ObjectiveTo study the serum transforming growth factorβ1 (TGF-β1) and interleukin-23 (IL-23) expression in the patients with chronic gastric ulcer or gastric cancer, and to investigate the clinical value of TGF-β1 and IL-23 on the prevention and treatment of gastric cancer. MethodsThe serum levels of TGF-β1 and IL-23 in cancer group (83 cases), gastric ulcer group (184 cases), and control group (58 cases) were detected by using ELISA assay method. The difference of serum TGF-β1 and IL-23 levels in patients with gastric cancer with different pathological parameters were compared. ResultsThe serum levels of TGF-β1〔(15.96±3.92) ng/mL〕and IL-23〔(645.25±234.18) ng/mL〕in gastric cancer group were higher than those of the gastric ulcer group〔(10.10±3.58) ng/mL, (496.10±108.32) ng/mL〕and normal control group〔(9.87±2.86) ng/mL, (372.75±89.27) ng/mL〕, the difference were statistically significant (P < 0.05). The levels of serum TGF-β1 in gastric cancer patients of stageⅠ-Ⅱ, ⅢandⅣwere successively increased, and the differences were statistically significant (P < 0.05). The levels of serum TGF-β1 in poorly differentiated gastric cancer or with lymph node metastasis patients were higher than those in high-middle differentiation or without lymph node metastasis patients, the difference were statistically significant (P < 0.05). There were no significant difference in the levels of serum TGF-β1 between different tumor diameter and different location (P > 0.05). The level of serum IL-23 in patients with stageⅠ-Ⅱwas higher than that in stageⅢandⅣ, the difference was statistically significant (P < 0.05). Ther were no significant difference in serum IL-23 levels between the different degree of differentiation, lymph node metastasis or not, different tumor diameter and different location of the tumor (P > 0.05). ConclusionTGF-β1 and IL-23 have important reference value in judging the stage and malignancy degree of gastric cancer.
Objective To study the pathology and possible mechanism of experimental hydrochloric acid（HCl） inhalation-indued pulmonary fibrosis in rats.Methods 120 male SD rats were randomly divided into a nomal control group，a bleomycin group，a high dose HCl group，a middle dose HCl group and a low dose HCl group.The bleomycin group was intratracheally injected with bleomycin once to induce pulmonary fibrosis.The three HCl groups were intratracheally injected with HCl once per week.The control group was given saline by the same way.Six rats of each group were randomly sacrificed on day 7，14，28 and 42 respectively.The histological changes of lung tissue were studied by HE and Masson’s trichrome staining.Hydroxyproline level in lung tissue was measured by digestion method.Protein and mRNA expression of transforming growth factor-β1（TGF-β1） were assayed by immunohistochemistry and RT-PCR respectively.Results Alveolitis in three HCl groups was significantl compared to control group，most severe at the second week，then remained at a high level which was equivalent to or exceeded the level of the bleomysin group after 28 days.Pulmonary fibrosis in three HCl groups was also significantly more severe than that in the control group，but milder than that in the bleomysin group.The high-dose and middle-dose HCl groups were not significantly different from the bleomysin group on day 42.There was no difference between three HCl groups in the earlier period，but the high-dose HCl group has a significantly difference from low-dose group on day 42.The content of hydroxyproline in high-dose and middle-dose HCl groups was also significantly higher than that in the control group.On day 42 hydroxyproline content in high-dose HCl dose rather middle –or low dose group was similiar with the level of bleomysin group.Content of TGF-β1 mRNA in three HCl groups was comparable to the level of bleomysin group on day 28 and exceeded on day 42.The expression of TGF-β1 in three HCl groups was not significantly different from the bleomysin group on day 42.Conclusion Experimental acid aspiration might contribute to pulmonary fibrosis in rats.Acid induced alveolar epithelial cell damage，abnormal proliferation and repair and fibrosis could be involved..
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.
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 investigate the effects of bursopentin ( BP5) on expression of extracellular matrix in human lung fibroblasts ( HLFs) and its mechanism.Methods HLFs were cultured in vitro and divided into five groups. The cells in the control group were cultured in DMEMwithout TGF-β1 or BP5. The cells in TGB-β1 treatment group were cultured in DMEMcontaining 5 μg/L TGF-β1 . While in three TGF-β1 + BP5 treatment groups, the cells were cultured in DMEM containing 5 μg/L TGF-β1 and simultaneously intervened with BP5 at three different concentrations ( 2. 5 μg/mL, 5 μg/mL, and 10 μg/mL respectively) . The expression of α-SMA was detected using a fluorescent-labeling strategy. The expressions of Collagen-Ⅰ, p-Smad2/3, p-Smad3, and Smad7 proteins were measured by Western blot. Results The cells in the TGF-β1 treatment group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In the TGF-β1 treatment group, the expressions of collagen-Ⅰ( 1. 402 ±0. 158 vs. 0. 605 ±0. 367) , p-Smad2/3 ( 1. 457 ±0. 111 vs. 0. 815 ±0. 039) , and p-Smad3 ( 1. 320 ±0. 147 vs. 0. 623 ±0. 128) increased with statistical significance ( P lt; 0. 01) . Meanwhile the expression of Smad7 reduced ( 0. 614 ±0. 107 vs. 0. 865 ±0. 063, P lt;0. 05) . But in the TGF-β1 + BP5 treatment groups, over-expressions of collagen-Ⅰ, α-SMA, p-Smad2 and p-Smad3 induced by TGF-β1 were obviously inhibited by BP5, especially at the BP5 concentration of 10 μg/mL ( collagen-Ⅰ: 0. 718 ±0. 049 vs. 1. 402 ±0. 158; p-Smad2 /3: 0. 696 ±0. 031 vs. 1. 457 ±0. 111; p-Smad3: 0. 766 ±0. 006 vs. 1. 320 ±0. 147; all P lt; 0. 01) . Otherwise, the up-regulation of Smad7 ( 1. 237 ±0. 173 vs. 0. 614 ±0. 107) was found.Conclusions Bursopentin can reduce the expressions of collagen-Ⅰ and α-SMA protein of fibroblast stimulated by TGF-β1 , maybe through inhibiting TGF-β1 /Smads transduction pathway. It is suggested that bursopentin may have intervention effect on pulmonary fibrosis.
Abstract： Objectives To determine the atrial expression of the collagen Ⅰ, collagen Ⅲ and the transforming growth factorbeta 1 (TGF-β1) in patients with rheumatic heart disease (RHD) and permanent atrial fibrillation(PAF) and to investigate the relationship between the extent of atrial fibrosis and the effectiveness of radiofrequency maze procedure in patients with RHD and PAF. Methods A total of 40 patients with RHD and PAF (≥6 months) who underwent a radiofrequency maze procedure with concomitant valvular surgery were collected for the experimental group. We acquired 100 mg of the left auricle tissue in each patient and followed up these patients after 3, 6 months of [CM(158mm]surgery. Then we assigned these patients to nonAF group and persistent AF group according to the results of the 6month followup. Another 10 patients with RHD and sinus rhythm(SR) who underwent valvular surgery alone were assigned to SR group and their left auricle tissue was also obtained. In order to determine the extent of atrial fibrosis, we observed the amount of collagen volume fraction Ⅰ，Ⅲ(CVF-Ⅰ,CVF-Ⅲ) by semiquantitative analysis with picrosirius red staining method. Using the β actin protein as the endogenous reference gene, we detected the expressions of TGF-β1 mRNA by semiquantitative reverse transcriptionpolymerase chain reaction(RT-PCR) technique. Results Each group has the same clinical baseline. At 6month follow-up, 28 among the 40 patients were categorized into the nonAF group and 12 into the AF group. (1) Patients in the nonAF group and the AF group had higher mRNA expressions of TGF-β1, CVF-Ⅰ and CVF-Ⅰ/CVF-Ⅲ compared with the SR group (F=6.487, P=0.003; F=3.711, P=0.032; F=3.697, P=0.032). The AF group had higher mRNA expressions of TGF-β1, CVF-Ⅰ and CVF-Ⅰ/CVF-Ⅲ than the nonAF group (t=4.372, P=0.043; t=4.603, P=0.038; t=4.776, P=0.035). But the CVFⅢ had no significant differences among the three groups (P＞0.05). (2) The patients whose left atrial function recovered after Maze procedure had lower mRNA expression than those patients whose left atrial function did not recover in the nonAF group (t=5.570, P=0.027). (3) The TGF-β1 mRNA expression has a positive correlation with both the contents of CVF-Ⅰ and left atrial diameter (r=0.786, Plt;0.05; r=0.858, Plt;0.05). Multiple logistic regression analysis revealed that the mRNA expression levels of TGF-β1, CVF-Ⅰ and left atrial diameter were independently associated with the postoperative persistence of atrial fibrillation. Conclusion The extent of atrial fibrosis in patients with RHD and PAF may be related to the sinus rhythm restoration and maintenance after AF surgical radiofrequency ablation and the resumption of atrial function.
Objective To investigate the expression of transforminggrowth factor β1(TGF-β1) and insulin-like growth factorⅠ(IGF-Ⅰ)in new bone after low frequency micromovement. Methods Fifteen female sheep from Shandong province were involved in the study and their bilateral tibias transversely osteotomized in the middle shafts with a defect of 2 mm.The hind limbs were fixed with unilateral external fixators connected to a controlled micromovement device. Ten days after osteotomy, one hind limb of each sheep randomlywas selected to perform micromovement at an amplitude of 0.25 mm and a frequency of 1 Hertz, 30 min a day for 4 weeks ( micromovement group). The other hindlimb served as the control group. Five sheep were sacrificed at 3,4 and 6 weeks after osteotomy, respectively, and specimens were harvested for detecting the expression of TGF-β1 and IGF-Ⅰby immunohistochemistry and RT-PCR. Results Immunohistochemistry: In the third postoperative week in the micromovement group, the expression of TGF-β1 was detected in different areas of new chondrocytes at the margin of callus, mainly in proliferating area, and IGF-Ⅰexpressed in osteoblasts at the margin of endochondral ossification area, calcified and mature chondrocytes and osteocytes. There was seldom expression ofIGF-Ⅰ and little expression of TGF-β1 in the corresponding area in the control one. In the 4th postoperative week in the micromovement group, theexpression of TGF-β1 diminished gradually with the mature of new bone and be located in extracellular matrix and osteoblasts around ossified areas; The expression ofIGF-Ⅰ reached the peak and be located mainly in osteoblasts of new bone surface, maturing osteocytes and calcifing osteoid. But there was little expression of them in the control group. In the sixth postoperative week in the micromovement group, there was a little expression of IGF-Ⅰ expression but little expression of TGF-β1; there was nearly no expression of them in the control group. In the micromovement group, the absorbance values of TGF-β1 at 3 and 4 weeksand of IGF-Ⅰat 3, 4 and 6 weeks were significantlyhigher than those in control group（P＜0.05）. RTPCR: In the third and fourth postoperative weeks in the micromovement group, there was higher expression of mRNA of TGF-β1 and TGF-I than those in control group; in the sixth postoperative week, the expression diminished gradually, but was higher than that in control group. The absorbance values of TGF-β1 at 3 and 4 weeks and IGF-Ⅰat 3, 4 and 6weeks were significantly higher than those of control group（P＜0.05）. Conclusion Low frequency and controlled micromovement in the early stage of the fracture healing can promote the expression of TGF-β1 and IGF-Ⅰ.They worked together to regulate the process of the endochondral ossification, while in the late stage the differentiation of osteocytes and mineralization of osteoid were regulated mainly by IGF-Ⅰ, which played an important role in regulating the cell biological behavior during micromovement.
ObjectiveTo investigate the effects of liver X receptor agonist, T0901317, on the proliferation, migration and hydroxyproline production of human embryonic lung fibroblasts (HELF). MethodsHELF cells were devided into a control group, a growth factor group, a T0901317 group and three growth factor+T0901317 groups. The cells of the control group were treated with Dulbecco's modified Eagle medium. The cells of T0901317 group were treated with 1.00 μmol/L T0901317. The growth factor+T0901317 groups were incubated with different doses of T0901317 (0.25 μmol/L, 0.50 μmol/L and 1.00 μmol/L) for 2 h. Then the cells of the growth factor+T0901317 groups and the growth factor group were incubated with basic fibroblast growth factor and transforming growth factor-β1 for 24 h. The proliferation, migration and collagen production of HELF were determined by cell counting kit-8 method, transwell chamber, and hydroxyproline method. ResultsCompared with the control group, T0901317 had no effect on the proliferation, migration and hydroxyproline production of HELF. Growth factors could promote the proliferation, migration and hydroxyproline production of HELF significantly. T0901317 could inhibit those effects of growth factors with a dosage-dependent manner. ConclusionT0901317 may inhibit the proliferation, migration and hydroxyproline production of HELF induced by growth factors.
Objective To construct the recombinant adenovirus bearing human transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) genes, and investigate its co-expression in the marrow stromalstemcells (MSCs) and bioactivity effect. Methods Using the replication defective adenovirus AdEasy as a carrier, MSCs were infected by the high-titer-level recombinant adenovirus taking TGF-β1 and BMP-7 genes. Immunocytochemistry, in situ hybridization，reverse transcription-polymerase chain reaction (RT-PCR), and hexuronic acid level test were used to detect the coexpression of the exogenous genes and to analyze their effect transfection on directive differentiation of MSCs. Results The immunocytochemistry staining showed that the brown coarse grains were situated in the cytoplasm of the most MSCs 72 h after infection. Procollagen ⅡmRNA in the cells was detected by the in situ hybridization, and the content of hexuronic acid in the culture mediumwas significantly increased 10 days after infection compared with the level before infecton (Plt;0.01). Conclusion The recombinant adenovirus bearing human TGF-β1 and BMP-7 genes can be constructed, and the exogenous gene can be coexpressed in MSCs, which may offer a novel approach to thelocal combination gene therapy for repairing joint cartilage defects.
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.