Objective To investigate the mechanismof lung injury caused by paraquat poisoning by observing the changes of fibrogenic cytokines in acute paraquat poisoned rats and the effects of pyrrolidine dithiocarbamate ( PDTC) . Methods Sprague-Dawley rats were randomly divided into three groups, ie. acontrol group ( n =6) , a PDTC group ( n =36) , a paraquat group ( n = 36) , and a paraquat + PDTC group( n =36) . The rats in the PDTC group, the paraquat group, and the paraquat + PDTC group were subdivided into 6 subgroups sacrificed respectively on 1st, 3rd,7th,14th, 28th and 56th day after the treatment. The levels of transforming growth factor-β1( TGF-β1 ) , platelet-derived growth factor ( PDGF) , insulin-like growthfactor-1 ( IGF-1) in serum were measured. Meanwhile the expression of connective tissue growth factor ( CTGF) and hydroxyproline in lung tissues were detected. The relationship of above cytokines with hydroxyproline was analyzed. Results The destructive phase in early ( 1 ～7 d) was characterized by hemorrhage, alveolar edema, and inflammatory cell infiltration. The proliferous phase in later stage ( 14 ～56 d) was characterized by diffused alveolar collapse with fibroblast proliferation and patchy distribution of collagen fibers. Compared with the control group, the level of TGF-β1 on all time points, the level of PDGF from7th to 56th day, the level of IGF-1 from3rd to 56th day in the paraquat group all significantly increased ( P lt;0. 01) . Immunohistochemistry results showed CTGF positive cells mainly located in aleolar epithelialcells, endothelial cells,macrophages in early stage, and fibroblasts were main positive cells on the 28th and the 56th day. The expression of CTGF in the paraquat group increased gradually compared with the control group on different time points ( P lt; 0. 05 or P lt; 0. 01) . Meanwhile, the levels of above cytokines were positively correlated with the level of hydroxyproline. Noteworthy, PDTC treatment led to significant decreases of above cytokines compared with the paraquat group in corresponding time points ( P lt;0. 05 or P lt;0. 01) .Conclusions Over expressions of IGF-1, TGF-β1 , PDGF, IGF-1 and CTGF may play important roles in lung fibrosis of paraquat poisoned rats. PDTC, as a b NF-κB inhibitor, may inhibits NF-κB activity and further significantly decreases expressions of cytokines, leading to significantly attenuated pulmonary inflammation and fibrosis. However, the mechanisms of PDTC intervention still remain to be explored.
Objective To investigate the expression of vascular endothelial growth factor-A （VEGF-A） and the phenotypic transition after the activation of fibroblasts by the supernatant of cultured tumor cells．Methods The growth tendency of fibroblasts was tested by the MTT assay．The expressions of alpha-smooth muscle actin （α-SMA） and VEGF-A mRNA were tested by RT-PCR．The expressions of α-SMA and VEGF-A protein were tested by immunohistochemistry and Western blot．Results The MTT assay indicated that the conditional medium which contained tumor cells supernatant could obviously promote the growth of the fibroblasts． RT-PCR and Western blot manifested that α-SMA expressed by the fibroblasts which cultured by normal medium reached its peak on day 5，then decreased to a low level on day 7．When the medium contained 2 ng/ml transforming growth factor-β1 （TGF-β1），the fibroblasts could steadily express more α-SMA．But the above two mediums could not make the fibroblasts express the VEGF-A. When using the conditional medium，the α-SMA peak advanced on the third day and maintained at a high level，so as the expression of the VEGF-A．Conclusions The results suggested that fibroblasts can be activated to be myofibroblasts when using the conditional medium．The best activation time of the fibroblasts is consistent with the time of the VEGF-A expression at the highest level by the activated fibroblasts．The fibroblasts which activated at the best time are expected to become a kind of cells which can be used for promoting revascularization．
Objective To observe the expression and distribution of transforming growth factor-β1 (TGF-β1) in the healing process of bile duct and discuss its function and significance in the process of benign biliary stricture formation. Methods An injury to bile duct of dog was made and then repaired. The expression and distribution of TGF-β1 in the tissue at different time of the healing process were studied after operation with immunohistochemical SP staining. Results TGF-β1 staining was observed in the granulation tissue, fibroblasts and endothelial cells of blood vessels. High expression of TGF-β1 was observed in the healing process lasting for a long time. Conclusion The high expression of TGF-β1 is related closely with the fibroblast proliferating activity, extracellular matrix overdeposition and scar proliferation in the healing process of bile duct.
Objective To explore the treatment effect of bone marrow mesenchymal stem cells( BMSCs)transplantation in ratmodel of bleomycin-induced pulmonary fibrosis. Methods BMSCs fromten-day-old SDmale rat were cultured and marked with 4, 6-diamidino-2-phenylindole( DAPI) . Seventy female SD rats were randomly divided into four groups. Group A( n = 21) was intratracheally injected with saline as control. Group B( n = 21)were intratracheally injected with BLMA5 to establish pulmonary fibrosis. Group C( n = 21) was injected with BLMA5 intratracheally and BMSCs intravenously via tail vein simultaneously. Group D( n = 7) was injected with BMSCs 14 days after BLMA5 injection. The rats were sacrificed on 7th, 14th and 28th day respectively( rats of group D were on28th) . HE and Masson stainings were performed to observe lung pathological changes. Fluorocyte marked with DAPI was analyzed by fluorescent microscope. Sex determining region Y( SRY) gene were detected by PCR. The lung levels of HYP, tumor necrosis factor-α( TNF-α) and transforming growth factor-β1 ( TGF-β1 ) were measured by ELISA. Results ( 1) In group C and D, BMSCs marked with DAPI were detected in lung frozen section on 7th, 14th and 28th day, and SRY gene of male rats were detected by PCR. ( 2) Alveolitis was most obvious on 7th day and pulmonary fibrosis was most severe on 28th day in group B compared to other three groups( P lt;0. 05 or 0. 01) . Alveolitis and pulmonary fibrosis in group C and D were significantly alleviated compared to group B( P lt; 0. 05) , but still more severe than group A( P lt; 0. 05 or 0. 01) , which in group D was more severe compared to group C( P lt;0. 05) . ( 3) HYP level in group B, coincided with fibrosis, began to increase on7th day and reached the peak on 28th day, significantly higher than other three groups( P lt;0.05 or 0. 01) . TNF-αlevel in group B was highest on 7th day, then descended, which was significantly higher than group A and C on 14th day and not obviously different from other three groups on 28th day. TGF-β1 level in group B was highest on 28th day which was different significantly fromother three groups. Conclusion BMSCs can colonize in the recipient lung tissue and effectively prevent the development of pulmonary fibrosis of rats induced by BLMA5, especially in the early stage.
Objective To observe the effects of cigarette smoke extract ( CSE) on the proliferation and secretion of hydrogen peroxide ( H2O2 ) in human lung fibroblasts ( HLFs) induced by transforming growth factor-β1 ( TGF-β1 ) . Methods Cultured HLFs were divided into a normal group and a model group induced by TGF-β1 ( 5 ng/mL) , then intervened with CSE at different concentrations ( 0% , 2. 5% , 5% ,10% , respectively) . Brdu ELISA assay was used to detect cell proliferation. H2O2 release from cultured cells was assayed using a fluorimetric method. Cellular localization of H2O2 and expression of α-SMA were performed using a fluorescent-labeling strategy. Results TGF-β1 stimulated group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In TGF-β1 stimulated group, 2. 5% and 5% CSE promoted cell proliferation ( P lt; 0. 01 or 0. 05) , while 10% CSE inhibited cell proliferation ( P lt; 0. 01) . In the normal group, both low and high concentration of CSE inhibited cell proliferation ( P lt; 0. 01 or P lt; 0. 05) , and the inhibition effect was dose-dependent. HLF induced by TGF-β1 generated low constitutive levels of extracellular H2O2 that was markedly enhanced by CSE treatment ( P lt; 0. 01) . Pretreatment with DPI, an inhibitor of NADPH oxidase, abolished secretion of H2O2 . Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast. Conclusions Low concentration of CSE can promote myofibroblast proliferation, and markedly increase extracellular secretion of H2O2 . CSE possibly take part in the development and progress of idiopathic pulmonary fibrosis by increasing oxidative stress.
Objective To study the inhibitory effects of curcumin on bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage and explore its possible mechanism.Methods 96 male SD rats were randomly divided into a normal control group，a fibrosis model group，a fibrosis model treated with prednisone group and a fibrosis model treated with curcumin group.Pulmonary fibrosis were induced by instilled bleomycin through tracheal.From day 15 after bleomycin administration，the curcumin group and prednisone group were given curcumin（300 mg/kg） or prednisone（5 mg/kg） per day by intragastric administration，respectively.The normal control group and fibrosis model group were given 1% sodium carboxymethyl cellulose（10 mL/kg） as control.Six rats of each group were randomly sacrificed on day 21，28，42 and 56 after bleomycin administration，respectively.The histological changes of the lung were evaluated by HE and Masson’s trichrome staining.Lung expressions of transforming growth factor-β1（TGF-β1） and hydroxyproline were assessed by immuno-histochemistry and digestion method，respectively.Results Pulmonary fibrosis and hydroxyproline level in the curcumin group were significantly reduced as compared with those in the model group on day 42 and 56.The expession of TGF-β1 in the curcumin group was significantly lower than that in the model group on day 28，42 and 56，and was not significantly different from the normal group on day 56.Conclusion Curcumin could alleviate bleomycin-induced pulmonary fibrosis in rats at the fibrosing stage by inhibiting the expressions of TGF-β1.
Objective To observe the effects of different doses of atorvastatin on bleomycin-induced pulmonary fibrosis in rats. Methods Seventy-five healthy female SD rats were randomly divided into five groups ( 15 rats in each group) , ie. a normal group , a model group, a 10 mg/ kg atorvastatin-treated group, a 20 mg/ kg atorvastatin-treated group, and a 40 mg/ kg atorvastatin-treated group. The rats in the model group and treatment groups were instilled with bleomycin in trachea( 5 mg/kg) , and the normal group were instilled with equal volume of normal saline. The treatment groups were gastric gavaged with different doses of atorvastatin each day from2 nd day on after instillation, and the normal group and model group were gavaged with normal saline. Blood samples were obtained from abdominal aorta in five rats in each group and blood gas analysis was performed on1st week, 2nd week and 4th week respectively after BLM instillation. Then the animals were killed and lung tissue samples were harvested for histopathology study. HE and Masson staining were used to determine the extent of alveolus inflammation and pulmonary fibrosis respectively.Histoimmunochemical stain were used to determine the protein levels of transforming growth factor-β1 ( TGF-β1 ) and connective tissue growth factor( CTGF) in pulmonary tissues. Results The arterial partial pressure of oxygenate ( PaO2 ) in the treatment groups were increased gradually with the increasing of therapeutic dose at each time point and decreased with prolongation of time in the same group. The protein levels of TGF-β1 and CTGF in pulmonary tissues were decreased gradually with prolongation of time. TGF-β1 and CTGF expressed obviously less in the treatment groups than those in the model group at each time point .The higher therapeutic doses were, the less the expressions of TGF-β1 and CTGF were. Conclusion Atorvastatin has remarkable inhibitory effects on BLM-induced pulmonary fibrosis of rats in a dose- and timedependentmanner.
ObjectiveTo investigate the effect of diammonium glycyrrhizinate (DG) plus bone marrow mesenchymal stem cells (MSCs) transplantation in the treatment of acute exacerbation of pulmonary fibrosis induced by bleomycin (BLM) in rats.MethodsMSCs were isolated from male Wistar rats and cultured in vitro. Twenty-four female Wistar rats were randomly divided into 4 groups. The NC group was intratracheally injected with normal saline; the BLM group, the MSC group and the DGMSC group were intratracheally injected with BLM for 7 days; then the MSC group was injected with 0.5 mL of MSCs solution (2.5×106 cells) into the tail vein; the DGMSC group was intraperitoneally injected with DG for 21 days in a dose of 150 mg·kg–1·d–1 on the base of the MSCs injection. The rats were sacrificed on the 28th day and the lung tissue was extracted. Pathological examination was performed to determine the degree of alveolitis and pulmonary fibrosis. Immunofluorescence was used to detect the number and distribution of alveolar type Ⅱ epithelial cells. Alkali hydrolysis method was used to determine the content of hydroxyproline (HYP) in lung tissue; thiobarbituric acid method was used to measure the content of malondialdehyde (MDA) in lung tissue; colorimetric method was used to determine the superoxide dismutase activity (SOD) and total antioxidant capacity (T-AOC); enzyme linked immunosorbent assay was used to detect the expression levels of tumor necrosis factor-α (TNF-α ) and transforming growth factor-β1 (TGF-β1) in lung tissue homogenates.ResultsThe DG combined with MSCs injection can reduce the degree of alveolitis and pulmonary fibrosis in BLM model rats. The content of HYP and TGF-β1 in lung tissue homogenate of the DGMSC group were significantly lower than those in the MSC group (P<0.05). Meanwhile, DG combined with MSCs injection significantly increased the antioxidant capacity of the BLM model rats. MDA content decreased, SOD activity and T-AOC ability improved significantly in the DGMSC group compared with the MSC group (P<0.05). The alveolar type Ⅱ epithelial cells were significantly increased and the cell morphology was maintained in the DGMSC group compared with the MSC group.ConclusionsDG has a synergistic effect with MSCs in treatment of acute exacerbation of pulmonary fibrosis. The mechanism may be related to reducing inflammatory factors during pulmonary fibrosis, attenuating oxidative stress and promoting MSCs migration into lung tissue and transformation to alveolar type Ⅱ epithelial cells.
OBJECTIVE: To explore the autocrine regulation for the transforming growth factor-beta 1(TGF-β1) by the fibroblasts in vitro. METHODS: Fibroblasts were cultured in vitro which isolated from the normal prepuce by circumcision. TGF-β1 concentration were determined with ELISA for the different passages and the different time at the same passage. RESULTS: The TGF-β1 concentration was different for the distinct passage of fibroblasts and achieved the peak (450 ng/L) at the sixth passage. Among the same fibroblasts, the TGF-β1 concentration was different in the various days and passage of the summit (680 ng/L) on the fifth day, which was 2.5 times as high as that of the first day. CONCLUSION: The fibroblasts possess the ability of autocrine of the TGF-β1 and have some regularity. It will provide the theory basis for the research about TGF-β1 regulation mechanism and the medical application about salvia miltiorrhiza.
【Abstract】Objective To investigate the apoptosis induced by TGF-β1 in human hepatic carcinoma cell lines and its relationship with p53 gene and Smad. Methods Three human hepatic carcinoma cell lines which involving in various status of the p53 gene were used in this study. TGF-β1-induced apoptosis in hepatic carcinoma cell lines was measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. To study the mechanism of TGF-β1-induced apoptosis, these cell lines were transfected with a TGF-β1-inducible luciferase reporter plasmid containing Smad 4 binding elements (SBE) and luciferase gene using Lipofectamine 2000, then treated with TGF-β1, relative luciferase activity was assayed. Results Of three cell lines studied with TUNEL assay, TGF-β1 induced apoptosis was observed in HepG2 cells (wild type p53). Huh-7 (mutant p53) and Hep3B (deleted p53) cell lines showed less apoptosis. Luciferase activity assay indicated that the response to TGF-β1 induction in HepG2 cells was increased dramatically but was not significant in Huh-7 and Hep3B cell lines. Conclusion HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh7 cell lines. Smad 4 is a central mediator of the TGF-β1 signal transduction pathway.