , Leukemia/pathology , Mice , Mice, Nude , Models, Chemical , Nitric Oxide/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Receptors, Retinoic Acid/metabolism , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
A series of cinchona alkaloid derivatives were used to catalyze the asymmetric anti-Mannich-type reaction of 3-methyl-2-oxindole with N-tosyl aryl aldimines. The resulting anti-3,3-disubstituted 2-oxindole products were obtained in good yields (up to 92%) with high diastereo- and enantioselectivities (anti/syn up to 97:3 and 91% ee).
Subject(s)
Cinchona Alkaloids/chemistry , Indoles/chemistry , Catalysis , Molecular Structure , Oxindoles , StereoisomerismABSTRACT
Determining cell quantity is a common problem in cytology research and anti-tumor drug development. A simple and low-cost method was developed to determine monolayer and adherent-growth cell quantities. The cell nucleus is located in the cytoplasm, and is independent. Thus, the nucleus cannot make contact even if the cell density is heavy. This phenomenon is the foundation of accurate cell-nucleus recognition. The cell nucleus is easily recognizable in images after fluorescent staining because it is independent. A one-to-one relationship exists between the nucleus and the cell; therefore, this method can be used to determine the quantity of proliferating cells. Results indicated that the activity of the histone deacetylase inhibitor Z1 was effective after this method was used. The nude-mouse xenograft model also revealed the potent anti-tumor activity of Z1. This research presents a new anti-tumor-drug evaluation method.
Subject(s)
Apoptosis/drug effects , Cell Nucleus , Histone Deacetylase Inhibitors/pharmacology , Xenograft Model Antitumor Assays/methods , Animals , Cell Count/methods , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Screening Assays, Antitumor/methods , HCT116 Cells , HeLa Cells , Humans , Hydroxamic Acids/pharmacology , Mice , Mice, Nude , VorinostatABSTRACT
OBJECTIVE: To investigate the pharmacokinetics and tissue distribution of Schisandra chinensis in mice. METHODS: Schisandrin in mice plasma and tissues including heart, liver, spleen, lung and kidney was quantitatively determined by HPLC. RESULTS: The concentration-time curve of Schisandra chinensis extract was described by a single compartment model, Cmax was (2.17 +/- 0.27) mg/ mL, t(max) was (1.00 +/- 0.32) h, AUC0-->infinity, was (4.07 +/- 0.62) mg x h/mL. The sequence of distribution of schisandrin in mice body was as follows: liver > plasma > kidney > lung > heart > spleen. CONCLUSION: The distribution of extract in the body is abroad. Liver has relative high concentration of schisandrin, which is beneficial to the treatment of hepatic disease.
Subject(s)
Cyclooctanes/blood , Cyclooctanes/pharmacokinetics , Lignans/blood , Lignans/pharmacokinetics , Liver/metabolism , Plant Extracts/pharmacokinetics , Polycyclic Compounds/blood , Polycyclic Compounds/pharmacokinetics , Schisandra/chemistry , Administration, Oral , Animals , Area Under Curve , Chromatography, High Pressure Liquid/methods , Cyclooctanes/administration & dosage , Fruit/chemistry , Kidney/metabolism , Lignans/administration & dosage , Lung/metabolism , Male , Mice , Plant Extracts/blood , Polycyclic Compounds/administration & dosage , Spleen/metabolism , Tissue DistributionABSTRACT
BACKGROUND: Adrenomedullin (ADM), a potent hypotensive small peptide, was recently found to inhibit the proliferation of glomerular mesangial cells (MsC) in vitro and to attenuate glomerular lesions in vivo, however the mechanisms remain poorly understood. In this study, we attempted to elucidate them using molecular signal transduction. METHODS: Cultured rat MsC were treated with ADM and several inhibitors of signalling molecules. Methyl thiazoleterazolium (MTT) assay and BrdU incorporation method were employed for examining MsC proliferation. Western blot analysis was used for detecting total mitogen activated protein kinases (t-MAPKs) and phosphorylated MAPKs (p-MAPKs) proteins. RESULTS: ADM suppressed MsC proliferation in a concentration- and time-dependent fashion. This response was inhibited by ADM receptor antagonist CGRP8-37 and a potent protein kinase-A (PKA) inhibitor, H89. Forskolin, a direct adenylate cyclase activator, also significantly inhibited MsC proliferation. SB203580, a P38MAPK inhibitor, and U0126, a MEK inhibitor, both completely blocked ADM mediated responses in MsC. However, curcumin, a SAPK/JNK inhibitor, and GF109203X, a potent protein kinase-C (PKC) inhibitor, had no effect on MsC growth. Western blot analysis showed that ADM did not change the expression of t-MAPKs but increased p-SAPK/JNK and p-P38MAPK levels and decreased p-ERK level. These responses were inhibited by CGRP8-37. All these kinase phosphorylations, except for the increase in p-SAPK/JNK, could be stimulated using forskolin. In addition, only ADM mediated changes in ERK and P38MAPK phosphorylations were inhibited by H89. GF109203X did not affect ADM induced changes in three p-MAPKs expressions. CONCLUSIONS: ADM inhibits MsC proliferation possibly through cAMP-PKA pathway. Both phosphorylations of ERK and P38MAPK pathways were necessary in mediating the antiproliferative response of ADM. It does not preclude the involvement of cAMP independent pathways in the ADM mediated responses.
Subject(s)
Glomerular Mesangium/drug effects , Peptides/pharmacology , Signal Transduction/physiology , Adrenomedullin , Animals , Cell Proliferation/drug effects , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/physiology , Glomerular Mesangium/cytology , JNK Mitogen-Activated Protein Kinases/physiology , Rats , p38 Mitogen-Activated Protein Kinases/physiologyABSTRACT
OBJECTIVE: To study the effect of aldose reductase (AR) on expression of fibronectin and collagen IV in cultured rat renal mesangial cells (MsC). METHODS: AR expression plasmid vector (pCDNA3-AR) was constructed by restriction endonuclease digestion and ligation procedures. Stable expression of AR in MsC was established by Lipofectin transfection. Western blot and immunofluorescence analyses were performed to verify the transfection efficiency. Expression of fibronectin and collagen IV proteins were analyzed using Western blot. RESULTS: Expression of fibronectin and collagen IV in naive MsC treated with TGF-beta1 was upregulated in comparison to that of the untreated naive MsC (P < 0.01). MsC transfected with pCDNA3-AR showed a remarkable increase of expression of fibronectin and collagen IV (P < 0.01). Aldose reductase inhibitors (Sorbinil and Zopolrestat) significantly inhibited the expression of fibronectin and collagen IV in naive MsC (P < 0.05). CONCLUSIONS: Overexpression or inhibition of AR activity significantly alters the expression of fibronectin and collagen IV proteins in cultured rat MsC, suggesting that AR plays a significant role in the pathogenesis of glomerulosclersis.
Subject(s)
Aldehyde Reductase/metabolism , Collagen Type IV/metabolism , Fibronectins/metabolism , Mesangial Cells/metabolism , Aldehyde Reductase/antagonists & inhibitors , Aldehyde Reductase/genetics , Animals , Benzothiazoles/pharmacology , Cells, Cultured , Genetic Vectors , Imidazolidines/pharmacology , Phthalazines/pharmacology , Plasmids , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection , Transforming Growth Factor beta1/pharmacologyABSTRACT
OBJECTIVE: To study the expressions of MMP-2 and TIMP-2 mRNA on cultured rat mesangial cells (MsC) and in human diseased glomeruli, and to explore their significance in the development of glomerulosclerosis. METHODS: The expressions of MMP-2, TIMP-2, and Col IV mRNA on cultured rat MsC stimulated by IL-1 or/and TGF-beta1 were investigated through Northern blot analysis. The levels of MMP-2 and TIMP-2 mRNA expressions and immunoreactivity of PCNA and Col IV in human diseased glomeruli from renal biopsies of lupus nephritis (LN) patients were examined by in situ hybridization and immunohistochemistry, respectively. RESULTS: The levels of MMP-2, TIMP-2, and Col IV mRNA expressions were markedly increased on cultured rat MsC stimulated by IL-1 or/and TGF-beta1. Meanwhile, upregulation of MMP-2 and TIMP-2 mRNA expressions was confirmed in diseased glomeruli from patients with various subtypes of LN, and was closely related to the positive cell number of PCNA presentation and deposition of Col IV in glomeruli. CONCLUSION: The results suggest that the over-expressions of MMP-2 and TIMP-2 mRNA on glomerular cells might play a critical role in the development of glomerulosclerosis.
Subject(s)
Kidney Glomerulus/metabolism , Lupus Nephritis/metabolism , Matrix Metalloproteinase 2/biosynthesis , Tissue Inhibitor of Metalloproteinase-2/biosynthesis , Animals , Cells, Cultured , Collagen Type IV/biosynthesis , Collagen Type IV/genetics , Glomerular Mesangium/metabolism , Humans , Lupus Nephritis/pathology , Matrix Metalloproteinase 2/genetics , Proliferating Cell Nuclear Antigen/biosynthesis , Proliferating Cell Nuclear Antigen/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Tissue Inhibitor of Metalloproteinase-2/genetics , Up-RegulationABSTRACT
OBJECTIVE: The expression of C/EBPalpha protein and mRNA during automatically activation process in primary cultures of HSCs were observed in order to explore its possible association with the proliferation and activation of HSCs. METHODS: Immunocytochemistry, Western blot and RT-PCR were used to evaluated the expression of C/EBPalpha protein and mRNA; as well as the expression of alpha-SMA, Desmin, MMP2, type I procollagen (alpha1). The eukaryotic vector harboring the full length cDNA of C/EBPalpha was transfected into activated HSC, then immunocytochemistry was applied to confirm the transfection and evaluate the effect of transfection on the proliferation of HSC by calculating the PCNA-positive cells. The morphological changes of HSC were observed by use of phase-contrast microscope. RESULTS: Constitutive expression of mRNA and protein of C/EBPalpha were detected in primarily cultured HSCs, and the protein was seen in both nuclei and cytoplasm with the latter being dominant. Their expression levels reached highest at day 2 of the culture, then decreased gradually when continually cultured to the day 4, 7, 10, on the other hand, the expression of alpha-SMA, MMP2 and ColI(alpha1) increased steadily. Transient transfection was verified by the fact that much more and stronger C/EBPalpha stain was observed in transfected HSCs than in void-vector transfected cells. In C/EBPalpha gene transfected HSCs, the number of PCNA-positive cells dramatically decreased compared with the void-vector transfected cells 24h after transfection. In addition, the C/EBPalpha gene transfected HSCs died 36 h after transfection, a few surviving cells became longer and thinner in morphology, however the void-vector transfected cells almost all remained alive. CONCLUSIONS: C/EBPalpha was likely involved in the HSCs activation, and over-expressed C/EBPalpha by transfection had inhibitory influence on the proliferation of cultured rat HSCs.
Subject(s)
CCAAT-Enhancer-Binding Protein-alpha/genetics , Liver/metabolism , Animals , Cells, Cultured , Collagen Type I/genetics , Liver/cytology , Male , Matrix Metalloproteinase 2/genetics , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , TransfectionABSTRACT
OBJECTIVES: To inject decorin-transfected mesangial cells (MsC) vector into the kidneys of rats with anti-thy-1 serum-induced nephritis via left renal artery and observe the survival condition of MsC vector and its influence on glomerular lesions in rats with anti-thy-1 serum induced nephritis. METHODS: Rat mesangio-proliferative glomerulonephritis was established by tail intravenous injection with rabbit anti-thy-1 serum (ATS). Decorin-transfected MsC was injected into rat kidneys via left renal artery. Primary culture, immunostaining for BrdU and decorin of transfected MsC lines were performed to observe their survival. Immunohistochemistry with image analysis was performed to detect the expression of BrdU, alpha-SMA, decorin, TGF-beta1, FN and ColIV in diseased glomeruli. RESULTS: Rat anti-thy-1 serum-induced nephritis identified by pathological examination was successfully established by injecting rabbit ATS, and decorin transfected MsC vector was transfused to rat glomeruli via left renal artery. The active growth and positive expressions of BrdU and decorin proteins on the nuclei and cytoplasms of ex vivo MsC were observed respectively. TGF-beta1, FN, ColIV expressions in diseased glomeruli of rats with ATS nephritis were decreased significantly at day 4 (TGF-beta1, P < 0.05) and day 2 (FN and ColIV, P < 0.01) respectively, compared to uninjected kidneys. CONCLUSIONS: MsC vector is successfully transferred to the glomeruli of experimental rats via left renal artery injection with no affect on cell survival. Decorin protein is expressed on the transfected MsC and shows antagonistic effect on the glomerular lesions of ATS rats. It suggests that the use of ex vivo MsC vector system can provide useful experimental basis for gene therapy of kidney disease in animal model.
Subject(s)
Genetic Therapy , Glomerular Mesangium/metabolism , Glomerulonephritis, Membranoproliferative/therapy , Proteoglycans/genetics , Thy-1 Antigens/immunology , Animals , Decorin , Disease Models, Animal , Extracellular Matrix Proteins , Glomerulonephritis, Membranoproliferative/pathology , Immune Sera/immunology , Kidney Glomerulus/pathology , Rats , TransfectionABSTRACT
OBJECTIVE: To investigate matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expressions in the cultured rat mesangial cells (MsC) transfected with Smad 7 vector and to elucidate the mechanism of Smad 7 in blocking tissue fibrosis. METHODS: Lipofectin method was used to transfect Smad 7 vector into MsC. Western blot and RT-PCR analyses were then used to detect Smad 7 protein and mRNA expression levels. The expressions of MMP-2 and TIMP-2 were determined by Western blot, RT-PCR and zymography assay. RESULTS: Two MsC clones (S-22, S-26) with Smad 7 overexpression were successfully established. The two clones showed an increased expression of MMP-2 protein and enhanced enzyme activity. The expressions of TIMP-2 protein and mRNA however were suppressed. CONCLUSIONS: It is possible that Smad 7 can alleviate the development of tissue fibrosis by upregulating the expression of MMP-2 and downregulating the expression of TIMP-2 in mesangial cells.
Subject(s)
DNA-Binding Proteins/metabolism , Glomerular Mesangium/metabolism , Matrix Metalloproteinase 2/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Trans-Activators/metabolism , Animals , Blotting, Western , Cells, Cultured , DNA-Binding Proteins/genetics , Gene Expression , Genetic Vectors/genetics , Glomerular Mesangium/cytology , Matrix Metalloproteinase 2/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Smad7 Protein , Tissue Inhibitor of Metalloproteinase-2/genetics , Trans-Activators/geneticsABSTRACT
OBJECTIVE: To explore the effect of transforming growth factor (TGF) beta1/Smad signaling pathway on the expression and enzymatic activity of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in cultured rat mesangial cells (MsC). METHODS: Lipofectin method was used to transfect Smad 2, Smad 3 and Smad 7 vectors into MsC; and immunofluorescence, RT-PCR and Western blot analysis were used to detect their transfection efficiency. The expression and enzymatic activity of MMP-2 and TIMP-2 were determined by Western blot, zymography or reverse zymography assay. RESULTS: MsC transfected with Smad 2 gene showed slightly increased expression and enzymatic activity of both MMP-2 and TIMP-2, which was more obvious upon stimulation by TGF-beta1. MsC transfected with Smad 3 gene showed a slight upregulation of TIMP-2 expression and its enzymatic activity, which was enhanced after TGF-beta1 stimulation. There was however no change in MMP-2 expression and its enzymatic activity. On the other hand, MsC transfected with Smad 7 gene showed a decrease in MMP-2 and TIMP-2 expression and enzymatic activity, which was especially obvious after stimulation by TGF-beta1. CONCLUSIONS: TGF-beta1/Smad signaling pathway may play an important role in the pathogenesis of glomerulosclerosis, probably via MMP-2 and TIMP-2 expression and the associated enzymatic activity.
Subject(s)
DNA-Binding Proteins/physiology , Matrix Metalloproteinase 2/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Trans-Activators/physiology , Transforming Growth Factor beta/pharmacology , Animals , Blotting, Western , Cells, Cultured , DNA-Binding Proteins/genetics , Fluorescent Antibody Technique , Gene Expression , Genetic Vectors/genetics , Glomerular Mesangium/cytology , Glomerular Mesangium/drug effects , Glomerular Mesangium/metabolism , Male , Matrix Metalloproteinase 2/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Smad2 Protein , Smad3 Protein , Smad7 Protein , Tissue Inhibitor of Metalloproteinase-2/genetics , Trans-Activators/genetics , Transfection , Transforming Growth Factor beta1ABSTRACT
OBJECTIVE: To study the effect of RAR-beta transfection plus treatment with the corresponding ligand ATRA on the proliferation and phenotype of platelet-derived growth factor (PDGF)-activated hepatic stellate cells (HSC). METHODS: PDGF-activated hepatic stellate cells of rats were transfected with eukaryotic expression vector pCMV-script-RAR-beta, which was verified by western blot. The proliferation of transfected HSC was assayed by BrdU incorporation as well as MTT methods. Their phenotype (alpha-SMA and desmin) was observed by immunocytochemistry assay with image analysis and RAR-beta protein expression was detected by western blot. RESULTS: Transfection of RAR-beta gene and treatment with ligand ATRA could increase the expression of RAR-beta protein for at least 144h and inhibit the proliferation and the expression of alpha-SMA and desmin in PDGF-activated HSC. Significant statistical differences were perceived comparing with sham-transfected, only-PDGF treated, non-ligand treated and irrelevant ligand-treated HSC. CONCLUSIONS: Transfected with RAR-beta gene as well as using related ligand ATRA could suppress the proliferation and reverse the activation phenotype of activated HSC.
Subject(s)
Liver/cytology , Receptors, Retinoic Acid/physiology , Animals , Blotting, Western , Cell Division , Phenotype , Platelet-Derived Growth Factor/pharmacology , Rats , Transfection , Tretinoin/pharmacologyABSTRACT
AIM:To investigate the morphological changes in the process of heteroserum induced rat liver fibrosis and the mechanism of fibrogenesis of this model.METHODS:A model of heteroserum-induced rat liver fibrosis was established by intraperitoneal injection of porcine serum. In addition to the observation of the morphological changes of this model, the infiltration of eosinophils and mast cells were measured quantitatively and the deposition of IgG and complement C(3) was detected by immunofluorescence.RESULTS:The rat liver fibrosis was induced successfully at the end of the 8th week after the injection of heteroserum.Besides the increase of hepatic stellate cells (HSC) during the process of liver fibrosis,proliferation and activation of primary mesenchyma cells (PMCs) were also found.In the early stage, the infiltration of eosinophils and mast cells was significantly increased and the deposition of IgG and complement C(3) was positive in the portal tracts and septa, while gradually reduced after the injection was stopped.CONCLUSION:This model is suitable for the research on liver fibrogenesis; the pathogenesis of this model may be related with the allergen-induced late phase reaction (LPR) caused by the injection of heteroserum, and the HSCs and the PMCs are important sources of ECM-producing cells.