Transforming growth factor-β1 and vascular endothelial growth factor levels in senile acute myeloid leukemia and correlation with prognosis

BACKGROUND Acute myeloid leukemia(AML)is a disease in which immature hematopoietic cells accumulate in the bone marrow and continuously expand,inhibiting hematopoiesis.The treatment and prognosis of this disease have always been unsatisfactory.AIM To investigate the correlation between vascular endothelial growth factor(VEGF)and transforming growth factor-β1(TGFβ1)expression and prognosis in older adults with AML.METHODS This study enrolled 80 patients with AML(AML group),including 36 with complete response(AML-CR),23 with partial response(AML-PR),and 21 with no response(AML-NR).The expression levels of VEGF and TGFβ1 were detected by reverse transcription polymerase chain reaction in bone marrow mononuclear cells isolated from 56 healthy controls.Kaplan-Meier analysis was performed to assess overall survival(OS)and progression-or disease-free survival(DFS).Prognostic risk factors were analyzed using a Cox proportional hazards model.RESULTS The AML group showed a VEGF level of 2.68±0.16.VEGF expression was lower in patients with AML-CR than those with AML-PR or AML-NR(P<0.05).TGFβ1 expression in the AML group was 0.33±0.05.Patients with AML-CR showed a higher TGFβ1 expression than those with AML-PR or AML-NR(P<0.05).VEGF and TGFβ1 expression in patients with AML was significantly correlated with the counts of leukocytes,platelets,hemoglobin,and peripheral blood immature cells(P<0.05);Kaplan-Meier survival analysis revealed that patients with high TGFβ1 expression had better OS and DFS than those with low TGFβ1 expression(P<0.05),whereas patients with low VEGF levels showed better OS and DFS than those with high VEGF levels(P<0.05).VEGF,TGFβ1,and platelet count were identified by the Cox proportional hazards model as independent risk factors for OS(P<0.05),while VEGF,TGFβ1,and white blood cell count were independent risk factors for DFS(P<0.05).CONCLUSION Decreased VEGF expression and increased TGFβ1 expression in patients with AML provide valuable references for determining and individualizing clinical treatment strategies.

Dihydroergotamine ameliorates liver fibrosis by targeting transforming growth factor β type Ⅱ receptor

BACKGROUND The transforming growth factor β(TGFβ) signaling pathway plays a crucial role in the development of liver fibrosis by activating TGFβ type Ⅱ receptor(TGFβR2), followed by the recruitment of TGFβR1 finally triggering downstream signaling pathway.AIM To find drugs targeting TGFβR2 that inhibit TGFβR1/TGFβR2 complex formation, theoretically inhibit TGFβ signaling pathway, and thereby ameliorate liver fibrosis.METHODS Food and Drug Administration-approved drugs were screened for binding affinity with TGFβR2 by virtual molecular docking. We identified 6 candidates and further explored their potential by Cell Counting Kit-8(CCK-8) cell cytotoxic experiment to validate toxicity and titrated the best cellular working concentrations. Next, we further demonstrated the detailed molecular working mechanisms using mutagenesis analysis. Finally, we used a mouse model to investigate its potential anti-liver fibrosis effect.RESULTS We identified 6 drug candidates. Among these 6 drugs, dihydroergotamine(DHE) shows great ability in reducing fibrotic gene expressions such as collagen, p-SMAD3, and α-SMA in TGFβ induced cellular model of liver fibrosis in LX-2 cells. Furthermore, we demonstrated that DHE binds to TGFβR2. Moreover, mutation of Leu27, Phe30, Thr51, Ser52, Ile53, and Glu55 of TGFβR2 disrupted the binding of TGFβR2 with DHE. In addition, DHE significantly improved liver fibrosis, as evidenced by Masson’s trichrome staining of liver sections. This is further supported by the width and the velocity of the portal vein, and serum markers of liver function. In line with those observations, DHE also decreased macrophages infiltration and extracellular matrix deposition in the liver.CONCLUSION DHE alleviates liver fibrosis by binding to TGFβR2 thereby suppressing TGFβ signaling pathway. We show here that as far as drug repurposing, DHE has great potential to treat liver fibrosis.

Plasma Levels of Transforming Growth Factor-Beta 1 in Women with Pelvic Organ Prolapse

Objective: In women with pelvic organ prolapse (POP), decreased expression of transforming growth factor-beta 1 (TGF-β1) has been shown in POP tissues. However, no studies have evaluated plasma TGF-β1 levels in patients with POP, so it is unknown whether they are also changed or not. Therefore, we compared plasma TGF-β1 levels in women with and without POP. Methods: Participants were 49 women with POP and 23 healthy control women. All participants were postmenopausal. We measured plasma TGF-β1 and compared data between patients with POP and controls, and between patients with uterine prolapse (UP, n = 19) and those with a cystocele (CC, n = 30). In addition, in patients, we assessed the POP quantification system (POP-Q) stage. Results: Plasma TGF-β1 levels were significantly lower in patients than in healthy controls. POP-Q stage was not significantly different between the UP and CC subgroups, but POP-Q stage IV was diagnosed in 63% of patients with UP and 7% of those with CC. Plasma TGF-β1 levels were significantly lower in the CC subgroup than in the UP subgroup. Conclusion: Plasma TGF-β1 is decreased in POP. It remains unclear whether the lower levels indicate a reduction in systemic TGF-β1 activity, but they can be assumed to reflect reduced TGF-β1 expression in POP tissues.

ROLE OF TRANSFORMING GROWTH FACTOR β(TGF-β)IN REPAIRING OF BONE DEFECTS

TGF-β is a multifunctional cytokine that regulates many aspects of cellular function, including periosteal mesenchymal cell proliferation, differentiation. This experiment is to study its effects on bone defect repair. A rabbit radial bone defect model was used to evaluate the effect of TGF-β, which was extracted and purified from bovine blood platelets, on the healing of a large segmental osteoperiosteal defect. A 1. 5-centimeter segmental defect was created in the mid-upper part of the radial shaft of adult rabbits. The defect was filled with implant containing TGF-β that consisted of carrier and bovine TGF-β. Limbs served as controls received carrier alone. The defectswere examined radiographically and histologically at 4, 8,12 , 16 and 20 weeks after implantation. The results showed that in TGF-β implant group . the defect areas at 12 weeks post operation were bridged by uniform new bone and the cut ends of cortex could not be seen;while in control group, the defects remained clear. Only a small amount of new bone formed as a cap on the cut bone ends. In the experimental group, new lamellar and woven bone formed in continuity with the cut ends of the cortex. An early medullar canal appears to be forming and contained normal-appearancing marrow elements; while the control group displayed entirely fibrous tissue within the defect site. Remnants of the cancellous bone carrier were observed in the control specimen. These data demonstrate that exogenous TGF-β initiate osteogenesis and stimulate the bone defects repair in animal model.

Downregulation of transforming growth factor-<i>β</i>(TGF-<i>β</i>) and vascular endothelial growth factor (VEGF) in ehrlich ascites carcinoma-bearing mice using stearic acid-grafted carboxymethyl chitosan (SA-CMC)

The present study was conducted to investigate the use of stearic acid-grafted carboxymethyl chitosan(SA-CMC) as a downregulator for trans- forming growth factor-β (TGF- β) and vascular endothelial growth factor (VEGF) in Ehrlich ascites carcinoma (EAC)-bearing mice. The antitumor effect of stearic acid-grafted carboxymethyl chitosan was assessed by the estimation of TGF- β and VEGF in serum in addition to the estimation of tumor volume, median survival time (MST), percentage of increase in life span (ILS%) as well as the contents of total lipid, DNA and RNA in liver tissues. Hematological profiles (hemoglobin, red blood cells, and platelets) were also assessed. In addition, liver function tests and the redox status were estimated. TGF- β, VEGF, DNA, RNA, and malondialdehyde (MDA) levels, in addition to serum alanine transaminase (ALT) and gamma glutamyl transferase (GGT) activities as well as total white blood cells counts and tumor volume were all highly significantly increased (P < 0.001) in untreated EAC-bearing mice compared to controls. However, hematological profiles, total lipid in liver tissues and serum albumin were highly decreased in EAC-bearing mice compared to controls. All these parameters were restored to the normal levels in SA-CMC treated EAC-bearing mice com- pared to the untreated EAC-bearing mice. It is thus concluded that stearic acid-grafted carboxymethyl chitosan has a remarkable antitumor activity against EAC in Swiss albino mice through downregulation of TGF-β and VEGF.

Targeting Transforming Growth Factor-<i>β</i>(TGF-<i>β</i>) in Cancer and Non-Neoplastic Diseases

Transforming growth factor-β?(TGF-β) superfamily is a key player in the regulation of a wide variety of physiological processes from development to pathogenesis. Since the discovery of the prototypic member, TGF-β, almost three decades ago, there have been tremendous advances in our understanding of its complex biology. TGF-β?misregulation has been implicated in the pathogenesis of a variety of diseases, including cancer with a direct role in facilitating metastasis, fibrosis and inflammation. Consequently, TGF-β?is currently explored as a prognostic candidate biomarker of tumor invasiveness and metastasis;and it offers an attractive target for cancer therapy. Several anti-TGF-β?approaches, such as TGF-β?antibodies, antisense oligonucleotides and small molecules inhibitors of TGF-β?type 1 receptor kinase, have shown great promise in the preclinical studies. Here, we consider why the TGF-βsignaling pathway is a drug target, the potential clinical applications of TGF-β?inhibition, the issues arising with anti-TGF-β?therapy and how these might be adopted using personalized approaches with a special care for patient selection and timing of therapy so that we may bring forward all the potentials of targeting this pathway for therapeutic uses in both cancer, preferentially in combination therapy, and non-neoplastic diseases.

Effect of intestinal ischemia-reperfusion on expressions of endogenous basic fibroblast growth factor and transforming growth factor β in lung and its relation with lung repair

AIM To study the changes of endogenoustransforming growth factor β(TGFβ)and basicfibroblast growth factor(bFGF)in lung followingintestinal ischemia and reperfusion injury andtheir effects on lung injury and repair.METHODS Sixty Wistar rats were divided intofive groups,which underwent sham-operation,ischemia(45 minutes),and reperfusion(6,24and 48 hours,respectively)after ischemia(45minutes).Immunohistochemical method wasused to observe the localization and amounts ofboth growth factors.RESULTS Positive signals of both growthfactors could be found in normal lung,mainly inalveolar cells and endothelial cells of vein.Afterischemia and reperfusion insult,expressions ofboth growth factors were increased and theiramounts at 6 hours were larger than those ofnormal control or of 24 and 48 hours after insult.CONCLUSION The endogenous bFGF and TGF βexpression appears to be up-regulated in thelung following intestinal ischemia andreperfusion,suggesting that both growth factorsmay be involved in the process of lung injury andrepair.

Transforming growth factor-β and fibrosis

Transforming growth factor-β （TGF-β）, a prototype of multifunctional cytokine, is a key regulator of extracellular matrix （ECM） assembly and remodeling. Specifically, TGF-β isoforms have the ability to induce the expression of ECM proteins in mesenchymal cells, and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. Elevated TGF-β expression in affected organs, and subsequent deregulation of TGF-β functions, correlates with the abnormal connective tissue deposition observed during the onset of fibrotic diseases. During the last few years, tremendous progress has been made in the understanding of the molecular aspects of intracellular signaling downstream of the TGF-β receptors. In particular, Smad proteins, TGF-β receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, have been studied extensively. The role of Smad3 in the transcriptional regulation of type I collagen gene expression and in the development of fibrosis, demonstrated both/n vitro and in animal models with a targeted deletion of Smad3, is of critical importance because it may lead to novel therapeutic strategies against these diseases. This review focuses on the mechanisms underlying Smad modulation of fibrillar collagen expression and how it relates to fibrotic processes.

Vascular endothelial growth factor A, secreted in response to transforming growth factor-β1 under hypoxic conditions, induces autocrine effects on migration of prostate cancer cells

Hypoxia and transforming growth factor-β1 （TGF-β1） increase vascular endothelial growth factor A （VEGFA） expression in a number of malignancies. This effect of hypoxia and TGF-β1 might be responsible for tumor progression and metastasis of advanced prostate cancer. In the present study, TGF-β1 was shown to induce VEGFA165 secretion from both normal cell lines （HPV7 and RWPE1） and prostate cancer cell lines （DU 145 and PC3）. Conversely, hypoxia-stimulated VEGFA165 secretion was observed only in prostate cancer cell lines. Hypoxia induced TGF-β1 expression in PC3 prostate cancer cells, and the TGF-β1 type I receptor （ALK5） kinase inhibitor partially blocked hypoxia-mediated VEGFA16s secretion. This effect of hypoxia provides a novel mechanism to increase VEGFA expression in prostate cancer cells. Although autocrine signaling of VEGFA has been implicated in prostate cancer progression and metastasis, the associated mechanism is poorly characterized. VEGFA activity is mediated via VEGF receptor （VEGFR） 1 （Fit-l） and 2 （FIk-I/KDR）. Whereas VEGFR-1 mRNA was detected in normal prostate epithelial cells, VEGFR-2 mRNA and VEGFR protein were expressed only in PC3 cells. VEGFA165 treatment induced phosphorylation of extracellular signal-regulated kinase 1/2 （ERKI/2） in PC3 cells but not in HPV7 cells, suggesting that the autocrine function of VEGFA may be uniquely associated with prostate cancer. Activation of VEGFR-2 by VEGFA165 was shown to enhance migration of PC3 cells. A similar effect was also observed with endogenous VEGFA induced by TGF-β1 and hypoxia. These findings illustrate that an autocrine loop of VEGFA via VEGFR-2 is critical for the tumorigenic effects of TGF-β1 and hypoxia on metastatic prostate cancers.

Stage-specific localization of transforming growth factor β1 and β3 and their receptors during spermatogenesis in men

Aim: To investigate the stage-specific localization of transforming growth factor (TGF) β1 and β3 during spermatogenesis in adult human testis. Methods: The localization of TGFβ1 and β3 was investigated by immunohis tochemical staining method employing specific polyclonal antibodies. Results: Both TGFβ1 and β3 and their recep tors were preponderant in the Leydig celis. TGFβ1 could not be detected in the seminiferous tubules. TGFβ3 and TGFβ-Receptor (R) I were mainly seen in the elongated spermatids, while TGFβ-RⅡ in the pachytene spermatocytes and weak in the spermatogonia, spermatids and Sertoli celis. Only TGFβ-RⅡ was detected in the Sertoli celis. TGFβ3, TGFβ-RⅠ and TGFβ-RⅡ showed a staining pattern dependent upon the stages of the seminiferous epithelium cycle. Conclusion: TGFβ isoforms and their receptors are present in the somatic and germ celis of the adult human testis, suggesting their involvement in the regulation of spermatogenesis.

Effect of matrine on transforming growth factor β1 and hepatocyte growth factor in rat liver fibrosis model

Objective:To observe the preventive and control effect of matrine on transforming growth factor(TCF- β1) and hepatocyte.growth factor(HCF) of liver fibrosis tissue in rals.Methods:A total of48 SD rats were randomly divided into A,B,C,D groups with 12 in each,group A as the normal control group and groups B.C,D as liver fibrosis models using composite modulus method with carbon tetrachloride(CCL_4).Group B was the model group,group C adopted γ— interferon lavage therapy in the second day of modeling,and group D adopted matrine lavage treatment,at 4 and8 weeks after treatment.Six rats were executed for detection of TGF- β1 and HGF,liver tissue histology and comparison fibrosis degree changes of rat liver tissue between groups.Results:Croups B,C,D showed a more significantly increased TCF- β1 at each time point compared with group A(P<0.05);Group B showed a more significantly increased TGF- β1 than groups C and D at weeks 4 and 8(P<0.05);group D showed a lowest level of TGF-β1,followed by groups C and B.HGF of group B decreased more significantly than A group at weeks 4 and 8(P<0.05);HGF of groups C and D was significantly elevated at 4 and 8 weeks than groups A and B(P<0.05),in which the group D showed the highest level of HGF.According to tissue histologic observation,rat liver tissue structure of group A was clear and normal,tissue structure of group B was destroyed with obvious fibrous tissue hyperplasia and fatty change of hepatic cells;groups C and D showed a slighter liver tissue damage,cell necrosis and connective tissue hyperplasia in collect abbacy than group B with a trend of obvious improvement.Conclusions:Matrine can reduce TGF- β1expression and enhance the activity of HGF,so as to realize the inhibition effect on liver fibrosis in rats.

Expression of transforming growth factors in hepatocellular carcinoma and its relations with clinicopathological parameters and prognosis

BACKGROUND: Transforming growth factors (TGF)-beta 1, TGF-beta R2 and Smad4 belong to the TGF family, and play important roles in carcinogenesis and the development of carcinoma, especially hepatocellular carcinoma (HCC). TGF-beta 1 is a multipotent polypeptide, which inhibits the growth of epithelial cells including hepatoma cell lines and hepatocytes by inducing apoptosis. TGF-beta R2 forms a heterodimeric complex upon binding to TGF-beta, and then generates the first step in the signal transduction pathway leading to growth inhibition in coordination with the type I receptor. Smad4 protein is an important mediator in the TGF-beta signaling pathway, and negatively regulates the growth of epithelial cells. This study aimed to detect the expression of TGF-beta 1, TGF-beta R2 and Smad4 in HCCs and their adjacent normal tissues, while assessing its relations with the clinicopathological parameters of HCC. METHODS: Forty-seven HCC specimens and their adjacent normal tissues were obtained surgically at the Affiliated Hospital of Medical College, Qingdao University. The expression of TGF-beta 1, TGF-beta R2 and Smad4 was separately detected by immunohistochemistry in all HCC specimens and their adjacent normal tissues, and its relations with the clinicopathological parameters of HCC were assessed. RESULTS: The positive expression of TGF-beta 1 was 72.34% in the HCC specimens, which was higher than that in the adjacent normal tissues (P < 0.001). The positive expression of Smad4 and TGF-beta R2 was 34.04% and 59.57% respectively in the carcinoma specimens. The expression of TGF-beta 1, TGF-beta R2 and Smad4 was significantly higher in groups with a tumor embolus of the portal vein, integrity of the amicula, and Edmondson's III-IV than that in other groups, but it was not related to tumor size (P < 0.05). CONCLUSIONS: TGF-beta 1 may play an important role in the occurrence and development of HCC. Combined detection of TGF-beta 1, TGF-beta R2 and Smad4 may be useful for the determination of the degree of malignancy and the prognosis of HCC.

Roles of Smad3 and Smad7 in rat pancreatic stellate cells activated by transforming growth factor-beta 1

BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the expression of the Smad3 and Smad7 genes in the process of PSC activation, and explore the mechanisms of chronic pancreatitis. METHODS: The expressions of Smad3 and Smad7 in PSCs before and after TGF-beta 1 treatment were detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Smad3 expression was detected in PSCs after treatment with 5 ng/ml of TGF-beta 1 for 24 hours. RESULTS: Smad7 expression was decreased in TGF-beta 1 -activated PSCs (P<0.05) in a dose-dependent manner. When TGF-beta 1 concentration reached 10 ng/ml, the expression of p-Smad3, Smad3, and Smad7 was inhibited (P<0.05). CONCLUSIONS: TGF-beta 1 promotes the expression of Smad3 and inhibits the expression of Smad7 during the activation of PSCs. In contrast, high-dose TGF-beta 1 downregulates the expression of Smad3 in completely activated PSCs.

The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1,Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.

Transforming growth factor beta 1, a cytokine with regenerative functions

We review the biology and role of transforming growth factor beta 1 （TGF-β1） in peripheral nerve injury and regeneration, as it relates to injuries to large nerve trunks （i.e., sciatic nerve, brachial plexus）, which often leads to suboptimal functional recovery. Experimental studies have suggested that the reason for the lack of functional recovery resides in the lack of sufficient mature axons reaching their targets, which is a result of the loss of the growth-supportive environment provided by the Schwann cells in the distal stump of injured nerves. Using an established chronic nerve injury and delayed repair animal model that accu- rately mimics chronic nerve injuries in humans, we summarize our key findings as well as others to better understand the pathophysiology of poor functional recovery. We demonstrated that 6 month TGF-β1 treat- ment for chronic nerve injury significantly improved Schwann cell capacity to support axonal regeneration. When combined with forskolin, the effect was additive, as evidenced by a near doubling of regenerated axons proximal to the repair site. We showed that in vivo application of TGF-β1 and forskolin directly onto chronically injured nerves reactivated chronically denervated Schwann cells, induced their proliferation, and upregulated the expression of regeneration-associated proteins. The effect of TGF-β1 and forskolin on old nerve injuries is quite impressive and the treatment regiment appears to mediate a growth-supportive milieu in the injured peripheral nerves. In summary, TGF-β1 and forskolin treatment reactivates chronical- ly denervated Schwann cells and could potentially be used to extend and prolong the regenerative responses to promote axonal regeneration.

Quantitative analysis of transforming growth factor beta 1 mRNA in patients with alcoholic liver disease

AIM: To investigate the expression of the transforming growth factor beta 1(TGF-beta 1) mRNA in different stages of alcoholic liver disease (ALD) and its clinical value. METHODS: One hundred and seven male alcoholics were grouped by clinical findings into four groups: alcohol abusers without liver impairment (n =22), alcoholic steatosis (n =30); alcoholic hepatitis (n=31); and alcoholic cirrhosis(n=24). Using peripheral blood mononuclear cells (PBMC) as samples the gene expression of TGF-beta 1 was examined quantitatively by reverse transcription polymerase chain reaction (RT-PCR) and dot blot. There are 34 healthy subjects served as control. RESULTS: The expression of TGF-beta 1 from all ALD patients was significantly greater than that in controls (1.320 +/- 1.162 vs 0.808 +/- 0.276, P【0.001). The differences of the expressions were significant between the patients from each groups (alcoholic steatosis, alcoholic hepatitis and alcoholic cirrhosis) and the controls (1.168 +/- 0.852, 1.462 +/- 1.657, 1.329 +/- 0.610 vs 0.808 +/- 0.276, P【0.050). No significant differences of TGF -beta 1 mRNA expression were observed between alcohol abusers without liver impairment and controls. The expressions in patients with alcoholic hepatitis and alcoholic cirrhosis were significantly greater than that in alcohol abusers respectively (1.462 +/- 1.657, 1.329 +/- 0.610 vs 0.841 +/- 0.706, P【0.050). No significant differences of TGF-beta 1 mRNA expression were observed between alcoholic fatty liver men and alcohol abusers. CONCLUSION: TGF-beta 1 expression level can be a risk factor for alcoholic liver disease and might be related to the inflammatory activity and fibrosis of the liver in patients.

Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts

AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.

Transforming growth factor-β1 induces intestinal myofibroblast differentiation and modulates their migration

AIM： To investigate the effects of transforming growth factor β1 （TGF-β1） on the differentiation of colonic lamina propria fibroblasts （CLPF） into myofibroblasts in vitro.METHODS： Primary CLPF cultures were incubated with TGF-β1 and analyzed for production of m-smooth muscle actin （α-SMA）, fibronectin （FN） and FN isoforms. Migration assays were performed in a modified 48-well Boyden chamber. Levels of total and phosphorylated focal adhesion kinase （FAK） in CLPF were analyzed after induction of migration.did not change α-SMA levels, while TGF-β1 treatment for 6 d significantly increased α-SIVlA production. Short term incubation （6 h） with TGF-β1 enhanced CLPF migration, while long term treatment （6 d） of CLPF with TGF-β1 reduced migration to 15%-37% compared to untreated cells. FN and FN isoform mRNA expression were increased after short term incubation with TGF-β1 （2 d） in contrast to long term incubation with TGF-β1 for 6 d. After induction of migration, TGF-β1-preincubated CLPF showed higher amounts of FN and its isoforms and lower levels of total and phosphorylated FAK than untreated cells.CONCLUSION： Long term incubation of CLPF with TGF-β1 induced differentiation into myofibroblasts with enhanced α-SMA, reduced migratory potential and FAK phosphorylation, and increased FN production. In contrast, short term contact （6 h） of fibroblasts with TGF-β1 induced a dose-dependent increase of cell migration and FAK phosphorylation without induction of α-SMA production.

LOW DOSE PIRFENIDONE SUPPRESSES TRANSFORMING GROWTH FACTOR BETA-1 AND TISSUE INHIBITOR OF METALLOPROTEINASE-1, AND PROTECTS RATS FROM LUNG FIBROSIS INDUCED BY BLEOMYCIN

Objective To investigate the optimal dosage of pirfenidone for the treatment of pulmonary fibrosis induced by bleomycin in Wistar rats, and the alteration of expressions of transforming growth factor beta-1 （ TGF-β1 ）, tissue inhibitor of metalloproteinase-1 （ TIMP-1 ）, and matrix metalloproteinase-13 （ MMP-13 ） in lung tissue. Methods Male Wistar rats were endotracheally instilled with bleomycin or normal saline. Pirfenidone （25-800 mg · kg^-l · d^-1 ）, dexamethasone （3 mg/kg）, or 1% carboxymethylcellulose sodium were given daily by feed 2 days before instillation of bleomycin. Groups T7 and T14 were fed pirfenidone 50 mg · kg^-1 · d^-1 at 7 days or 14 daYs after bleomycin instillation. Lungs were harvested at 28 days after bleomycin instillation. Patholological changes in luffg tissues were evaluated with HE staining. Lung collagen was stained by sirius red and measured by content of hydroxypro- line. Expression of proteins of TGF-β1 TIMP-1, and MMP-13 were detected by Western blotting. Results At doses of 25, 50, and 100 mg· kg^- 1 · d ^- 1, pirfenidone had significant anti-fibrotic effects for bleomy- cin-induced rat pulmonary fibrosis, and these effects were most significantly attenuated at the dosage of 50 mg · kg^-1 ·d^ -1（ HE： P 〈 0. 01, P 〈 0.01, and P = 0.064; sirius red： P 〈0.05, P 〈 0.01, and P 〈 0.05 ; hydroxyproline： P = 0.595, P 〈 0.01, and P = 0.976）. Pirfenidone at a dosage of 50 mg · kg^- l · d^-1 inhibited protein expression of TGF-131 and TIMP-1 in lung tissue in the early phase （0.79 and 0.75 times of control group）, but had no effect on ex- nr^eelnn nf MMP-13. Conclusion Low dose pirfenidone, especially at dosage of 50 mg · kg^-1 · d^-1, has significant anti-fibrotic effects on bleomycin-induced rat pulmonary fibrosis. Pirfenidone partially inhibits the enhancement of the expression of TGF-131 and TIMP-β1 in lung tissue.

Role of transforming growth factor-beta signaling pathway in pathogenesis of benign biliary stricture

AIM: To characterize the expression of members of the transforming growth factor-beta (TGF-β)/Smad/ connective tissue growth factor (CTGF) signaling pathway in the tissue of benign biliary stricture, and to investigate the effect of TGF-β signaling pathway in the pathogenesis of benign biliary stricture. METHODS: Paraffin embedded materials from 23 cases of benign biliary stricture were analyzed for members of the TGF-β/Smad/CTGF signaling pathway. TGF-β_1, TβRⅠ, TβRⅡ, Smad4, Smad7 and CTGF protein were detected by immunohistochemical strepto-advidinbiotin complex method, and CTGF mRNA was evaluated by hybridization in situ, while 6 cases of normal bile duct served as controls. The percentages of positive cells were counted. The correlation between TGF-β_1, Smad4 and CTGF was analyzed. RESULTS: The positive expression ratios of TGF-β_1, TβRⅠ , TβRⅡ , Smad4, CTGF and CTGF mRNA in 23 cases with benign biliary stricture were 91.3%, 82.6%, 87.0%, 78.3%, 82.6% and 65.2%, respectively, signifi cantly higher than that in 6 cases of normal bile duct respectively (vs 33.3%, 16.7%, 50.0%, 33.3%, 50.0%, 16.7%, respectively, P < 0.05). The positiveexpression ratio of Smad7 in cases with benign biliary stricture was 70.0%, higher than that in normal bile duct, but this difference is not statistically signifi cant 70.0% vs 50%, P > 0.05). There was a positive correlation between positive expression of TGF-β_1, Smad4 and CTGF in cases with benign biliary stricture. CONCLUSION: The high expression of TGF-β/Smad/ CTGF signaling pathway plays an important role in the pathogenesis of benign biliary stricture.