Objective To investigate the effects of matrine on cell proliferation and expression of connective tissue growth factor( CTGF) and hypoxia inducible factor-1α( HIF-1α) of human lung fibroblast ( WRC-5) in normoxia ( 21% O2, 74% N2 , 5% CO2 ) and hypoxia ( 1% O2, 94% N2 , 5% CO2 )conditions. Methods MRC-5 cells were cultured and divided into differrent groups interfered with different dose of Matrine ( final concentration of 0 ～3. 2 mmol / L) in normoxia or hypoxia for 24 h. Cells were dividedinto 8 groups according to culture conditions, ie. normoxiagroup( N0 group) , normoxia + matrine 0. 2 mmol / L group( N0. 2 group) , normoxia + matrine 0. 4 mmol / L group( N0. 4 group) , normoxia + matrine 0. 8 mmol / L group( N0. 8 group) , hypoxia group( H0 group) , hypoxia + matrine 0. 2 mmol /L group( H0. 2 group) , hypoxia +matrine 0. 4 mmol /L group( H0. 4 group) , and hypoxia + matrine 0. 8 mmol / L group( H0. 8 group) . The MTT assay was used to measure the cell proliferation activity. Western-blot assay was used to examine the expression of CTGF and HIF-1α. Results Hypoxia promoted the cell proliferation in all groups( P lt;0. 05) .Matrine inhibited the proliferation of WRC-5 cells in a concentration-dependent manner in hypoxia or normoxia conditions( P lt;0. 05) . The expression of CTGF andHIF-1αwas lower in normoxia and higher in hypoxia( P lt;0. 01) . Matrine inhibited the expression of CTGF and HIF-1αin a concentration-dependent manner in hypoxiaand normoxia( P lt;0. 05) . Conclusion Matrine can inhibit the cell proliferation and the expression of CTGF and HIF-1αof WRC-5 cells in normoxia and hypoxia in a concentration-dependent manner.
Objective To explore the effect of connective tissue growth factor on the pathogenesis of hypertrophic scar and keloid tissue. Methods The content of hydroxyproline was determined and the expression of connective tissue growth factor gene was detected by the reverse transcription-polymerase chain reaction and image analysis technique in 5 normal skins, 15 hypertrophic scars and 7 keloid tissues. Results The contents of hydroxyproline in the hypertrophic scar(84.10±1.76) and keloid tissue (92.38±2.04) were significantly higher than that of normal skin tissue (26.52 ± 4.10) (P lt; 0.01). The index of connective tissue growth factor mRNA in the hypertrophic scar (0.78 ± 0.63) and keloid tissue (0.84 ± 0.04) were higher than that of normal skin tissue ( 0.09 ± 0.25) (P lt; 0.01). Conclusion Connective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar and keloid tissue.
Objective To observe the influence of the transforming growth factor β1(TGF-β1) on the denervated mouse musclederived stem cells（MDSCs） producing the connective tissue growth factor（CTGF）at different time points in vitro. Methods MDSCs from the primarycultureof the denervated mouse skeletal muscle were isolated and purified by the preplate technique, and they were identified before the culture and after the culturein vitro with TGF-β1 (10 ng/ml) for 24 hours. Then, MDSCs were randomlydivided into 6 groups (Groups A, B, C, D, E and F) according to the different time points, and were cultured in vitro with TGF-β1 (10 ng/ml) for 0, 3, 6, 12, 24 and 48 hours, respectively. The levels of CTGF mRNA in MDSCs were measured by the real time RT-PCR and the expression of CTGF protein was detected by the CTGF Western blot. Results The immunohistochemistry revealed that before the adding of TGF-β1, MDSCs highly expressed Sca-1, with a positivityrate of 96%; however, after the adding of TGF-β1, the positive expression of Sca-1 decreased greatly, with a negativity rate gt;99%. The Western blot test showed that the ratios of CTGF to the average absorbance of βactin in Groups A-F were 0.788±0.123, 1.063±0.143, 2.154±0.153, 2.997±0.136, 3.796±0.153 and 3.802±0.175, respectively. In Groups AD，the absorbance increased gradually, with a significant difference between the abovementioned groups (Plt;0.05). However, in Groups D-F, there was no significant difference between the groups as the promotive tendency became less significant (P＞0.05). The RT-PCR test showed that the △Ct values in GroupsA-F were 1.659±0.215, 1.897±0.134, 2.188±0.259, 2.814±0.263,2.903±0.125 and 3.101±0.186, respectively. In Groups A-D, the increase in the △Ct value was gradual, but the differences were significant between the groups (Plt;0.05). But in Groups E and F, the promotive tendency became less significant（Pgt;0.05）. Conclusion TGF-β1 can promote the production of CTGF inthe mouse MDSCs cultured in vitro and the time-dependent relation exists for 3-12 hours.
ObjectiveTo investigate the effect of transforming growth factor β1 (TGF-β1) induced proliferation of ligamentum flavum cells and ligamentum flavum hypertrophy and its effect on connective tissue growth factor (CTGF) expression.MethodsThe ligamentum flavum tissue in lumbar intervertebral disc herniation was extracted and the ligamentum flavum cells were isolated and cultured by collagenase pre-digestion method. Morphological observation, immunofluorescence staining observation, and MTT assay were used for cell identification. The 3rd generation ligamentum flavum cells were divided into 5 groups. The cells of groups A, B, C, and D were respectively sealed with 3 ng/mL TGF-β1, 50 ng/mL CTGF, 3 ng/mL TGF-β1+CTGF neutralizing antibody, and 50 ng/mL CTGF+CTGF neutralizing antibody. Serum free DMEM was added to group E as the control. MTT assay was used to detect the effects of TGF-β1 and CTGF on the proliferation of ligamentum flavum cells. Western blot was used to detect the expression of CTGF protein. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of collagen type Ⅰ, collagen type Ⅲ, and CTGF genes.ResultsThe morphological diversity of cultured ligamentum flavum cells showed typical phenotype of ligamentum flavum fibroblasts; all cells expressed collagen type Ⅰ and vimentin, and some cells expressed collagen type Ⅲ; MTT identification showed that with the prolongation of culture time, the absorbance (A) value of each generation of cells increased gradually, and the A value of the same generation of cells at each time point was significantly different (P<0.05), there was no significant difference in A value between the cells of each generation at the same time point (P>0.05). After cultured for 24 hours, MTT assay showed that the A value of cells in groups A and B was significantly higher than that of group E (P<0.05). After adding CTGF neutralizing antibody, the A value of cells in groups C and D decreased, but it was still higher than that of group E (P<0.05). There were also significant differences among groups A, C and groups B, D (P<0.05). Western blot analysis showed that the relative expression of CTGF protein in groups A and B was significantly higher than that in group E (P<0.05), while the relative expression of CTGF protein in groups C and D was significantly lower than that in group E (P<0.05), and the difference between groups A, C and groups B, D was also significant (P<0.05). qRT-PCR detection showed that the mRNA relative expression of CTGF, collagen type Ⅰ, and collagen type Ⅲ in group A was significantly higher than that in group E (P<0.05). After adding neutralizing antibody, the mRNA relative expression of genes in group C was inhibited and were significantly lower than that in group A, but still significantly higher than that in group E (P<0.05). The mRNA relative expressions of collagen type Ⅰ and collagen type Ⅲ in group B was significantly higher than that in group E (P<0.05), but the mRNA relative expression of CTGF was not significantly different from that in group E (P>0.05); after neutralizing antibody was added, the mRNA relative expression of collagen type Ⅰ and collagen type Ⅲ in group D was inhibited and was significantly lower than that in group B, but still significantly higher than that in group E (P<0.05); there was no significant difference in the mRNA relative expression of CTGF between group D and groups B, E (P>0.05).ConclusionTGF-β1 can promote CTGF, collagen typeⅠ, collagen type Ⅲ gene level and protein expression in ligamentum flavum cells, and TGF-β1 can synergistically promote proliferation of ligamentum flavum cells through CTGF.
ObjectiveTo explore the expression of periostin in bronchoalveolar lavage fluid (BALF) of patients with dermatomyositis-related interstitial lung disease (DM-ILD) and rheumatoid arthritis-related interstitial lung disease (RA-ILD).MethodsA total of 44 patients with DM-ILD and 28 patients with RA-ILD were underwent bronchoalveolar lavage. Cells in BALF were collected and analyzed by absolute different cell counts. The level of periostin and Krebs von den Lungen-6 (KL-6) were tested by enzyme linked immunosorbent assay. Results of high resolution CT of patients were scored. Thirty patients without interstitial lung disease (ILD) served as a control group.ResultsLevels of periostin and KL-6 were significantly increased in BALF of patients with DM-ILD and RA-ILD compared with control group (all P<0.05). Levels of periostin were positively correlated with lymphocyte counts and levels of KL-6 in BALF (allP<0.05). Furthermore, levels of periostin were significantly correlated with high resolution CT scores (P<0.05).ConclusionsLevels of periostin are increased in patients with DM-ILD and RA-ILD. Periostin might be served as an indicator of CTD-ILD.
ObjectiveTo investigate the role of transforming growth factor β1(TGF-β1) and connective tissue growth factor (CTGF) in pathogenesis and progression of human intervertebral disc degeneration by detecting the expressions of these two factors in different degrees of degenerative discs. MethodsThe lumbar intervertebral discs were collected from 33 patients with lumbar disc herniation and 12 patients with lumbar vertebral fracture between November 2012 and April 2013.All samples were observed under the microscope after HE staining,and then were divided into different subgroups according to the degenerative degree.The expressions of TGF-β1 and CTGF were detected by Western blot. ResultsAccording to the pathological features,10 discs were defined as normal discs,10 as mild degenerative discs,9 as moderate degenerative discs,and 16 as severe degenerative discs.The histological observation showed that rounded nucleus pulposus cells with similar size evenly distributed in the cartilage-like matrix,and no hyperplastic collagenous fiber was seen in normal discs;mild degenerative discs characterized by slightly larger nucleus pulposus cells in the matrix,but cells did not decrease,a small quantity of inflammatory cells infiltrated in the matrix,hyperplasia of collagenous fiber was not seen;most of the nucleus pulposus cells became bigger,some showed a bulb form,the number of nucleus pulposus cells was significantly reduced,low grade hyperplasia of collagenous fiber emerged in the matrix,new vessels and inflammatory cells were both found in some specific areas of discs in moderate degenerative discs;there was no nucleus pulposus cells in the matrix of severe degenerative discs,the hyperplasia of collagenous fiber was obvious.The relative expression of TGF-β1 in 3 degeneration discs was significantly higher than that in normal discs (P<0.05),and the expression of TGF-β1 was significantly higher in severe degenerative discs than in moderate and mild degenerative discs (P<0.05),but no significant difference between moderate and mild degenerative discs (P>0.05).The relative expression of CTGF in moderate and severe degeneration discs was significantly higher than that in normal discs (P<0.05);and the expression of CTGF in mild degenerative discs was higher than that in normal discs,but there was no significant difference (P>0.05);and significant difference in CTGF expression was found among 3 degeneration discs (P<0.05). ConclusionThe expressions of TGF-β1 and CTGF are closely related to the degree of human lumbar disc degeneration,these two factors may play an important role in promoting lumbar intervertebral disc degeneration.
Objective To investigate the role of IFN-γ in suppressing bleomycin-induced pulmonary fibrosis in rats.Methods Seventy-five SD rats were randomly divided into five groups (15 rats in each group)，ie.a normal group，a bleomycin-induced pulmonary fibrosis model group，a dexamethasone-treated group，a high-dose IFN-γ-treated group (150 000 U/kg) and a low-dose IFN-γ-treated group (50 000 U/kg).Five rats in each group were randomly killed in 7th day，14th day and 28th day after relative treatment respectively，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 method were adapted to determine protein levels of TGF-β1，CTGF，type Ⅰcollagen and type Ⅲ collagen in pulmonary tissues.Results Histopathological study showed that treatment with either dexamethasone or IFN-γ (both high dose and low dose) remarkably meliorated the extent of alveolus inflammation and suppressed pulmonary fibrosis (compared with model group，all Plt;0.05).Histoimmunochemical study suggested that both dexamethasone and IFN-γ could inhibit the expression of TGF-β1，CTGF，type Ⅰand type Ⅲ collagen (compared with model group，all Plt;0.05)，and the suppression of TGF-β1，type Ⅰand type Ⅲ collagen expression was more obvious in high-dose IFN-γ-treated group than those in low-dose group (Plt;0.05).Conclusions INF-γ possesses apparent anti-fibrosis effect that is similar to dexamethasone but with less side effect.Such effect may resulted from reduced production of type Ⅰand type Ⅲ collagen through expression inhibition of cytokines such as TGF-β1 and CTGF.
Objective To explore the clinical effect and safety of sildenafil combined with bosentan in the treatment of connective tissue disease associated moderate-severe pulmonary arterial hypertension (CTD-MS-PAH ). Methods Seventy-six patients with CTD-MS-PAH during January 2013 to January 2017 were collected and divided into group A (41 cases) and group B (35 cases) using a stratified random sampling approach. The patients in group A received 25 mg sildenafil tablet therapy, three times a day. The patients group B received 25 mg sildenafil and 62.5 mg bosentan tablet therapy, twice a day. Both groups were treated for 12 weeks. Before and after the trial, all patients undertook six-minute walk test. Meanwhile the Borg dyspnea index score, the pulmonary artery systolic pressure (PASP), right ventricular diameter (RVD), B-type natriuretic peptide (BNP), the partial pressure of oxygen in artery (PaO2), blood pressure, heart rate, liver and kidney function were all measured. Results After the therapy, six-minute walking distance increased, Borg dyspnea index score decreased, PASP, RVD and plasma BNP decreased, and PaO2 increased in both groups (all P<0.05), but group B was superior to group A (allP<0.05). There were no significant differences in blood pressure, heart rate, liver or kidney function compared with those before the treatment in both groups (allP>0.05). Conclusion Sildenafil combined with bosentan can significantly decrease the level of pulmonary arterial pressure and effectively improve the cardiopulmonary function in CTD-MS-PAH patients with good safety.
Objective To investigate the effect of cryopreservation (CP) on the expression of connective tissue growth factor (CTGF) in the renal tubular epithel ial cells. Methods A total of 40 male Wistar rats (weighing 230-250 g) were used in this study. En bloc removal with in situ cooling both kidneys and hypertonic citrate adenine preservation solution were adopted. The rat kidney was be preserved 0, 12, 24, 36 and 48 hours at 0-4℃ (n=8), respectively. The expression of CTGF of renal tubularepithel ial cells was detected by using immunohistochemistry and in situ hybridization analysis. Results The expression of CTGF was less in CP 0 hour group and CP 12 hours group, the positive unit (PU) values of CTGF protein were 5.91 ± 2.30 and 5.57 ± 2.40 (P gt; 0.05), respectively, and the PU values of CTGF mRNA were 6.24 ± 2.79 and 6.51 ± 2.43 (P gt; 0.05), respectively. The PU values of CTGF protein increased at CP 24 hours group (10.25 ± 2.92), CP 36 hours group (14.31 ± 2.83) and CP 48 hours group (18.11 ± 3.94, P lt; 0.05), respectively, and the PU values of CTGF mRNA increased at CP 24 hours group (15.24 ± 3.95), CP 36 hours group (19.20 ± 4.73) and CP 48 hours group (23.09 ± 4.40, P lt; 0.05), respectively; showing significant differences when compared with CP 0 hour group and CP 12 hours group (P lt; 0.05). Conclusion CTGF expression may increase with severe cold ischemia injury, and might play an important role in regeneration and repair of renal tubular epithel ial cell injury.
ObjectiveTo explore the therapeutic effects of spleen aminopeptide on connective tissue disease-related interstitial lung disease (CTD-ILD) and its mechanism for anti-fibrosis. MethodsNinety patients with CTD-ILD admitted between February 2014 and May 2015 were recruited in the study. The CTD-ILD patients were randomly divided into group A (conventional therapy alone) and group B (conventional therapy plus spleen aminopeptide). Peripheral blood collected from CTD-ILD patients were subjected to performance of flow cytometric analysis and cytokine/chemokines profiling by liquid Chip and ELISA assay. Pulmonary function test and high resolution CT (HRCT) scan were performed before and after the treatments for 12 weeks. Human cytomegalovirus (HCMV) DNA in the patients' blood was tested by Q-PCR. ResultsSignificantly improved lung function and HRCT score were observed in group B, but not in group A. The levels of Treg and IFN-γ were significantly increased in group B, compared with those in group A where markedly increased IL-6, IL-10 and IL-17 were detected (P < 0.05). There was higher virus negative reversal rate in group B than that in group A (P < 0.05). ConclusionSpleen aminopeptid can effectively regulate deregulated immune microenvironment in CTD-ILD patients and inhibit HCMV replication, thereby block pulmonary fibrotic development.