Objective To investigate the effects of sodium ferulate on lung mRNA expression of TGF-β1 signal transduction molecule in rats with pulmonary fibrosis，and explore the mechanism of sodium ferulate on pulmonary fibrosis.Methods A rat model of pulmonary fibrosis was induced by intratracheal injection of bleomycin (5 mg/kg).Thirty SD rats were randomly divided into three groups (n=10 in each group)，ie.a control group，a pulmonary fibrosis model group，and a sodium ferulate group.The lung histopathology and the expression of collagen was examined by HE staining and collagen fibril staining respectively.The expressions of TGF-βRII and Smad4 mRNA in the lung tissue were detected by situ hybridization.And the expression of TGF-β1 mRNA was detected by real-time fluorescence-quantification RT-PCR.Results Collagen fibril staining indicated that the expression of pulmonary collagen in the model group was significantly higher than that in the control group and sodium ferulate group (Plt;0.001).The mRNA expressions of pulmonary TGF-β1，TGF-βRII and Smad4 were significantly higher in the model group than those in the control group (all Plt;0.01)，and were significantly lower in the sodium ferulate group than those in the model group (all Plt;0.05).Conclusions Sodium ferulate can effectively reduce pulmonary fibrosis through inhibition of the mRNA expression of TGF-β1，TGF-βRII and Smad4 in the lung tissue，thus influence the TGF-β1/Smad4 signal transduction way and inhibit the target gene activation.
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 investigate the effect of transforming growth factor-β1 (TGF-β1) gene transfer on the biological characteristics of osteoblasts. Methods The expression of TGF-β1 in the transfected osteoblasts was detected by in situ hybridization and assay of TGF-β1 activity in the supernatant (minklung epithelium cell growth -inhibition test). The effects of gene transfer andsupernatant of the transfected osteoblasts on the proliferation and alkaline phosphatase(ALP) activity of osteoblasts were detected by 3 H-TdR and MTT. Results The results of in situ hybridization analysis suggested that the osteoblasts transfected by TGF-β1 gene could express TGF-β1 obviously. The complex medium, which was the mixture of serum-free DMEM and the activated supernatant according to 1∶1, 1∶2, 1∶4, could inhibit growth of Mv-1-Lu evidently and the ratios ofinhibition were 16.3%, 22.7%, 28.2% respectively. TGF-β1 gene transfer hadno effect on the biological characteristics of osteoblasts, but the activated supernatant of transfected osteoblasts stimulated proliferation and inhibited ALPactivity of osteoblasts. Conclusion TGF-β1 gene transfer promotes the expression of TGF-β1 and the biological characteristics of trasfected osteoblasts are stable, which is helpful for gene therapy of bone defects in vivo.
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 investigate the effects of adenovirus-mediated melanoma differentiation-associated gene-7 (mda-7)/IL-24 and/or adriamycin (ADM) on transplanted human hepatoma in nude mice and to explore a new way for hepatoma gene therapy combined with chemotherapy. Methods The recombinant adenovirus vector carrying Ad.mda-7 was constructed; Ad.mda-7 and/or ADM were injected into the tumor-bearing mice. Their effects on the growth of the tumor and the survival time of the mice were observed. The expressions of VEGF and TGF-β1 were detected by an immunohistochemistry method. Results Ad.mda-7 was constructed and expressed in vivo successfully. Compared with other three groups 〔control group (43.4±1.67) d, ADM group (64.2±4.14) d, Ad.mda-7 group (61.4±1.67) d〕, the mice treated with Ad.mda-7 combined with ADM had longer average survival time 〔(83.8±4.82) d, P＜0.01〕; the average size of tumor treated with Ad.mda-7 combined with ADM diminished significantly compared with that treated with ADM or Ad.mda-7 separately (P＜0.01). VEGF and TGF-β1 expressions of Ad.mda-7 group were (56.2±7.7)%, (35.2±4.5)%, respectively, and were lower than those in ADM group (VEGF: P＜0.05; TGF-β1: P＜0.01). VEGF expression of Ad.mda-7+ADM group was (37.3±5.0)%, and was significantly lower than that in other three groups (P＜0.01). TGF-β1 expression of Ad.mda-7+ADM group was (31.2±3.1)% and significantly lower than that in control group and ADM group (P＜0.01), however, there was no significant difference compared with Ad.mda-7 group (Pgt;0.05). Conclusion Ad.mda-7 combined with ADM has b antitumor potency and synergistic effects and suppresses the growth of human HCC xenograft in nude mice, possibly by inducing the apoptosis of hepatoma cell lines and suppressing tumor angiogenesis.
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 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 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.