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 fin...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.展开更多
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 v...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.展开更多
AIM: Transforming growth factor-β (TGF-β) plays a regulatory role in tissue repair. In a previous study, we found that TGF-β and its receptors were expressed in gastric mucosa of patients with well-healed gastric u...AIM: Transforming growth factor-β (TGF-β) plays a regulatory role in tissue repair. In a previous study, we found that TGF-β and its receptors were expressed in gastric mucosa of patients with well-healed gastric ulcers, as demonstrated by immunohistochemistry. To further characterize the role of TGF-β and its receptors in repairing gastric ulcers, we investigated the expression patterns of TGF-β and its receptors in gastric mucosa by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR).METHODS: Seventy-four patients with endoscopically proven gastric ulcers were eligible for participation in this study. All patients had routine biopsies on initial endoscopy and were then treated for 12 wk with an H2 blocker. Repeat endoscopy was then performed. There were 8 patients with poorly healed ulcers, and biopsies were taken from the margin of the residual ulcers. These tissue samples, along with biopsy of gastric mucosa near the original ulcers from 8 randomly selected patients with well-healed ulcers were examined for TGF-β and TGF-β receptor Ⅱ mRNA by RT-PCR and in situ hybridization, as well as immunohistochemistry.RESULTS: TGF-β and TGF-β receptor Ⅱ were strongly expressed in tissues from patients with well-healed ulcers.Four of the 8 patients with poor healing had low or absent expression of TGF-β or TGF-β receptor Ⅱ mRNA. All cases positive by RT-PCR assay were confirmed by in situ hybridization as well as immunohistochemistry.CONCLUSION: It is suggested that TGF-β and its receptors are important for gastric ulcer healing. These results may have implications for further investigation of the healing process and in predicting response to therapy.展开更多
OBJECTIVE To study the relationship among microsatellite instability (MSI), frameshift mutations (FM) of the transforming growth factor p receptorⅡ(TGFpRⅡ), methylation state of the hMLH1 promoter and hMLH1 protein ...OBJECTIVE To study the relationship among microsatellite instability (MSI), frameshift mutations (FM) of the transforming growth factor p receptorⅡ(TGFpRⅡ), methylation state of the hMLH1 promoter and hMLH1 protein expression level in gastric cancers, and to explore their relationship to gastric carcinogenesis. METHODS DMA was isolated from 101 gastric specimens and 5 microsatellite loci were detected. PCR, electrophoresis on denatured polyacrylamide gels and silver staining were performed to detect the MSI. The FMs of TGFβRⅡwere also screened with the same method. HMLH1 methylation was analyzed by methylation specific PCR (MSP) and sequencing. HMLH1 protein expression was detected using immunohistochemistry. RESULTS The incidence of MSIs was 53.7% and 0% in the cancers and normal tissues, respectively, with the frequency of MSIs being significantly higher in the gastric cancers compared to the normal gastric tissues (P<0.05). The frequency of hMLH1 methylation was 41.5%(17/41) in the gastric cancers and 0%(0/60) in the normal group. Decreased hMLH1 expression was observed in 94.1%(16/17) of cases exhibiting methylation. FMs of TGFβRⅡwere identified in 5 (62.5%) of the 8 samples with MSIH. In contrast, FMs were not found in MSI -L or microsatellite stable (MSS) cases. CONCLUSION MSIs and FMs of TGFβRⅡmay play an important role in gastric carcinogenesis. HMLH1 methylation is an important modification of the DNA which results in inactivation of hMLH1 and mismatch repair defects which lead to MSIs and FMs of TGFβRⅡ.展开更多
Objective: To study the transforming growth factor β receptor Ⅱ (TGFβ-R Ⅱ) expression in experimental cryptorchidism and apoptosis in spermatogenic cells in rats. Methods: The apoptosis of spermatogenic cells was ...Objective: To study the transforming growth factor β receptor Ⅱ (TGFβ-R Ⅱ) expression in experimental cryptorchidism and apoptosis in spermatogenic cells in rats. Methods: The apoptosis of spermatogenic cells was detected by means of the terminal deoxynucleotldyl transferase mediated dUTP nick end lableling method (TUNEL) and the TGFβ-R Ⅱ expression was observed with the immunohistochemistry SABC methods. Results: There was a significant increase in the TGFβ-R Ⅱ expression in unilateral undescended testes (UUTs) compared with that in contralateral descended testes (CDTs, P<0.01). However, there was a significant and time-dependent increase in the mean apoptotic index in UUTs than in CDTs. Conclusion: TGFβ-R Ⅱ may play an important role in spermatogenic cell apoptosis.展开更多
AIM:To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis.METHODS:A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was...AIM:To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis.METHODS:A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied,with all subjects having liver biopsy data and DNA available for testing.This study assessed the association of eight single nucleotide polymorphisms(SNPs)in a total of six genes including toll-like receptor 4(TLR4),transforming growth factor-beta(TGF-β),oxoguanine DNA glycosylase,monocyte chemoattractant protein 1,chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity.Genotyping was performed using high resolution melt analysis and sequencing.The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration.RESULTS:There were significant associations between the cofactors of male gender(P=0.0001),increasing age(P=0.006),alcohol consumption(P=0.0001),steatosis(P=0.03),hepatic iron concentration(P<0.0001)and the presence of hepatic fibrosis.Of the candidate gene polymorphisms studied,none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors.We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied.Importantly,in this large,well characterised cohort of patients there was no association between SNPs for TGF-βor TLR4and the presence of fibrosis,cirrhosis or increasing fibrosis stage in multivariate analysis.CONCLUSION:In our large,well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis.展开更多
Background Repeated attacks of bronchial asthma lead to different degrees of airway remodeling,the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some gr...Background Repeated attacks of bronchial asthma lead to different degrees of airway remodeling,the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some growth promotion factors. Angiotensin Ⅱ is a polypeptide that may be involved in airway remodeling. To evaluate its role in airway remodeling in asthma,we observed the effects of an angiotensin Ⅱ type 1 receptor antagonist (valsartan) on the expression of collagen Ⅲ,collagen Ⅴ,and transforming growth factor β_1 (TGF-β_1) mRNA and protein in the airway walls of sensitized rats.Methods Forty Wistar rats were randomly divided into 5 groups: control group,sensitized group,and valsartan groups 1,2,and 3. The rats in the sensitized group and in valsartan groups 1,2,and 3 were sensitized and challenged with ovalbumin. Rats in control group were sensitized and challenged with 0.9% NaCl. Rats from valsartan groups 1,2,and 3 were drenched with valsartan (10 μg, 20 μg,or 30 μg,respectively) at the time of the ovalbumin challenges. The expression of collagen Ⅲ,collagen Ⅴ,and TGF-β_1 protein were detected using immunohistochemical method in combination with image analysis methods. The expression of TGF-β_1 mRNA was detected by in situ hybridization. Results The expression in the airways of collagen Ⅲ and collagen Ⅴ was significantly higher in rats from the sensitized group (7.73±0.81, 1.34±0.28) and from valsartan groups 1,2,and 3 (5.73±0.64, 1.13±0.15; 4.96±0.51, 0.98±0.08; 4.43±0.35, 0.93±0.06,respectively) than those in the control group (2.65±0.38, 0.67±0.08,P <0.05). In addition,collagen levels were significantly lower in valsartan groups 1,2,and 3 than those from the sensitized group ( P <0.05). The expression of TGF-β_1 mRNA and protein in the airways was significantly higher in rats from the sensitized group (20.49%±3.46%,29.73%±3.25%) and from valsartan groups 1,2,and 3 (16.47%±1.94%, 19.41%±1.87%; 14.38%±1.58%, 18.29%±1.43%; 12.96%±1.73%, 18.63%±1.11%,respectively) than that from the control group (7.84%±1.61%, 5.63%±1.07%,P <0.05). TGF-β_1 mRNA and protein levels were significantly lower in valsartan groups 1,2,and 3 than that in the sensitized group ( P <0.05). Conclusions Angiotensin Ⅱ receptor antagonist valsartan can suppress synthesis of collagen Ⅲ and collagen Ⅴ by downregulating TGF-β_1 mRNA and protein expression. Valsartan can decrease airway remodeling and could play a role in asthma therapy.展开更多
Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering ...Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.展开更多
Objective: To investigate the effect of substance P (SP) on gene expression of transforming growth factor β-1 (TGFβ-1), transforming growth factor receptor-1 (TGFR-1) and transforming growth factor receptor-2 (TGFR-...Objective: To investigate the effect of substance P (SP) on gene expression of transforming growth factor β-1 (TGFβ-1), transforming growth factor receptor-1 (TGFR-1) and transforming growth factor receptor-2 (TGFR-2) in fibroblasts cultured in vitro from rat’s granulation tissues. Methods: The fibroblasts from the granulation tissues in the skeletal muscle of rat’s hind limbs injured by formaldehyde were cultured in vitro. When different concentrations (10 -9-10 -5 mol/L) of SP were added into the culture medium, the changes of gene expression of TGFβ-1, TGFR-1 and TGFR-2 in the cultured fibroblasts were observed with reverse transcription polymerase chain reaction at different intervals (0, 3, 6, 12 and 24 hours after incubation). Results: The gene expression of TGFβ-1, TGFR-1 and TGFR-2 in the fibroblasts cultured from rat’s granulation tissues was up-regulated by SP. The peak level of the mRNA expression was found at 10 -8 mol/L SP and the up-regulation effect was not found at 10 -5 mol/L and 10 -6 mol/L. The peak levels of gene expression of TGFβ-1, TGFR-1 and TGFR-2 in the fibroblasts treated with SP were achieved at 6 and 12 hours, respectively. Conclusions: SP has up-regulation effect on the gene expression of TGFβ-1, TGFR-1 and TGFR-2 in fibroblasts from rat’s granulation tissues in vitro, and the effect is related to different stimulating concentrations of SP. It may be concerned with proliferation and differentiation of fibroblasts and formation of scar tissues during wound healing.展开更多
OBJECTIVE: To investigate the effect of Sini decoction on rats with myocardial fibrosis induced by banding the abdominal aorta, and explore the mechanism underlying its actions on angiotensin Ⅱ(Ang Ⅱ), transforming ...OBJECTIVE: To investigate the effect of Sini decoction on rats with myocardial fibrosis induced by banding the abdominal aorta, and explore the mechanism underlying its actions on angiotensin Ⅱ(Ang Ⅱ), transforming growth factor-β_1(TGF-β_1)and connective tissue growth factor(CTGF).METHODS: Forty-eight male Sprague-Dawley rats were randomly divided into sham operation, model, Captopril, and Sini decoction groups. The models were established by the partial banding of the abdominal aorta according to Doering's method.Eight weeks later, heart weight indexes were calculated; hemodynamic changes of the hearts were tested; changes in myocardial tissue morphology were observed by Masson staining; and myocardial collagen volume fraction was calculated. Enzyme-linked immunosorbent assay was used to measure the concentration of Ang Ⅱ in serum. The expression of TGF-β_1 and CTGF were determined by immunohistochemistry and Western blotting.RESULTS: Compared with the sham operation group, the heart weight index, collagen volume fraction of the myocardium, serum levels of Ang Ⅱ,and the expression of myocardial TGF-β_1 and CTGF in the model group were significantly increased(P < 0.05). Compared with the model group, the heart weight index, collagen volume fraction of the myocardium, serum levels of Ang Ⅱ, and the expression of myocardial TGF-β_1 and CTGF in all treatment groups were significantly reduced(P < 0.05).CONCLUSION: Sini decoction reduced AngⅡ level and inhibited the expression of myocardial TGF-β_1 and CTGF, which may explain the mechanism of its protective effect on myocardium with fibrosis.展开更多
Background Acute lung infection due to Pseudomonas aeruginosa (P. Aeruginosa) is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming thre...Background Acute lung infection due to Pseudomonas aeruginosa (P. Aeruginosa) is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. Aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P.aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. Aeruginosa in vitro. We used flagellin protein from P. Aeruginosa, a key initiator of acute P.aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. Aeruginosa can cause fibrogenesis/remodeling.Methods We studied the effect of flagellin from P. Aeruginosa (flagellin for short) on the transforming growth factor beta 1 (TGF-β1) and interleukin-8 (IL-8) expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6)/mitogen activated protein kinase (MAPK) pathway. Flagellin was purified from the P. Aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay (ELISA). Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH2-terminal kinase (JNK) and extracellular regulated kinase (ERK).Results Expression of TGF-β1 in BEAS-2B cells was elevated by flagellin vs. Control groups ((104.3±20.8) vs.(44.6±4.4) pg/ml (P 〈0.01)) and was ablated by either p38 or JNK inhibitors compared with flagellin treatment ((45.1±18.8)vs. (104.3±20.8) pg/ml and (48.1±20.8) vs. (104.3±20.8) pg/ml, respectively (P 〈0.05)). Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. The control groups ((554.9±57.7) vs. (51.4±2.2.9) pg/ml (P 〈0.01)), and p38 MAPK inhibitors weaken the expression by flagellin ((301.1 ±155.1) vs. (554.9±57.7) pg/ml (P 〈0.05)). Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes (P 〈0.01), 30 minutes (P 〈0.01) and 15 minutes (P 〈0.01). TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-β1 and IL-8 has no statistical significance.Conclusions Flagellin from P. Aeruginosa PAO1 induces TGF-β1 expression in normal bronchial epithelial cells,BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P. Aeruginosa.展开更多
基金Supported by the Special Research Project for Capital Health Development,No.2022-2-2174the Beijing Municipal Science and Technology Commission,No.Z191100007619037.
文摘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.
基金Shaanxi Province Science and Technology Gongguan Program, China (No.2011-K14-02-03)
文摘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.
基金Supported by a grant to Shou-Chuan Shih from Mackay Memorial Hospital(project No:9137)and in part by grants to Chung-Liang Chien (NSC 91-2320-B-002-114)from the National Science Council,Taiwan
文摘AIM: Transforming growth factor-β (TGF-β) plays a regulatory role in tissue repair. In a previous study, we found that TGF-β and its receptors were expressed in gastric mucosa of patients with well-healed gastric ulcers, as demonstrated by immunohistochemistry. To further characterize the role of TGF-β and its receptors in repairing gastric ulcers, we investigated the expression patterns of TGF-β and its receptors in gastric mucosa by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR).METHODS: Seventy-four patients with endoscopically proven gastric ulcers were eligible for participation in this study. All patients had routine biopsies on initial endoscopy and were then treated for 12 wk with an H2 blocker. Repeat endoscopy was then performed. There were 8 patients with poorly healed ulcers, and biopsies were taken from the margin of the residual ulcers. These tissue samples, along with biopsy of gastric mucosa near the original ulcers from 8 randomly selected patients with well-healed ulcers were examined for TGF-β and TGF-β receptor Ⅱ mRNA by RT-PCR and in situ hybridization, as well as immunohistochemistry.RESULTS: TGF-β and TGF-β receptor Ⅱ were strongly expressed in tissues from patients with well-healed ulcers.Four of the 8 patients with poor healing had low or absent expression of TGF-β or TGF-β receptor Ⅱ mRNA. All cases positive by RT-PCR assay were confirmed by in situ hybridization as well as immunohistochemistry.CONCLUSION: It is suggested that TGF-β and its receptors are important for gastric ulcer healing. These results may have implications for further investigation of the healing process and in predicting response to therapy.
文摘OBJECTIVE To study the relationship among microsatellite instability (MSI), frameshift mutations (FM) of the transforming growth factor p receptorⅡ(TGFpRⅡ), methylation state of the hMLH1 promoter and hMLH1 protein expression level in gastric cancers, and to explore their relationship to gastric carcinogenesis. METHODS DMA was isolated from 101 gastric specimens and 5 microsatellite loci were detected. PCR, electrophoresis on denatured polyacrylamide gels and silver staining were performed to detect the MSI. The FMs of TGFβRⅡwere also screened with the same method. HMLH1 methylation was analyzed by methylation specific PCR (MSP) and sequencing. HMLH1 protein expression was detected using immunohistochemistry. RESULTS The incidence of MSIs was 53.7% and 0% in the cancers and normal tissues, respectively, with the frequency of MSIs being significantly higher in the gastric cancers compared to the normal gastric tissues (P<0.05). The frequency of hMLH1 methylation was 41.5%(17/41) in the gastric cancers and 0%(0/60) in the normal group. Decreased hMLH1 expression was observed in 94.1%(16/17) of cases exhibiting methylation. FMs of TGFβRⅡwere identified in 5 (62.5%) of the 8 samples with MSIH. In contrast, FMs were not found in MSI -L or microsatellite stable (MSS) cases. CONCLUSION MSIs and FMs of TGFβRⅡmay play an important role in gastric carcinogenesis. HMLH1 methylation is an important modification of the DNA which results in inactivation of hMLH1 and mismatch repair defects which lead to MSIs and FMs of TGFβRⅡ.
文摘Objective: To study the transforming growth factor β receptor Ⅱ (TGFβ-R Ⅱ) expression in experimental cryptorchidism and apoptosis in spermatogenic cells in rats. Methods: The apoptosis of spermatogenic cells was detected by means of the terminal deoxynucleotldyl transferase mediated dUTP nick end lableling method (TUNEL) and the TGFβ-R Ⅱ expression was observed with the immunohistochemistry SABC methods. Results: There was a significant increase in the TGFβ-R Ⅱ expression in unilateral undescended testes (UUTs) compared with that in contralateral descended testes (CDTs, P<0.01). However, there was a significant and time-dependent increase in the mean apoptotic index in UUTs than in CDTs. Conclusion: TGFβ-R Ⅱ may play an important role in spermatogenic cell apoptosis.
基金Supported by NHMRC Medical Postgraduate Scholarship and the Royal Brisbane and Women’s Hospital Research Foundation to Wood MJthe National Health and Medical Research Council(NHMRC)to Ramm GA and Powell LW+1 种基金the recipient of an NHMRC Senior Research Fellowship,1024672 to Subramaniam VNan NHMRC Senior Research Fellowship,No.552409 to Ramm GA
文摘AIM:To investigate the role of genetic polymorphisms in the progression of hepatic fibrosis in hereditary haemochromatosis.METHODS:A cohort of 245 well-characterised C282Y homozygous patients with haemochromatosis was studied,with all subjects having liver biopsy data and DNA available for testing.This study assessed the association of eight single nucleotide polymorphisms(SNPs)in a total of six genes including toll-like receptor 4(TLR4),transforming growth factor-beta(TGF-β),oxoguanine DNA glycosylase,monocyte chemoattractant protein 1,chemokine C-C motif receptor 2 and interleukin-10 with liver disease severity.Genotyping was performed using high resolution melt analysis and sequencing.The results were analysed in relation to the stage of hepatic fibrosis in multivariate analysis incorporating other cofactors including alcohol consumption and hepatic iron concentration.RESULTS:There were significant associations between the cofactors of male gender(P=0.0001),increasing age(P=0.006),alcohol consumption(P=0.0001),steatosis(P=0.03),hepatic iron concentration(P<0.0001)and the presence of hepatic fibrosis.Of the candidate gene polymorphisms studied,none showed a significant association with hepatic fibrosis in univariate or multivariate analysis incorporating cofactors.We also specifically studied patients with hepatic iron loading above threshold levels for cirrhosis and compared the genetic polymorphisms between those with no fibrosis vs cirrhosis however there was no significant effect from any of the candidate genes studied.Importantly,in this large,well characterised cohort of patients there was no association between SNPs for TGF-βor TLR4and the presence of fibrosis,cirrhosis or increasing fibrosis stage in multivariate analysis.CONCLUSION:In our large,well characterised group of haemochromatosis subjects we did not demonstrate any relationship between candidate gene polymorphisms and hepatic fibrosis or cirrhosis.
基金ThisworkwassupportedbyagrantfromtheShanxiProvinceFoundationforReturnedOverseasChineseScholars (No 9913 -95 )
文摘Background Repeated attacks of bronchial asthma lead to different degrees of airway remodeling,the mechanism of which is not yet clear. Some evidences indicate that it is related to the excessive expression of some growth promotion factors. Angiotensin Ⅱ is a polypeptide that may be involved in airway remodeling. To evaluate its role in airway remodeling in asthma,we observed the effects of an angiotensin Ⅱ type 1 receptor antagonist (valsartan) on the expression of collagen Ⅲ,collagen Ⅴ,and transforming growth factor β_1 (TGF-β_1) mRNA and protein in the airway walls of sensitized rats.Methods Forty Wistar rats were randomly divided into 5 groups: control group,sensitized group,and valsartan groups 1,2,and 3. The rats in the sensitized group and in valsartan groups 1,2,and 3 were sensitized and challenged with ovalbumin. Rats in control group were sensitized and challenged with 0.9% NaCl. Rats from valsartan groups 1,2,and 3 were drenched with valsartan (10 μg, 20 μg,or 30 μg,respectively) at the time of the ovalbumin challenges. The expression of collagen Ⅲ,collagen Ⅴ,and TGF-β_1 protein were detected using immunohistochemical method in combination with image analysis methods. The expression of TGF-β_1 mRNA was detected by in situ hybridization. Results The expression in the airways of collagen Ⅲ and collagen Ⅴ was significantly higher in rats from the sensitized group (7.73±0.81, 1.34±0.28) and from valsartan groups 1,2,and 3 (5.73±0.64, 1.13±0.15; 4.96±0.51, 0.98±0.08; 4.43±0.35, 0.93±0.06,respectively) than those in the control group (2.65±0.38, 0.67±0.08,P <0.05). In addition,collagen levels were significantly lower in valsartan groups 1,2,and 3 than those from the sensitized group ( P <0.05). The expression of TGF-β_1 mRNA and protein in the airways was significantly higher in rats from the sensitized group (20.49%±3.46%,29.73%±3.25%) and from valsartan groups 1,2,and 3 (16.47%±1.94%, 19.41%±1.87%; 14.38%±1.58%, 18.29%±1.43%; 12.96%±1.73%, 18.63%±1.11%,respectively) than that from the control group (7.84%±1.61%, 5.63%±1.07%,P <0.05). TGF-β_1 mRNA and protein levels were significantly lower in valsartan groups 1,2,and 3 than that in the sensitized group ( P <0.05). Conclusions Angiotensin Ⅱ receptor antagonist valsartan can suppress synthesis of collagen Ⅲ and collagen Ⅴ by downregulating TGF-β_1 mRNA and protein expression. Valsartan can decrease airway remodeling and could play a role in asthma therapy.
基金supported by the Foundation of Stomatology Hospital,Xi'an Jiaotong University
文摘Objective To study the effect of baicalin on the expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in cultured human periodontal ligament(HPDL)cells.Methods Small interfering RNA(siRNA)eukaryotic expression vector targeted transforming growth factor βⅡ receptor(TGF-β RⅡ)was constructed and transfected into T cells.HPDL cells with T cells transfected with siRNA or not were placed in the culture medium that had been added with lipopolysaccharide(LPS)and baicalin.The obtained solution was divided into six groups according to the components(group Ⅰ:HPDL cells+LPS+T cells transfected with siRNA1+baicalin;group Ⅱ:HPDL cells+LPS+T cells transfected with siRNA1;group Ⅲ:HPDL cells+LPS+T cells+baicalin;group Ⅳ:HPDL cells+LPS+T cells;group Ⅴ:HPDL cells+baicalin;group Ⅵ:HPDL cells)and was cultured for 48 hours.RT-PCR was used to observe the effect of baicalin on the expression of OPG-RANKL in HPDL cells.Results The ratio of RANKL/OPG in group Ⅰ was lower than that in group Ⅱ(P<0.01)and higher than that in group Ⅲ(P<0.01);The ratio of RANKL/OPG in group Ⅲ was lower than that in group Ⅳ(P<0.01);the ratio of RANKL/OPG in group Ⅳ was higher than that in group Ⅵ(P<0.01);the ratio of RANKL/OPG in group Ⅴ was lower than that in group Ⅵ(P<0.05).Conclusion ① Baicalin could decrease the ratio of RANKL/OPG in HPDL cells.② The TGF-β signaling transduction plays an important role in the effect of baicalin on the RANKL/OPG ratio in HPDL cells.③ Baicalin acts not only through TGF-β to regulate RANKL/OPG in HPDL cells,but also through other pathways.
基金ThisworkwassupportedbyMajorStateBasicResearchDevelopmentProgramofChina (No .G19990 5 4 2 0 4 )
文摘Objective: To investigate the effect of substance P (SP) on gene expression of transforming growth factor β-1 (TGFβ-1), transforming growth factor receptor-1 (TGFR-1) and transforming growth factor receptor-2 (TGFR-2) in fibroblasts cultured in vitro from rat’s granulation tissues. Methods: The fibroblasts from the granulation tissues in the skeletal muscle of rat’s hind limbs injured by formaldehyde were cultured in vitro. When different concentrations (10 -9-10 -5 mol/L) of SP were added into the culture medium, the changes of gene expression of TGFβ-1, TGFR-1 and TGFR-2 in the cultured fibroblasts were observed with reverse transcription polymerase chain reaction at different intervals (0, 3, 6, 12 and 24 hours after incubation). Results: The gene expression of TGFβ-1, TGFR-1 and TGFR-2 in the fibroblasts cultured from rat’s granulation tissues was up-regulated by SP. The peak level of the mRNA expression was found at 10 -8 mol/L SP and the up-regulation effect was not found at 10 -5 mol/L and 10 -6 mol/L. The peak levels of gene expression of TGFβ-1, TGFR-1 and TGFR-2 in the fibroblasts treated with SP were achieved at 6 and 12 hours, respectively. Conclusions: SP has up-regulation effect on the gene expression of TGFβ-1, TGFR-1 and TGFR-2 in fibroblasts from rat’s granulation tissues in vitro, and the effect is related to different stimulating concentrations of SP. It may be concerned with proliferation and differentiation of fibroblasts and formation of scar tissues during wound healing.
基金Supported by Liaoning Province Nature Science Foundation project:The Mechanic of Wenyangtongmai Method on Myocardial Injury Rats(No.201102128)
文摘OBJECTIVE: To investigate the effect of Sini decoction on rats with myocardial fibrosis induced by banding the abdominal aorta, and explore the mechanism underlying its actions on angiotensin Ⅱ(Ang Ⅱ), transforming growth factor-β_1(TGF-β_1)and connective tissue growth factor(CTGF).METHODS: Forty-eight male Sprague-Dawley rats were randomly divided into sham operation, model, Captopril, and Sini decoction groups. The models were established by the partial banding of the abdominal aorta according to Doering's method.Eight weeks later, heart weight indexes were calculated; hemodynamic changes of the hearts were tested; changes in myocardial tissue morphology were observed by Masson staining; and myocardial collagen volume fraction was calculated. Enzyme-linked immunosorbent assay was used to measure the concentration of Ang Ⅱ in serum. The expression of TGF-β_1 and CTGF were determined by immunohistochemistry and Western blotting.RESULTS: Compared with the sham operation group, the heart weight index, collagen volume fraction of the myocardium, serum levels of Ang Ⅱ,and the expression of myocardial TGF-β_1 and CTGF in the model group were significantly increased(P < 0.05). Compared with the model group, the heart weight index, collagen volume fraction of the myocardium, serum levels of Ang Ⅱ, and the expression of myocardial TGF-β_1 and CTGF in all treatment groups were significantly reduced(P < 0.05).CONCLUSION: Sini decoction reduced AngⅡ level and inhibited the expression of myocardial TGF-β_1 and CTGF, which may explain the mechanism of its protective effect on myocardium with fibrosis.
基金This study was supported by a grant from the National Natural Science Foundation of China (No. 30872719).
文摘Background Acute lung infection due to Pseudomonas aeruginosa (P. Aeruginosa) is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P. Aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P.aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P. Aeruginosa in vitro. We used flagellin protein from P. Aeruginosa, a key initiator of acute P.aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P. Aeruginosa can cause fibrogenesis/remodeling.Methods We studied the effect of flagellin from P. Aeruginosa (flagellin for short) on the transforming growth factor beta 1 (TGF-β1) and interleukin-8 (IL-8) expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6)/mitogen activated protein kinase (MAPK) pathway. Flagellin was purified from the P. Aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-β1 and IL-8 were detected by enzyme-linked immunosorbant assay (ELISA). Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH2-terminal kinase (JNK) and extracellular regulated kinase (ERK).Results Expression of TGF-β1 in BEAS-2B cells was elevated by flagellin vs. Control groups ((104.3±20.8) vs.(44.6±4.4) pg/ml (P 〈0.01)) and was ablated by either p38 or JNK inhibitors compared with flagellin treatment ((45.1±18.8)vs. (104.3±20.8) pg/ml and (48.1±20.8) vs. (104.3±20.8) pg/ml, respectively (P 〈0.05)). Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. The control groups ((554.9±57.7) vs. (51.4±2.2.9) pg/ml (P 〈0.01)), and p38 MAPK inhibitors weaken the expression by flagellin ((301.1 ±155.1) vs. (554.9±57.7) pg/ml (P 〈0.05)). Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes (P 〈0.01), 30 minutes (P 〈0.01) and 15 minutes (P 〈0.01). TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-β1 and IL-8 has no statistical significance.Conclusions Flagellin from P. Aeruginosa PAO1 induces TGF-β1 expression in normal bronchial epithelial cells,BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P. Aeruginosa.