• Clinical and Experimental Reproductive Medicine
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• v.47 no.2
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• pp.108-113
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• 2020

Objective: Endometrial fibrosis, the primary pathological feature of intrauterine adhesion, may lead to disruption of endometrial tissue structure, menstrual abnormalities, infertility, and recurrent pregnancy loss. At present, no ideal therapeutic strategy exists for this fibrotic disease. Eupatilin, a major pharmacologically active flavone from Artemisia, has been previously reported to act as a potent inducer of dedifferentiation of fibrotic tissue in the liver and lung. However, the effects of eupatilin on endometrial fibrosis have not yet been investigated. In this study, we present the first report on the impact of eupatilin treatment on transforming growth factor beta (TGF-β)-induced endometrial fibrosis. Methods: The efficacy of eupatilin on TGF-β-induced endometrial fibrosis was assessed by examining changes in morphology and the expression levels of fibrosis markers using immunofluorescence staining and quantitative real-time reverse-transcription polymerase chain reaction. Results: Eupatilin treatment significantly reduced the fibrotic activity of TGF-β-induced endometrial fibrosis in Ishikawa cells, which displayed more circular shapes and formed more colonies. Additionally, the effects of eupatilin on fibrotic markers including alpha-smooth muscle actin, hypoxia-inducible factor 1 alpha, collagen type I alpha 1 chain, and matrix metalloproteinase-2, were evaluated in TGF-β-induced endometrial fibrosis. The expression of these markers was highly upregulated by TGF-β pretreatment and recovered to the levels of control cells in response to eupatilin treatment. Conclusion: Our findings suggest that suppression of TGF-β-induced signaling by eupatilin might be an effective therapeutic strategy for the treatment of endometrial fibrosis.

• Development and Reproduction
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• v.3 no.1
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• pp.95-100
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• 1999

Growth differentiation factor-9 (GDF-9) is a member of the transforming growth factor $\beta$ (TGF-$\beta$) superfamily. It has been known that GDF-9 is a growth factor having a crucial role in normal folliculogenesis and its expression is oocyte-specific. The present study was aimed to elucidate the expression of GDF-9 mRNA in the mouse primordial follicles as well as in the other developmental stages. The semiquantitative analysis of GDF-9 mRNA expression was conducted. Total RNA was extracted from the ICR mice ovaries at gestational day 19, postnatal day 1, day 10, day 21, and day 28, and RT-PCR was performed to measure GDF-9 and $\beta$-actin mRNA levels. Level of GDF-9 mRNA were normalized against the level of $\beta$-actin mRNA, and compared among different stages. GDF-9 mRNA was detected in all samples including the fetal ovaries that mainly consists of primordial follicles. The highest level of mRNA was observed in ovaries obtained at day 10 that mainly consists of growing follicles. The present result suggests that GDF-9 may play an important role in the early stage of folliculogenesis.

• Archives of Pharmacal Research
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• v.25 no.6
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• pp.903-909
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• 2002

Research in the cytokine field has grown exponentially in recent years, and the validity of such studies relies heavily on the appropriate measurement of levels of cytokines in various biological samples. Transforming growth factor (TGF)-$\beta$, a hormonally active polypeptide found in normal and transformed tissue, is a potent regulator of cell growth and differentiation. The most widely used bioassay for TGF-$\beta$ is the inhibition of the proliferation of mink lung epithelial cells. Though detection of [$^3$H]thymidine incorporation is more sensitive than the MTT assay, it presents some disadvantages due to the safety and disposal problems associated with radioisotopes. In this study, we attempted to ascertain the experimental conditions which could be used for measuring the in vitro biological activity of TGF-$\beta$ in a safer and more sensitive way compared with the currently available methods. We compared the commonly used method, the MTT assay, to the XTT assay using different parameters including cell number, incubation time and the wave length used for detecting the product. We examined the anti-proliferative activities of TGF-$\beta$ in three different cell lines: Mv-1-Lu mink lung epithelial cells, MCF10A human breast epithelial cells and H-ras-transformed MCF10A cells. Herein, we present an experimental protocol which provides the most sensitive method of quantifying the biological activity of TGF-$\beta$, with a detection limit of as low as 10 pg/ml: Mv-1-Lu or H-ras MCF10A cells ($1{\times}10^5/well$) were incubated with TGF-$\beta$ at $37^{\circ}C$ in a humidified $CO_2$ incubator for 24 hr followed by XTT treatment and determination of absorbance at 450 or 490 nm. Our results may contribute to the establishment of an in vitro bioassay system, which could be used for the satisfactory quantitation of TGF-$\beta$.

• BMB Reports
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• v.29 no.5
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• pp.405-410
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• 1996

Previous studies have shown that transforming growth factor beta ($TGF-{\beta}$) is a potent regulator of cell growth and differentiation. To study the effects of $TGF-{\beta}$ on cell morphology and cytoskeleton reorganization, we conducted a survey using Mv1Lu mink lung epithelial cells with antibodies to cytoskeletal proteins and an extracellular matrix protein. While the untreated cells showed a cuboidal shape of typical epithelia, the Mv1Lu cells displayed a drastic shape change in the presence of $TGF-{\beta}$. This alteration was most prominent when near-confluent cells were treated with $TGF-{\beta}$. Since the morphology alteration is known to be accompanied by the reorganization of cytoskeletal proteins in other cell types, we investigated the intracellular distribution of the three major cytoskeletal structures: microfilaments, microtubules, and intermediate filaments. In the microfilament system, $TGF-{\beta}$ induced new stress fiber formation, which was caused primarily by the polymerization of cytoplasmic G-actin. However, $TGF-{\beta}$ appeared not to induce any significant changes in microtubular structures and vimentin filaments as determined by indirect fluorescence microscopy. Finally we confirmed the rapid accumulation of fibronectin by immunoblot analysis and chased the protein locations by immunofluorescence microscopy.

• Clinical and Experimental Pediatrics
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• v.52 no.5
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• pp.594-602
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• 2009

Purpose : Transforming growth factor (TGF)-${\beta}1$ reportedly increases neuronal survival by inhibiting the induction of inducible nitric oxide synthase (NOS) in astrocytes and protecting neurons after excitotoxic injury. However, the neuroprotective mechanism of $TGF-{\beta}1$ on hypoxic-ischemic (HI) brain injury in neonatal rats is not clear. The aim of this study was to determine whether $TGF-{\beta}1$ has neuroprotective effects via a NO-mediated mechanism and N-methyl-D-aspartate (NMDA) receptor modulation on perinatal HI brain injury. Methods : Cortical cells were cultured using 19-day-pregnant Sprague-Dawley (SD) rats treated with $TGF-{\beta}1$ (1, 5, or 10 ng/mL) and incubated in a 1% O2 incubator for hypoxia. Seven-day-old SD rat pups were subjected to left carotid occlusion followed by 2 h of hypoxic exposure (7.5% $O_2$). $TGF-{\beta}1$ (0.5 ng/kg) was administered intracerebrally to the rats 30 min before HI brain injury. The expressions of NOS and NMDA receptors were measured. Results : In the in vitro model, the expressions of endothelial NOS (eNOS) and neuronal NOS (nNOS) increased in the hypoxic group and decreased in the 1 ng/mL $TGF-{\beta}1-treated$ group. In the in vivo model, the expression of inducible NOS (iNOS) decreased in the hypoxia group and increased in the $TGF-{\beta}1$-treated group. The expressions of eNOS and nNOS were reversed compared with the expression of iNOS. The expressions of all NMDA receptor subunits decreased in hypoxia group and increased in the $TGF-{\beta}1$-treated group except NR2C. Conclusion : The administration of $TGF-{\beta}1$ could significantly protect against perinatal HI brain injury via some parts of the NO-mediated or excitotoxic mechanism.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.79-79
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• 1995

Although synthetic antisense oligodeoxynucleotides (ODNs) have been used to dissect gene function in vitro, technical difficulties of targeted delivery prevented the use of this approach for investigating the effect of gene products in vivo. Here we report the use of local delivery of antisense transforming growth factor-${\beta}$l (TGF-${\beta}$1) oligonucleotides to decrease the fibrosis in the skin wound. Adult wounds heal with scar-tissue formation, whereas fetal wounds heal without scarring and with a lesser inflammatory and cytokine response. We reasoned that strategy emptying antisense TGF-${\beta}$1 ODNs complementary to TGF-${\beta}$1 mRNA might decrease the scarring in dermal wound of mouse. To evaluate this concept, we tested the effects of antisense ODNs targeted to TGF-${\beta}$1 mRNA by topical application of the chemical on the skin wound. Phosphorothioate antisense ODNs was employed to retard their degradation. When antisense TGF-${\beta}$1 ODNs were applied into the wound site, there was a maked reduction of scar compared with control wound site, These effects of antisense TGF-${\beta}$1 ODNs on the scar formation were associated with decreased expression of TGF-${\beta}$1 gene. However sense TGF-${\beta}$l ODNs had no effect on expression of TGF-${\beta}$1 gene. Also, control wounds healed with excessive fibrosis, whereas the antisense treated wounds healed with less fibrosis. In conclusion, our results indicate that antisense TGF-${\beta}$1 ODNs could be used for amelioating scar formation during wound healing.

• IMMUNE NETWORK
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• v.1 no.3
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• pp.244-249
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• 2001

The transforming growth $factor-{\beta}$ ($TGF-{\beta}$) is a multifunctional cytokine modulating the onset and course of autoimmune disease as shown in experimental models. In synovial inflammation, there is a potential role for $TGF-{\beta}$ in repairment, the inhibition of cartilage and bone destruction, and the down-regulation of immune response. The biologic effects of $TGF-{\beta}$ depend on the cell type, the isoform and the availability of active $TGF-{\beta}$. We investigated $TGF-{\beta}$ expression in patients with rheumatoid arthritis (RA) and compared to those of osteoarthritis (OA). And we determined a correlation between $TGF-{\beta}1$ and $TGF-{\beta}2$, and also the relationships between each $TGF-{\beta}$ isoform and the parameters for disease activity of RA. Methods: The study population consisted of 20 patients with RA and 20 patients with OA. The commercial ELISA kit was used to study $TGF-{\beta}1$ and $TGF-{\beta}2$ levels in peripheral blood (PB) and synovial fluids (SF). Results: 1) While PB $TGF-{\beta}1$ level was of no difference between RA and OA patient groups, SF $TGF-{\beta}1$ level was higher in RA group than OA group. Similarly, PB $TGF-{\beta}2$ levels of RA and OA groups was not different, but SF $TGF-{\beta}2$ levels was higher in RA group than OA group. 2) In patients with RA, the $TGF-{\beta}1$ levels were higher than $TGF-{\beta}2$ in both the PB and SF, while in patients with OA, there showed higher readings for $TGF-{\beta}1$ than $TGF-{\beta}2$ in SF but no difference between $TGF-{\beta}1$ and $TGF-{\beta}2$ levels in PB. 3) In patients with RA, there were no correlations between PB $TGF-{\beta}1$ and PB $TGF-{\beta}2$ levels, nor between SF $TGF-{\beta}1$ and SF $TGF-{\beta}2$ levels. At the same way, there was no correlation between PB $TGF-{\beta}1$ and SF $TGF-{\beta}1$ levels, nor between each levels of $TGF-{\beta}2$ in patients with RA. 4) There was also no correlation between each $TGF-{\beta}$ isoform and the parameters for disease activity such as ESR, CRP, tender joint count, swollen joint count, rheumatoid factor, and the duration of morning stiffness except between in PB $TGF-{\beta}1$ and disease duration of RA (r=0.637, p<0.01). Conclusion: Each $TGF-{\beta}$ isoforms were higher in synovial fluid of patients with RA than that of patients with OA. The data from the RA patients demonstrated different patterns of expressions of the isoforms depending on which compartment (PB or SF) was investigated. The quantification of different $TGF-{\beta}$ isoform is thought to be important when $TGF-{\beta}$ is measured under disease conditions of RA.

• Journal of Life Science
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• v.11 no.2
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• pp.181-189
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• 2001

Cytokines are hormone-like proteins which mediate and regulast inflammatory and immune responses. Transforming growth factor -$\beta$1(TGF-$\beta$) plays an important role in the control of the immune response and wound healing, and in the development o various tissues and organs, Nitric oxide(NO) is major messenger molecule regulating immune function and blood vessel dilation and serving as a neurotransmitter in the brain and peripheral nervous system. Also, NO is to be a potent mutagen that cause mutation in the p53 tumor suppressor gene in early phases of human gastric carcinogenesis. The purpose of this study was to investigate the effect of Helicobacter phlori lystes, lipopolysaccharide (LPS), and Staphylococcus enterotoxin B(SEB) on production of TGF-$\beta$1 and NO by human fibroblasts. Primary cultured human fibroblasts were incubated with H. pylori lysates(Hp), LPs, SEB, Hp+LPS, Hp+SEB, Hp+LPS+SEB. Cultured supernatants that were collected at 24, 48 and 72 hr were assessed for TGF-$\beta$1 by enzyme-linked immunosorbent assay and NO production by quantification of nitrite ion. TGF-$\beta$1 production in fibroblasts exposed with Hp, LPS or SEB for 48 hrs was enhanced, but for 72 hrs inhibited. Its production by doble exposure such as Hp+LPS, Hp+SEB, Hp+LPS+SEB was lowered in comparison with single exposure of Hp in cases of 24 and 48 hrs incubation, but for 72 hrs decreased in Hp vaculoating toxin(+), increased in Hp vacuolating toxin(-). No production in fibroblasts increaed at all doses of LPS. But its production by exposure of SEB increased or decreased according to dose and incubation time. Also, NO production by Hp vacuolating toxin(+) increased at all doses, but its production by Hp vacuolating toxin(-) decreased. Its production by doble exposure such as Hp+LPS, Hp+SEB, Hp+LPS+SEB decreased in comparison with single exposure Hp Therefore, quantities pf TGB-$\beta$1 and NO released by human fibroblasts shows differences according to kinds of stimulants. Also, in care stimulated with same kinds of stimulants, its productions exhibit quantitative differences according to exposure times. These results suggest that the decreased of TGF-$\beta$1 in fibroblasts by mixed exposure with Hp producing vacuolating toxin and bacterial toxins such as LPS and SEB may effect negatively in healing of host tissue and increased of NO by infection oh H. pylori may related to the increased susceptibility for human gastric carcinogenesis.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.73-80
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• 1998

This study was undertaken to evaluate the interaction between cumulus cells and TGF $\beta$1 on in vitro maturation in porcine oocytes. No differ ences were found in maturation rates when follicular oocytes were cultured in medium with various concentrations of TGF $\beta$. At 24 h after maturation, the oocytes matured to metaphase-II were found in medium with TGF $\beta$ regardless of cumulus cells. On the other hand, the maturation rates were significantly(P < 0.01 higher cumulus-enclosed(70 and 52%) than cumulus-denuded oocytes(35 and 26%) in medium with or without TGF $\beta$ at 48 h after culture. In a another experiment, the same maturation rates (54-71%) were observed when cumulus-enclosed oocytes were cultured with various addition time of TGF $\beta$. However, the maturation rates in cumulus-denuded oocytes were significantly (P < 0.05) higher in medium added at 0~24 h (59%) or 24-48 h(57%) after culture than in medium with(27%) and without(38%) TGF $\beta$ for 48 h. These results indicated that cumulus cells is essential for in vitro maturation in porcine oocytes but TGF $\beta$ can promote oocytes maturation in cumulus-free oocytes.

• Molecules and Cells
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• v.40 no.3
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• pp.211-221
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• 2017

Transforming growth factor ${\beta}1$ $(TGF{\beta}1)/Smad4$ signaling plays a pivotal role in maintenance of the dynamic balance between bone formation and resorption. The microRNA miR-155 has been reported to exert a significant role in the differentiation of macrophage and dendritic cells. The goal of this study was to determine whether miR-155 regulates osteoclast differentiation through $TGF{\beta}1/Smad4$ signaling. Here, we present that $TGF{\beta}1$ elevated miR-155 levels during osteoclast differentiation through the stimulation of M-CSF and RANKL. Additionally, we found that silencing Smad4 attenuated the upregulation of miR-155 induced by $TGF{\beta}1$. The results of luciferase reporter experiments and ChIP assays demonstrated that $TGF{\beta}1$ promoted the binding of Smad4 to the miR-155 promoter at a site located in 454 bp from the transcription start site in vivo, further verifying that miR-155 is a transcriptional target of the $TGF{\beta}1/Smad4$ pathway. Subsequently, TRAP staining and qRT-PCR analysis revealed that silencing Smad4 impaired the $TGF{\beta}1$-mediated inhibition on osteoclast differentiation. Finally, we found that miR-155 may target SOCS1 and MITF to suppress osteoclast differentiation. Taken together, we provide the first evidence that $TGF{\beta}1/Smad4$ signaling affects osteoclast differentiation by regulation of miR-155 expression and the use of miR-155 as a potential therapeutic target for osteoclast-related diseases shows great promise.