• Journal of Nutrition and Health
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• v.55 no.6
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• pp.617-629
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• 2022

Purpose: Osteoporosis is characterized by structural deterioration of the bone tissue because of the loss of osteoblastic activity or the increase in osteoclastic activity, resulting in bone fragility and an increased risk of fractures. Hederagenin (Hed) is a pentacyclic triterpenoid saponin isolated from Dipsaci Radix, the dried root of Dipsacus asper Wall. Dipsaci Radix has been used in Korean herbal medicine to treat bone fractures. In this study, we attempted to demonstrate the potential anti-osteoporotic effect of Hed by examining its effect on osteoblast differentiation in MC3T3-E1 cells. Methods: Osteoblastic MC3T3-E1 cells were cultured in 0, 1, and 10 ㎍/mL Hed for 3 and 7 days. The activity of alkaline phosphatase (ALP), bone nodule formation and level of expression of bone-related genes and proteins were measured in MC3T3-E1 cells exposed to Hed. The western blot test was used to detect the activation of the bone morphogenetic protein-2 (BMP2)/ Suppressor of Mothers against Decapentaplegic (SMAD)1 pathway. Results: Hed significantly increased the proliferation of MC3T3-E1 cells. Intracellular ALP activity was significantly increased in the 1 ㎍/mL Hed-treated group. Hed significantly increased the concentration of calcified nodules. Furthermore, Hed significantly upregulated the expression of genes and proteins associated with osteoblast proliferation and differentiation, such as Runt-related transcription factor 2 (Runx2), ALP, osteopontin (OPN), and type I procollagen (ProCOL1). Induction of osteoblast differentiation by Hed was associated with increased BMP2. In addition, Hed induced osteoblast differentiation by increasing the activity of SMAD1/5/8. These results suggest that Hed has the potential to prevent osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells via the modulation of the BMP2/SMAD1 pathway. Conclusion: The results presented in this study indicate that Hed isolated from Dipsaci Radix has the potential to be developed as a healthcare food and functional material possessing anti-osteoporosis effects.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.2083-2087
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• 2014

Colorectal cancers remain to be a common cause of cancer-related death. Early-onset cases as well as those of various ethnic origins have aggressive clinical features, the basis of which requires further exploration. The aim of this work was to examine the expression patterns of $p15^{INK4b}$ and SMAD4 in colorectal carcinoma of different ethnic origins. Fifty-five sporadic colorectal carcinoma of Egyptian origin, 25 of which were early onset, and 54 cancers of Finnish origin were immunohistochemically stained with antibodies against $p15^{INK4b}$ and SMAD4 proteins. Data were compared to the methylation status of the $p15^{INK4b}$ gene promotor. $p15^{INK4b}$ was totally lost or deficient (lost in ${\geq}50%$ of tumor cell) in 47/55 (85%) tumors of Egyptian origin as compared to 6/50 (12%) tumors of Finnish origin (p=7e-15). In the Egyptian cases with $p15^{INK4b}$ loss and available $p15^{INK4b}$ promotor methylation status, 89% of cases which lost $p15^{INK4b}$ expression were associated with $p15^{INK4b}$ gene promotor hypermethylation. SMAD4 was lost or deficient in 25/54 (46%) tumors of Egyptian origin and 28/48 (58%) tumors of Finnish origin. 22/54 (41%) Egyptian tumors showed combined loss/deficiency of both $p15^{INK4b}$ and SMAD4, while $p15^{INK4b}$ was selectively lost/deficient with positive SMAD4 expression in 24/54 (44%) tumors. Loss of $p15^{INK4b}$ was associated with older age at presentation (>50 years) in the Egyptian tumors (p=0.04). These data show for the first time that $p15^{INK4b}$ loss of expression marks a subset of colorectal cancers and ethnic origin may play a role in this selection. In a substantial number of cases, the loss was independent of SMAD4 but rather associated with $p15^{INK4b}$ gene promotor hypermethylation and old age which could be related to different environmental exposures.

• Tuberculosis and Respiratory Diseases
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• v.82 no.1
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• pp.42-52
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• 2019

Background: Transforming growth factor ${\beta}$ (TGF-${\beta}$), retinoic acid (RA), p38 mitogen-activated protein kinase (MAPK), and MEK signaling play critical roles in cell differentiation, proliferation, and apoptosis. We investigated the effect of RA and the role of these signaling molecules on the phosphorylation of Smad2/3 (p-Smad2/3) induced by TGF-${\beta}1$. Methods: A549 epithelial cells and CCD-11Lu fibroblasts were incubated and stimulated with or without all-trans RA (ATRA) and TGF-${\beta}1$ and with MAPK or MEK inhibitors. The levels of p-Smad2/3 were analyzed by western blotting. For animal models, we studied three experimental mouse groups: control, bleomycin, and bleomycin+ATRA group. Changes in histopathology, lung injury score, and levels of TGF-${\beta}1$ and Smad3 were evaluated at 1 and 3 weeks. Results: When A549 cells were pre-stimulated with TGF-${\beta}1$ prior to RA treatment, RA completely inhibited the p-Smad2/3. However, when A549 cells were pre-treated with RA prior to TGF-${\beta}1$ stimulation, RA did not completely suppress the p-Smad2/3. When A549 cells were pre-treated with MAPK inhibitor, TGF-${\beta}1$ failed to phosphorylate Smad2/3. In fibroblasts, p38 MAPK inhibitor suppressed TGF-${\beta}1$-induced p-Smad2. In a bleomycin-induced lung injury mouse model, RA decreased the expression of TGF-${\beta}1$ and Smad3 at 1 and 3 weeks. Conclusion: RA had inhibitory effects on the phosphorylation of Smad induced by TGF-${\beta}1$ in vitro, and RA also decreased the expression of TGF-${\beta}1$ at 1 and 3 weeks in vivo. Furthermore, pre-treatment with a MAPK inhibitor showed a preventative effect on TGF-${\beta}1$/Smad phosphorylation in epithelial cells. As a result, a combination of RA and MAPK inhibitors may suppress the TGF-${\beta}1$-induced lung injury and fibrosis.

• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.747-760
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• 2005

Periodontal therapy has dealt primarily with attempts at arresting progression of disease. however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. Recombinant human bone morphogenetic protein-7(rhBMP-7) can differentiate the osteoprogenitor cells and induce bone formation. The purpose of this study was to evaluate the effect of BMP-7 on rat periodontal ligament cells differentiation, in vitro. In the control group, cells was cultured with DMEM media. In the experimental groups, cells were cultured with rhBMP-7 in concentration of 10, 25, 50 and 100 ng/ml. Each group was characterized by examining alkaline phosphatase activity at 3 and 5 days of culture and the ability to produce mineralized nodules of rat calvarial cells at 14 days of culture. Synthesis of type I collagen(COL-I), osteocalcin(OCN), and bone sialoprotein(BSP) was evaluated by RT-PCR at 7 days of culture. Activation of Smad proteins and p38 MAP kinase was determined by western blot analysis of the cell lysates. Alkaline phosphatase activity was significantly increased in the concentration of BMP-7 50 ng/ml and 100 ng/ml compared to the control(p<0.05). The mineralized bone nodule formation was greater with addition of 50 ng/ml and 100 ng/ml BMP-7 than the control(p<0.01). In 7 days' culture, the expressions of COL-I, BSP, and OCN was increased by BMP-7 in concentration of 10 $ng/ml{\sim}100$ ng/ml. In western blot analysis, BMP-7 treated culture cells expressed Smad 1,5,8 in dose-dependent manner, whereas BMP-7 did not activate phosphorylated form of p38 MAP kinase. These result suggested that BMP-7 stimulate rat periodontal ligament cells to differentiate toward osteoblast phenotype and increase bone matrix production by activation of BMP-Smad pathway.

• BMB Reports
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• v.55 no.8
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• pp.401-406
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• 2022

Ahnak, a large protein first identified as an inhibitor of TGF-β signaling in human neuroblastoma, was recently shown to promote TGF-β in some cancers. The TGF-β signaling pathway regulates cell growth, various biological functions, and cancer growth and metastasis. In this study, we used Ahnak knockout (KO) mice that underwent a 70% partial hepatectomy (PH) to investigate the function of Ahnak in TGF-β signaling during liver regeneration. At the indicated time points after PH, we analyzed the mRNA and protein expression of the TGF -β/Smad signaling pathway and cell cycle-related factors, evaluated the cell cycle through proliferating cell nuclear antigen (PCNA) immunostaining, analyzed the mitotic index by hematoxylin and eosin staining. We also measured the ratio of liver tissue weight to body weight. Activation of TGF-β signaling was confirmed by analyzing the levels of phospho-Smad 2 and 3 in the liver at the indicated time points after PH and was lower in Ahnak KO mice than in WT mice. The expression levels of cyclin B1, D1, and E1; proteins in the Rb/E2F transcriptional pathway, which regulates the cell cycle; and the numbers of PCNA-positive cells were increased in Ahnak KO mice and showed tendencies opposite that of TGF-β expression. During postoperative regeneration, the liver weight to body weight ratio tended to increase faster in Ahnak KO mice. However, 7 days after PH, both groups of mice showed similar rates of regeneration, following which their active regeneration stopped. Analysis of hepatocytes undergoing mitosis showed that there were more mitotic cells in Ahnak KO mice, consistent with the weight ratio. Our findings suggest that Ahnak enhances TGF-β signaling during postoperative liver regeneration, resulting in cell cycle disruption; this highlights a novel role of Ahnak in liver regeneration. These results provide new insight into liver regeneration and potential treatment targets for liver diseases that require surgical treatment.

• The Korea Journal of Herbology
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• v.39 no.4
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• pp.19-28
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• 2024

Objectives : Ephedrae Herba has been used in the East Asian traditional medicine, for treatment of asthma, cold and influenza. Silymarin is an effective antioxidant and its anti-fibrogenic, anti-inflammatory, and hepatoprotective properties have been reported. This study was performed to explore an anti-fibrogenic potential of Ephedrae Herba extract (EHE) + silymarin on immortalized human hepatic stellate cell line, LX-2 cells. Methods : We studied the anti-fibrogenic effects of EHE + silymarin on transforming growth factor β1 (TGF-β1) signaling pathway in LX-2 cells. Cell viability was measured using the MTT assay. mRNA levels were detected by real-time PCR. TGF-β1 signaling-related proteins expression were detected by Western blot. Results : Silymarin 30 ㎍/mL and EHE 100 ㎍/mL showed cytotoxicity on LX-2 cells. Therefore, the concentrations of silymarin and EHE were studied at 10 ㎍/mL, respectively. Silymarin significantly reduced PAI-1 protein expression, Smad binding element (SBE) luciferase activity, and mRNA (PAI-1, MMP2 and 9) expression compared to TGF-β1. EHE significantly reduced SBE luciferase activity and mRNA (PAI-1, MMP2 and 9) expression compared to TGF-β1. More importantly, EHE + silymarin significantly reduced all parameters compared to TGF-β1, and also significantly reduced compared to EHE alone and silymarin alone. Conclusion : The results indicate that EHE + silymarin has anti-fibrogenic effect in LX-2 cells induced by TGF-β1. Additionally, EHE + silymarin shows more effective anti-fibrogenic effect than EHE alone and silymarin alone.

• The Korea Journal of Herbology
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• v.34 no.2
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• pp.75-82
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• 2019

Objectives : In this study, various biological effects of Curcuma longa L. have been studied, however, beneficial effect of Curcuma longa L. in skin health remain still unclear. In this study, Curcuma longa L. water extract (CLE) was prepared. Inhibitory effect of CLE on melanin accumulation of B16F10 cells and expression levels of skin fibril-related proteins of human skin fibroblasts (HSF) were evaluated. Methods : The cytotoxic effect of CLE in B16F10 cells and HSF were examined by MTT assay. Inhibitory effect of CLE on the ${\alpha}-MSH-$ and IBMX-induced melanin accumulation and tyrosinase activity were evaluated in B16F10 cells. The expression levels of connective tissue growth factor (CCN2), Smad2, procollagen $1{\alpha}2$, collagen $1{\alpha}2$, and fibronectin in CLE-treated HSF were analyzed by western blotting. Results : The CLE treatment (concentrations 10 to $400{\mu}g/ml$) for 72 h did not affect to the B16F10 viability. However, 200 and $400{\mu}g/ml$ of CLE treatment for 24 h showed cytotoxic effect in HSF. Therefore, the concentrations 10, 50, and $100{\mu}g/ml$ of CLE were chosen in this study. The CLE treatment for 72 h dose dependently and significantly suppressed melanin accumulation and tyrosinase activity of B16F10 cells. In addition, the CLE treatment up-regulated expression levels of skin fibril-related proteins such as CCN2, Smad2, procollagen $1{\alpha}2$, collagen $1{\alpha}2$, and fibronectin. Conclusions : In conclusion, these results suggest that the CLE could be used as a natural material for skin health.

• Proceedings of the KACD Conference
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• 2007.11a
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• pp.643-643
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• 2007

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.36 no.6
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• pp.460-465
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• 2010

Introduction: A cleft palate is a common birth defect in humans with an incidence of 1/500 to 1/1,000 births. It appears to be caused by multiple genetic and environmental factors during palatogenesis. Many molecules are involved in palate formation but the biological mechanisms underlying the normal palate formation and cleft palate are unclear. Accumulating evidence suggests that transforming growth factor $\beta$/bone morphogenetic proteins (TGF-$\beta$/BMP) family members mediate the epithelial-mesenchymal interactions during palate formation. However, their roles in palatal morphogenesis are not completely understood. Materials and Methods: To understand the roles of TGF-$\beta$/BMP signaling in vivo during palatogenesis, mice with a palatal mesenchyme- specific deletion of Smad4, a key intracellular mediator of TGF-$\beta$/BMP signaling, were generated and analyzed using the Osr2Ires-Cre mice. Results: The mutant mice were alive at the time of birth with open eyelids and complete cleft palate but died within 24 hours after birth. In skeletal preparation, the horizontal processes of the palatine bones in mutants were not formed and resulted in a complete cleft palate. At E13.5, the palatal shelves of the mutants were growing as normally as those of theirwild type littermates. However, the palatal shelves of the mutants were not elevated at E14.5 in contrast to the elevated palatal shelves of the wild type mice. At E15.5, the palatal shelves of the mutants were elevated over the tongue but did not come in contact with each other, resulting in a cleft palate. Conclusion: These results suggest that mesenchymal Smad4 mediated signaling is essential for the growth of palatal processes and suggests that TGF-$\beta$/BMP family members are essential regulators during palate development.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.2
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• pp.105-113
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• 2006

Objective: To investigate the role of ERK and $PPAR{\gamma}$ on the $TGF-{\beta}1$ induced human endometrial stromal cell decidualization in vitro. Method: Endometrial stromal cells are cultured under the following condition: DMEM/F12 (10% FBS, 1 nM E2 and 100 nM P4). $TGF-{\beta}1$ (5 ng/ml), Rosiglitazone (50 nM), and PD98059 ($20{\mu}M$) were added according to experimental purposes. Trypan-Blue and hematocytometer were utilized to count cell number. Enzyme-linked immunosorbent assay (ELISA) and western blotting were utilized to detect proteins. Result: $TGF-{\beta}1$ inhibited proliferation of cultured human endometrial stromal cells and induced expression of PGE2 and prolactin. This effect was mediated by Smad and ERK activation. Administration of rosiglitazone, $PPAR{\gamma}$ agonist, prevented $TGF-{\beta}1$ effect on cell proliferation. Furthermore, Rosiglitazone inhibited $TGF-{\beta}1$ induced activation of ERK, consequently reduced PGE2 and prolactin production. Conclusion: $TGF-{\beta}1$ induced decidualization of endometrial stromal cell through Smad and ERK phosphorylation. $PPAR{\gamma}$ acts as a negative regulator of human ndometrial cell decidualization in vitro.