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.
【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.
ObjectiveTo explore the expressions of prostaglandin F2α receptor (PTGFR) and cyclooxygenase-2 (COX-2) in tissues of benign bile duct scar and their significances, and investigate the regulating effect of transforming growth factor-β1 (TGF-β1) on the expression of PTGFR in human bile duct fibroblasts cultured in vitro. MethodsThe samples of common bile duct (CBD) scars were collected from 18 patients with benign bile duct scar stricture and 6 cases of normal CBD tissues from liver transplantation donor were collected as control. The expressions of PTGFR and COX-2 were detected by immunohistochemical strept-avidin-biotin complex (SABC) method. Semiquantitative RT-PCR and ELISA methods were used to detect the mRNA and protein levels of PTGFR in bile duct fibroblasts which were effected by TGF-β1 with different concentrations (0, 10, 20, and 30 ng/ml) for 24 h. ResultsThe positive rates of PTGFR and COX-2 were 88.9% (16/18) and 83.3% (15/18) in tissues of benigh CBD scar and 33.3% (2/6) and 0 (0/6) in normal CBD tissues (Plt;0.05). The expressions of the PTGFR mRNA and protein levels became upregulated when the concentrations of the TGF-β1 became higher in human bile duct fibroblasts (Plt;0.05). And the effect was concentration dependant to some extent. ConclusionsThe high expressions of PTGFR and COX-2 play important roles in the process of benign bile duct stricture formation. TGF-β1 is able to induce higher expressions of PTGFR mRNA level and the PTGFR protein level in a concentration dependent manner, and regulate the formation of benign bile duct stricture.
OBJECTIVE: To explore the expression of alpha-smooth muscle actin (alpha-SMA) induced by transforming growth factor beta 1 (TGF-beta 1). METHODS: Five samples of hypertrophic scars and three samples of normal mature scars were collected as the experimental and control groups respectively. The fibroblasts were isolated from scars, and cultured in 2-dimension or 3-dimension culture system. The immunohistochemical staining method of LSAB were used to investigate the expression of alpha-SMA in fibroblasts in the different concentration of TGF-beta 1. RESULTS: The expression of alpha-SMA in 3-dimension culture system were markedly lower than those in 2-dimension culture system with respect to the fibroblasts in the experimental group. The expression of alpha-SMA in fibroblasts were different in response to various TGF-beta 1 concentration, it was more effective at the concentration of 5 ng/ml. The expression of alpha-SMA in the fibroblasts from hypertrophic scars seemed to be more sensitive to TGF-beta 1 compared to that of the normal mature scars. CONCLUSION: There are concentration-dependent in the expression of alpha-SMA induced by TGF-beta 1 in scar fibroblasts in vitro. The biological characteristics of the fibroblasts from hypertrophic scars and normal mature scars and their sensitivity to the inducement of TGF-beta 1 were different. The inducement of TGF-beta 1 may be depressed by extracellular matrix components and that may decrease the expression of alpha-SMA.
Objective To investigate the effects of exogenous bone morphogenetic protein(BMP) and transforming growth factor-β(TGF-β) on biomechanical property for ulna of fracture healing.Methods Thirty-six adult rabbits were made the model of right ulnar fracture and treated locally with TGF-β/PLA, BMP/PLA，TGF-β+BMP/PLA or PLA(as control group). Fracture healing was evaluated by measurement of the mechanical parameters and geometric parameters.Results As compared with control group, the geometric parameters, the bending broken load, the ultimatebending strength, the bending elastic modulus, the ultimate flexural strength, the flexural elastic modulus, the ultimate compressing strength, the compressingelastic modulus, and the ultimate tensile strength for ulna of fracture healingincreased significantly in the treatment groups(P＜0.01). These parameters were higher in TGF-β+BMP/PLA group than in TGF-β/PLA group or in BMP/PLA group andin TGF-β/PLA group than in BMP/PLA group(P＜0.05). There was no significant difference in bone density between the treatment groups and control group. Conclusion Local application of exogenous TGF-β and BMP canincrease the callus formation and enhance biomechanical strength of bone after fracture healing. A combination of TGF-β and BMP has synergetic effect in enhancing fracture healing.
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 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 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.
Objective To examine the effects of TGF-β1 on epithelial-myofibroblast transition ( EMT) of A549 cells and its relationship with extracellular regulating kinase1/2 ( ERK1/2) signaling system. Methods Cultured A549 cells were divided into one negative control group and four groups incubated with TGF-β1 for 48 hours at different concentration ( 0.05, 0. 5, 5, 10 μg/L, respectively) . The protein expressions of E-cadherin, α-smooth muscle actin ( α-SMA) , vimentin and fibronectin were assessed by indirect immunofluorescence and Western blot. In the other experiment, cultured A549 cells were incubated with TGF-β1 for different time. The protein and mRNA expressions of E-cadherin and α-SMA were assessed by Western blot and RT-PCR. The protein expressions of vimentin, fibronectin, ERK1 /2, and p-ERK1 /2 were detected by Western blot. Results By indirect immunofluorescence, Western blot, and RT-PCR analysis, E-cadherin expression significantly decreased and α-SMA expression significantly increased in A549 cells treated with TGF-β1 compared with negative controls in a time- and concentrationdependent manner ( Plt;0.05 ) . Vimentin and fibronectin protein expressions significantly increased simultaneously ( Plt;0.05) . The concentration of 5 ng/mL of TGF-β1 was most effective. The ratio of p-ERK1 /2 and ERK1/2 was significantly increased in the TGF-β1 treated cells in a time-dependent manner ( P lt;0. 05) . Conclusions TGF-β1 can induced EMT in A549 cells in vitro in a time- and concentrationdependant manner. This effect may involve in upregulation of ERK1/2 signaling system.
ObjectiveTo investigate the effect of dust fine particles on tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP), transforming growth factor-β1 (TGF-β1), and collagens in the lung tissue of rats.MethodsAccording to random number table method, 96 male Wistar rats were divided into an untreated control group, a treated control group and an experimental group, with 32 rats in each group. The experimental group was exposed to the wind tunnel simulation of sandstorm (5 days per week, 5 hours per day); the untreated control group was put in the standard living environment next to the wind tunnel; the treated control group was exposed to the same wind tunnel simulation of sandstorm for 5 hours every day, the speed of wind was the same as the experimental group, but without dust; On the 30th, 60th, 90th, and 120th day, the levels of TNF-α, MMP-2, MMP-9, TGF-β1, lung collagen type Ⅰ and Ⅲ in the lung tissue of rats were determined by enzyme linked immunosorbent assay.ResultsCompared with the untreated control group and the treated control group, the content of TNF-α was higher in the experimental group on 30th, 60th, 90th and 120th day (all P<0.05). The contents of MMP-9 and MMP-2 in the experimental group on 60th and 90th day were significantly higher than those in the untreated group and the treated control group, respectively (all P<0.05). On the 30th, 60th, 90th, and 120th day, the content of TGF-β1 in the experimental group was significantly higher compared with the two control groups (all P<0.05). The contents of lung collagen type Ⅰ and type Ⅲ were higher in the experimental group on 60th, 90th and 120th day, respectively, compared with the two control groups (all P<0.05).ConclusionsThe strong sandstorm environmental exposure to a certain period of time can promote lung interstitial collagen deposition in rat. With the prolonged exposure time, the deposition of collagen increases. TNF-α, MMP-2, MMP-9 and TGF-β1 may all participate and induce the process of pulmonary fibrosis.