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.

Detection of Frameshift Mutations of the Transforming Growth Factor p ReceptorⅡin Gastric Cancers with Microsatellite Instability

OBJECTIVE To study the relationship among microsatellite instability （MSI）, frameshift mutations （FM） of the transforming growth factor β receptor Ⅱ （TGF β R Ⅱ）, methylation state of the hMLH1 promoter and hMLH1 protein expression level in gastric cancers, and to explore their relationship to gastric carcinogenesis. METHODS DNA 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 TGFβR Ⅱ were 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 TGFβR Ⅱ were 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 TGFβR Ⅱ may 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 MSls and FMs of TGFβR Ⅱ.

Transforming growth factor β receptor Ⅱ expression inexperimental cryptorchidism and apoptosisin spermatogenic cells in rats

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.

Correlation between expression of two transforming growth factor-beta 1 receptors and microvascular density in a rat model of cerebral ischemia and reperfusion injury

The effects of transforming growth factor-β1 （TGF-β1） are currently controversial. Whether TGF-β1 promotes or inhibits revascularization under different conditions remains poorly understood. Based on previous studies, the current experiment established rat models of cerebral ischemia and reperfusion injury （IRI）, and demonstrated that pathological and functional damage was also increased after IRI. The most serious damage was observed at 3 days after reperfusion, at which time microvascular density fell to its lowest level. Soon afterwards, microvascular density increased, new collateral circulation was gradually established at 4 to 7 days after reperfusion, and pathological damage and neurological deficits were improved. TGF-β1, activin receptor-like kinase 5 （ALK5） mRNA and protein expression levels increased gradually over time. In contrast, ALK1 mRNA and protein expression decreased over the same period. A significant negative correlation was detected between microvascular density and expression of the ALK5 gene transcript. There was no correlation between microvascular density and ALK1 gene transcriptional expression following cerebral IRI in a rat model. These findings suggest that ALK5, rather than ALK1, is the critical receptor in the TGF-β1 signal pathways after cerebral IRI.

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.

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.

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.

Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and transdifferentiation

AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

Transforming growth factor-β and toll-like receptor-4 polymorphisms are not associated with fibrosis in haemochromatosis

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.

Bone morphogenetic protein-4 and transforming growth factor-beta1 mechanisms in acute valvular response to supra-physiologic hemodynamic stresses

AIM:To explore ex vivo the role of bone morphogenetic protein-4(BMP-4) and transforming growth factorbeta1(TGF-β1) in acute valvular response to fluid shear stress(FSS) abnormalities.METHODS:Porcine valve leaflets were subjected ex vivo to physiologic FSS,supra-physiologic FSS magnitude at normal frequency and supra-physiologic FSS frequency at normal magnitude for 48 h in a double-sided cone-and-plate bioreactor filled with standard culture medium. The role of BMP-4 and TGF-β1 in the valvular response was investigated by promoting or inhibiting the downstream action of those cytokines via culture medium supplementation with BMP-4 or the BMP antagonist noggin,and TGF-β1 or the TGF-β1 inhibitor SB-431542,respectively. Fresh porcine leaflets were used as controls. Each experimental group consisted of six leaflet samples. Immunostaining and immunoblotting were performed to assess endothelial activation in terms of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expressions,paracrine signaling in terms of BMP-4 and TGF-β1 expressions and extracellular matrix(ECM) remodeling in terms of cathepsin L,cathepsin S,metalloproteinases(MMP)-2 and MMP-9 expressions. Immunostained images were quantified by normalizing the intensities of positively stained regions by the number of cells in each image while immunoblots were quantified by densitometry. R E S U LT S :Regardless of the culture medium,physiologic FSS maintained valvular homeostasis. Tissue exposure to supra-physiologic FSS magnitude in standard medium stimulated paracrine signaling(TGF-β1:467% ± 22% vs 100% ± 6% in freshcontrols,BMP-4:258% ± 22% vs 100% ± 4% in fresh controls; P < 0.05) and ECM degradation(MMP-2:941% ± 90% vs 100% ± 19% in fresh controls,MMP-9:1219% ± 190% vs 100% ± 16% in fresh controls,cathepsin L:1187% ± 175% vs 100% ± 12% in fresh controls,cathepsin S:603% ± 88% vs 100% ± 13% in fresh controls; P < 0.05),while BMP-4 supplementation also promoted fibrosa activation and TGF-β1 inhibition reduced MMP-9 expression to the native tissue level(MMP-9:308% ± 153% with TGF-β1 inhibition vs 100% ± 16% in fresh control; P > 0.05). Supra-physiologic FSS frequency had no effect on endothelial activation and paracrine signaling regardless of the culture medium but TGF-β1 silencing attenuated FSS-induced ECM degradation via MMP-9 downregulation(MMP-9:302% ± 182% vs 100% ± 42% in fresh controls; P > 0.05).CONCLUSION:Valvular tissue is sensitive to FSS abnormalities. The TGF-β1 inhibitor SB-431542 is a potential candidate molecule for attenuating the effects of FSS abnormalities on valvular remodeling.

Effects of RNA interference targeting transforming growth factor-beta 1 on immune hepatic fibrosis induced by Concanavalin A in mice

BACKGROUND: Previous studies have shown that transforming growth factor-beta 1 (TGF-beta 1) is the most potent means of stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells. Thus, TGF-beta 1 could be a target for treating hepatic fibrosis. This study aimed to investigate the inhibitory effects of specific TGF-beta 1 small interference RNA (siRNA) on immune hepatic fibrosis induced by Concanavalin A (Con A) in mice. METHODS: Three short hairpin RNAs targeting different positions of TGF-beta 1 were designed and cloned to the plasmid pGenesil-1 to obtain three recombinant expression vectors (pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 and pGenesil-TGF-beta 1-m3). Thirty male Kunming mice were randomly divided into 6 groups: normal, model, control, and three treatment groups. The immune hepatic fibrosis models were constructed by injecting Con A via the tail vein at 8 mg/kg per week for 6 weeks. At weeks 2, 4 and 6, pGenesil-TGF-beta 1-ml, pGenesil-TGF-beta 1-m2 or pGenesi1-TGF-beta 1-m3 was injected by a hydrodynamics-based transfection method via the tail vein at 0.8 ml/10 g within 24 hours after injection of Con A in each of the three treatment groups. The mice in the control group were injected with control plasmid pGenesil-HK at the same dose. All mice were sacrificed at week 7. The levels of hydroxyproline in liver tissue were determined by biochemistry. Liver histopathology was assessed by Van Gieson staining. The expression levels and localization of TGF-beta 1, Smad3, and Smad7 in liver tissue were detected by immunohistochemistry. The expression of TGF-beta 1, Smad3, Smad7 and alpha-smooth muscle actin (alpha-SMA) mRNAs in the liver were assessed by semi-quantitative RT-PCR. RESULTS: The levels of hydroxyproline in the liver tissue of the treatment groups were lower than those of the model group (P<0.01). Histopathologic assay showed that liver fibrogenesis was clearly improved in the treatment groups compared with the model group. The expression levels of TGF-beta 1 and Smad3 of liver tissue were also markedly lower in the treatment groups than in the model group (P<0.01), while the levels of Smad7 were higher in the treatment groups than in the model group (P<0.01). RT-PCR further showed that the expression of TGF-beta 1, Smad3 and alpha-SMA mRNA was significantly inhibited in the treatment groups compared with the model group, while the levels of Smad7 were increased. There was no difference in the above parameters among the three treatment groups or between the control and model groups (P>0.05), but the inhibitory effect of pGenesil-TGF-beta 1-ml was the highest among the treatment groups. CONCLUSIONS: Specific siRNA targeting of TGF-beta 1 markedly inhibited the fibrogenesis of immune hepatic fibrosis induced by Con A in mice. The anti-fibrosis mechanisms of siRNAs may be associated with the down-regulation of TGF-beta 1, Smad3 and alpha-SMA expression and up-regulation of Smad7 expression in liver tissue, which resulted in suppressing the activation of hepatic stellate cells. (Hepatobiliary Pancreat Dis Int 2009; 8: 300-308)

Platelet-rich plasma increases transforming growth factor-beta1 expression at graft-host interface following autologous osteochondral transplantation in a rabbit model

AIM: To explore the effect of platelet-rich plasma on protein expression patterns of transforming growth factor-beta1(TGF-β1) in cartilage following autologous osteochondral transplantation(AOT) in a rabbit knee cartilage defect model.METHODS: Twelve New Zealand white rabbits received bilateral AOT. In each rabbit, one knee was randomized to receive an autologous platelet rich plasma(PRP) injection and the contralateral knee received saline injection. Rabbits were euthanized at 3, 6 and 12 wk post-operatively. Articular cartilage sections were stained with TGF-β1 antibody. Histological regions of interest(ROI)(left, right and center of the autologous grafts interfaces) were evaluated using Meta Morph. Percentage of chondrocytes positive for TGF-β1 was then assessed.RESULTS: Percentage of chondrocytes positive for TGF-β1 was higher in PRP treated knees for selected ROIs(left; P = 0.03, center; P = 0.05) compared to control and was also higher in the PRP group at each post-operative time point(P = 6.6 × 10^(-4), 3.1 × 10^(-4) and 7.3 × 10^(-3) for 3, 6 and 12 wk, respectively). TGF-β1 expression was higher in chondrocytes of PRP-treated knees(36% ± 29% vs 15% ± 18%)(P = 1.8 × 10^(-6)) overall for each post-operative time point and ROI. CONCLUSION: Articular cartilage of rabbits treated with AOT and PRP exhibit increased TGF-β1 expression compared to those treated with AOT and saline. Our findings suggest that adjunctive PRP may increase TGF-β1 expression, which may play a role in the chondrogenic effect of PRP in vivo.

Effects of Tanshinone ⅡA on Transforming Growth Factor β1-Smads Signal Pathway in Renal Interstitial Fibroblasts of Rats

The effects of tanshinone ⅡA （TSN） on transforming growth factor β1 （TGFβ1） signal transduction in renal interstitial fibroblasts of rats were studied in order to investigate its mechanism in prevention of renal interstitial fibrosis. Rat renal fibroblasts of the line NRK/49F were cultured in vitro, stimulated with 5 ng/mL TGFβ1 and pretreated with 10-6, 10-5, 10-4 mol/L TSN respectively. The mRNA levels of fibronectin （FN） were examined by RT-PCR. The protein expression of FN and Smads was detected by Western blot. TGFβ1 induced the expression of FN mRNA and Smads in a time-dependent manner in a certain range. Compared with pre-stimulation, the FN mRNA and protein levels were increased by 1.1 times and 1.5 times respectively （P〈0.01, P〈0.01）, and the protein expression of phosphorylated Smad2/3 （p-Smad2/3） increased by 7 times at the end of TGFβ1 stimulation （P〈0.01）. TSN pretreatment may down-regulate the FN and p-Smad2/3 expression in a dose-dependent manner. 10-6 mol/L TSN pretreatment had no effect on the FN and p-Smad2/3 expression （both P〉0.05）. After pretreatment with 10-5 and 10-4 mol/L TSN, the FN mRNA levels were decreased by 28.1% and 43.8% respectively （P〈0.05, P〈0.01）, the FN protein levels were decreased by 40% and 44% respectively （P〈0.05, P〈0.05）, and the p-Smad2/3 protein expression were decreased by 40% and 65% respectively （P〈0.05, P〈0.01）. The inhibitory effect of TSN on renal interstitial fibrosis may be related to its blocking effect on TGFβ1-Smads signal pathway in renal intersti- tial fibroblasts.

Uncovering the profile of mutations of transforming growth factor beta-induced gene in Chinese corneal dystrophy patients

AIM： To uncover the mutations profile of transforming growth factor beta-induced （TGFBI） gene in Chinese corneal dystrophy patients and further investigate the characteristics of genotype-phenotype correlations. METHODS： Forty-two subjects （6 unrelated families including 15 patients and 8 unaffected members, and 19 sporadic patients） of Chinese origin were subjected to phenotypic and genotypic characterization. The corneal phenotypes of patients were documented by slit lamp photography. Mutation screening of the coding regions of TGFBI was performed by direct sequencing. RESULTS： We detected four corneal dystrophy types. The most frequent phenotypes were granular corneal dystrophy （GCD） （including 3 families and 8 sporadic patients） and lattice corneal dystrophy （LCD） （including 2 families and 9 sporadic patients）. The next phenotypes were corneal dystrophy of Bowman layer （CDB） （1 family and 1 sporadic patient） and epithelial basement membrane dystrophy （EBMD） （1 sporadic patient）. Six distinct mutations responsible for TGFBI corneal dystrophies were identified in 30 individuals with corneal dystrophies. Those were, p.R124H mutation in 1 family and 2 sporadic patients with GCD, p.R555W mutation in 2 families and 3 sporadic patients with GCD, p.R124C mutation in 2 families and 7 sporadic patients with LCD, p.A620D mutation in 1 sporadic patient with LCD, p.H626R mutation in 1 sporadic patient with LCD, and p.R555Q in 1 family and 1 sporadic patient with CDB. No mutation was detected in the remaining 3 atypical GCD patients and 1 EBMD patient, CONCLUSION： GCD and LCD are the most frequent phenotypes in Chinese population. R555W was the most common mutation for GCD; R124C was the most common mutation for LCD, Our findings extend the mutational spectrum of TFGBI , and this is the extensively delineated TGFBI mutation profile associated with the various corneal dystrophies in the Chinese population.

Pre-ischemia electro-acupuncture potentiates the expression of Bcl-2 and transforming growth factor-beta 1 in rat brains

The expression of the anti-apoptotic molecules Bcl-2 and transforming growth factor-beta 1 is known to confer protective effects on the cerebral ischemia-reperfusion injury.The current study investigated the expression levels of Bcl-2 and transforming growth factor-beta 1 in response to multiple pre-ischemia electro-acupuncture at acupoints Zusanli（ST36）and Fengchi（GB20） stimulation.Rats were divided into five groups：uninjured,control,non-acupoint,GB20 and ST36. Rats in the non-acupoint,GB20 and ST36 groups received 30 minutes（3 times or 18 times）of electro-acupuncture stimulation before experimental cerebral ischemia was induced.Bcl-2 and transforming growth factor-beta 1 were found to be significantly increased in the ST36 groups with either 3 or 18 electro-acupuncture treatments（P〈0.05）.The production was higher with 18 electro-acupuncture treatments in the ST36 groups（P〈0.05）.In the GB20 groups,significant increase was only observed in transforming growth factor-beta 1 with 18 electro-acupuncture treatments（P〈0.05）.No significant elevation of the level of transforming growth factor-beta 1 was observed in the non-acupoint groups.However,the production of Bcl-2 increased with 18 treatments in the non-acupoint groups（P〈0.05）.The data suggest that multiple pre-ischemia electro-acupuncture at ST36 was effective in conferring neuroprotective effect on the brain by means of upregulation of Bcl-2 and transforming growth factor-beta 1 and the effect was increase with the number of treatment.

Dab2 attenuates brain injury in APP/PS1 mice via targeting transforming growth factor-beta/SMAD signaling

Transforming growth factor-beta （TGF-β） type II receptor （TβRⅡ） levels are extremely low in the brain tissue of patients with Alzheimer＇s disease. This receptor inhibits TGF-β1/SMAD signaling and thereby aggravates amyolid-beta deposition and neuronal injury. Dab2, a specific adapter protein, protects T RII from degradation and ensures the effective conduction of TGF-β 1/SMAD signaling. In this study, we used an adenoviral vector to overexpress the Dab2 gene in the mouse hippocampus and investigated the regulatory effect of Dab2 protein on TGF-β1/SMAD signaling in a mouse model of Alzheimer＇s disease, and the potential neuroprotective effect. The results showed that the TβRⅡ level was lower.in APP/PS1 mouse hippocampus than in normal mouse hippocampus. After Dab2 expression, hippocampal TβRⅡ and p-SMAD2/3 levels were signifi- cantly increased, while amyloid-beta deposition, microglia activation, tumor necrosis factor- and interleulin-6 levels and neuronal loss were significantly attenuated in APP/PS1 mouse brain tissue. These results suggest that Dab2 can exhibit neuroprotective effects in Alzheimer＇s disease by regulating TGF-β1/SMAD signaling.

Two mutations in the transforming growth factor beta-induced gene associated with familial Lattice corneal dystrophy

AIM：To report a phenotypic variant pedigree of lattice corneal dystrophy（LCD）associated with two mutations,R124C and A546 D,in the transforming growth factor betainduced gene（TGFBI）.METHODS：A detailed ocular examination was taken for all participants of a LCD family. Peripheral blood leukocytes from each participant were extracted to obtain the DNA. Polymerase chain reaction（PCR）of all seventeen exons of TGFBI gene was performed. The products were sequenced and analyzed. Histological examination was carried out after a penetrating keratoplasty from the right eye of proband. RESULTS：Genetic analysis showed that the proband and all 6 affected individuals harbored both a heterozygous CGC to TGC mutation at codon 124 and a heterozygous GCC to GAC mutation at codon 546 of TGFBI. None of the 100 control subjects and unaffected family members was positive for these two mutations. Ocular examination displayed multiple refractile lattice-like opacities in anterior stroma of the central cornea and small granular deposits in the peripheral cornea. The deposits were stained positively with Congo red indicating be amyloid in nature and situated mainly in the anterior and middle stroma. CONCLUSION：We observed a novel LCD family which carried two pathogenic mutations（R124C and A546D）in the TGFBI gene. The phenotypic features were apparently different from those associated with corresponding single mutations. The result reveals that although the definite mutation is the most important genetic cause of the disease,some different modifier alleles may influence the phenotype.

Expression of transforming growth factor β receptors in CCI_4, -induced cirrhotic liver in rats

Objective: To investigate the expression and the role of transforming growth factor 6 (TGF-β) receptors in CCI4 --Induced cirrhotic liver. Methods: In situ hybridization was used. Results: The TGF--β type I receptors mRNA was mainly expressed in Ito cells, endothelial cells and myofibroblasts. Only a few hepatocytes expressed it. However. the TGF-β type Ⅱ receptors was mostly localized in endothelial cells and Ito cells but few hepatocytes. The expression of mRNA of both the 2 types of receptors were significantly increased in the cirrhotic liver than in the control. Conclusion: The autocrine and paracrine effects of TGF-β on matrix production and activation of Ito cells might be an important factor of fibrogenesis in CCI4 cirrhotic ever.