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ObjectiveTo investigate the effect and mechanism of pachymic acid （PA） in Poria on the invasion and metastasis of renal carcinoma cells. MethodThe effect of PA （0， 20， 40， 80， 160 μmol·L-1） on cell viability was detected by cell counting kit-8（CCK-8）， and the dose of PA was selected for subsequent experiments. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell proliferation was evaluated by colony formation assay. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell adhesion ability was observed by cell adhesion assay. The effect of PA （0， 20， 40， and 80 μmol·L-1） on cell invasion and metastasis was investigated by Wound healing assay and Transwell invasion assay. The inhibitory effect of PA （0， 20， 40， 80 μmol·L-1） on cell motility was further observed and verified by high-content imaging technology. The effects of PA （0， 20， 40， 80 μmol·L-1） on the expression of matrix metalloproteinase （MMP）/tissue inhibitor of metalloproteinasas （TIMP） related to invasion and metastasis and Smads were detected by Western blot. ResultCCK-8 results showed that compared with the blank group， the PA groups showed decreased cell viability（P<0.01）， with the half-maximal inhibitory concentration （IC50） of ACHN cells of 70.42 μmol·L-1 at 24 h. Colony formation assay showed that the number of cell clonal groups in the PA groups was reduced compared with that in the blank group（P<0.01）. Cell adhesion assay showed that compared with the blank group， the PA groups displayed reduced cell adhesion（P<0.01）. Wound healing assay showed that the wound healing rate of cells in the PA groups was lower than that in the blank group （P<0.05，P<0.01）. Transwell invasion assay showed that compared with the blank group， the number of transmembrane cells in PA groups was reduced（P<0.01）. High-content imaging showed that the cumulative migration distance of cells in the PA groups was shorter than that in the blank group（P<0.01）. The results of Western blot showed that the protein expression of MMP-2 and MMP-9 in the PA groups decreased （P<0.01）， and TIMP-1 protein expression increased （P<0.01） compared with those in the blank group. In addition， compared with the blank group， the PA groups showed decreased protein expression of Smad2 and Smad3 （P<0.01）. ConclusionPA can inhibit the invasion and metastasis of renal carcinoma cells presumably through regulating the homeostasis of MMP/TIMP by Smad2/3.
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Long noncoding RNAs (lncRNAs) are expressed in different species and different tissues, and perform different functions, but little is known about their involvement in the synthesis or secretion of follicle-stimulating hormone (FSH). In general, we have revealed lncRNA‒microRNA (miRNA)‒messenger RNA (mRNA) interactions that may play important roles in rat primary pituitary cells. In this study, a new lncRNA was identified for the first time. First, we analyzed the gene expression of lncRNA-m18as1 in different tissues and different stages by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and observed the localization of lncRNA-m18as1 with fluorescence in situ hybridization, which indicated that this lncRNA was distributed mainly in the cytoplasm. Next, we used RT-qPCR and enzyme-linked immunosorbent assay (ELISA) to analyze the regulation of FSH synthesis and secretion after overexpression or knockdown of lncRNA-m18as1 and found that lncRNA-m18as1 was positively correlated with FSH synthesis and secretion. In addition, mothers against decapentaplegic homolog 2 (Smad2) was highly expressed in our sequencing results. We also screened miR-18a-5p from our sequencing results as a miRNA that may bind to lncRNA-m18as1 and Smad2. We used RNA immunoprecipitation-qPCR (RIP-qPCR) and/or dual luciferase assays to confirm that lncRNA-m18as1 interacted with miR-18a-5p and miR-18a-5p interacted with Smad2. Fluorescence in situ hybridization (FISH) showed that lncRNA-m18as1 and miR-18a-5p were localized mainly in the cytoplasm. Finally, we determined the relationship among lncRNA-m18as1, miR-18a-5p, and the Smad2/3 pathway. Overall, we found that lncRNA-m18as1 acts as a molecular sponge of miR-18a-5p to regulate the synthesis and secretion of FSH through the Smad2/3 pathway.
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Animais , Ratos , Linhagem Celular Tumoral , Proliferação de Células , Hormônio Foliculoestimulante/metabolismo , Regulação Neoplásica da Expressão Gênica , Hibridização in Situ Fluorescente , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismoRESUMO
Long non-coding RNA (lncRNA) Dnm3os plays a critical role in peritendinous fibrosis and pulmonary fibrosis, but its role in the process of cardiac fibrosis is still unclear. Therefore, we carried out study by using the myocardial fibrotic tissues obtained by thoracic aortic constriction (TAC) in an early study of our group, and the
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Humanos , Fibroblastos , Fibrose , Miocárdio/patologia , RNA Longo não Codificante , Transdução de Sinais , Fator de Crescimento Transformador beta1RESUMO
OBJECTIVE@#To evaluate the effects of Huoxin Pill (, HXP) on cardiac fibrosis and heart failure (HF) in isoproterenol (ISO)-induced HF rats.@*METHODS@#Thirty Wistar rats were randomly divided into 5 groups including control, HF, isosorbide mononitrate (ISMN), HXP low (HXP-L), and HXP high (HXP-H) groups (n=6 for each group) according to the complete randomization method. Rats were pretreated with ISMN (5 mg/kg daily), low concentration of HXP (10 mg/kg daily) or high concentration of HXP (30 mg/kg daily) or equal volume of saline by intragastric administration for 1 week, followed by intraperitoneal injection of ISO (10 mg/kg, 14 days), and continually intragastric administrated with above medicines or saline for additional 6 weeks. The effects of HXP treatment on the cardiac function, heart weight index (HWI), pathological changes, and collagen content were further assessed. Moreover, the role of HXP on activation of transforming growth factor- β 1 (TGF-β 1)/Smads pathway was further explored using immunohistochemistry (IHC) and Western-blot assay.@*RESULTS@#HXP treatment significantly alleviated the decrease of ejection fraction (EF) and fractional shortening (FS), while decreased the elevation of left ventricular end-systolic volume (LVESV) in ISO-induced HF rats (P<0.05). Moreover, HXP treatment obviously attenuated the increase of HWI and serum level of creatine kinase MB (CK-MB, P<0.05), as well as pathological changes in ISO-induced HF rats. Further determination indicated that HXP treatment alleviated the elevation of collagen I and collagen III protein expression in cardiac tissues of ISO-induced HF rats. Furthermore, HXP treatment significantly down-regulated the increase of TGF-β 1 and p-Smad2/3 protein expression in cardiac tissues of HF rats (P<0.05), while did not affect the expression of total Smad2/3.@*CONCLUSIONS@#HXP attenuated heart failure and cardiac fibrosis in ISO-induced HF rats by suppression of TGF-β 1/Smad2/3 pathway.
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Aim To explore the effect of beta-sitosterol (BS) on liver fibrosis induced by CCL4 in mice and the mechanisms. Methods Fifty C57BL/6 male mice were randomly divided into five groups; control group (CG) , carbon tetrachloride group (CTG), low/medium/high dose of BS group ( BS-L/M/H), with 10 mice in each group. The model of hepatic fibrosis was established by injecting CCL4 in peritoneal cavity, the study lasted 30 days, and different doses of BS were given from 1st day to 15 th day. All mice were sacrificed for the observation of morphological changes and the measurement of liver index. Liver collagenous fibers were observed by HE and Masson staining, the changes of serum ( ALT and AST) were assessed by Elisa, the expressions of a-SMA and Collagen I were detected by Western blot and immunohistochemistry, and the changes of TpRl-Smad2/3 and TNF-a-NF∗kB were detected by Elisa and Western blot. Results Compared to control group, different doses of BS markedly inhibited the increase of liver index, A .T, AST, a-SMA and Collagen I in a dose-dependent n an-ner ( P < 0. 05 or P < 0. 01 ). Liver morphology, inflammatory cell infiltration and collagenous fiber irj BS groups were better than those in CCL4 group, meanwhile BS-M decreased the expression of TgKl, Smad2/3, TNF-a and p-NF-KB (P <0. 01). Conclusions BS dose-dependently inhibits mouse liver f bro-sis induced by CCL4, and its mechanism may be related to inhibiting TpRl-Smad2/3 and TNF-a-N •-kB signaling pathways.
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OBJECTIVE@#To study the anti- fibrotic effect of human umbilical cord mesenchymal stem cell-derived exosomes (hUCMSC-EXOs) and explore the mechanism.@*METHODS@#Twenty-four C57 BL/6 mice were divided into 4 groups (=6), including the control group treated with intratracheal injection of saline (3 mg/kg); lung fibrosis model group with intratracheal injection of 1.5 mg/mL bleomycin solution (prepared with saline, 3 mg/kg); EXOs1 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the next day after modeling); and EXOs2 group with intratracheal injection of 1.5 mg/mL bleomycin solution (3 mg/kg) and hUCMSC-EXOs (100 μg/250 μL, given by tail vein injection on the 10th day after modeling). At 21 days after modeling, pulmonary index, lung tissue pathology and collagen deposition in the mice were assessed using HE staining and Masson staining. The expression level of TGF-β1 was detected using ELISA, and vimentin, E-cadherin and phosphorylated Smad2/3 (p-Smad2/3) were detected using immunohistochemical staining. CCK8 assay was used to evaluate the effect of hUCMSCEXOs on the viability of A549 cells, and Western blotting was used to detect the expression levels of p-Smad2/3, vimentin, and E-cadherin in the cells.@*RESULTS@#Compared with those in the model group, the mice treated with hUCMSC-EXOs showed significantly reduced the pulmonary index ( < 0.05), collagen deposition, lung tissue pathologies, lowered expressions of TGF-β1 ( < 0.05), vimentin, and p-Smad2/3 and increased expression of E-cadherin. hUCMSC-EXOs given on the second day produced more pronounced effect than that given on the 11th day ( < 0.05). CCK8 assay results showed that hUCMSC-EXOs had no toxic effects on A549 cells ( > 0.05). Western blotting results showed that hUCMSC-EXOs treatment significantly increased the expression of E-cadherin and decreased the expressions of p-Smad2/3 and vimentin in the cells.@*CONCLUSIONS@#hUCMSC-EXOs can alleviate pulmonary fibrosis in mice by inhibiting epithelialmesenchymal transition activated by the TGF-β1/Smad2/3 signaling pathway, and the inhibitory effect is more obvious when it is administered on the second day after modeling.
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Animais , Humanos , Camundongos , Transição Epitelial-Mesenquimal , Exossomos , Células-Tronco Mesenquimais , Fibrose Pulmonar , Fator de Crescimento Transformador beta1 , Cordão UmbilicalRESUMO
OBJECTIVE@#To investigate the ameliorate effect and underlying mechanism of Xueshuantong for Injection (Lyophilized, , XST) in streptozocin (STZ)-induced diabetic retinopathy (DR) rats.@*METHODS@#Diabetes mellitus (DM) model was induced by intraperitoneal (i.p.) injection of STZ (60 mg/kg) in Sprague-Dawley rats. Diabetic rats were randomized into 3 groups (n=10) according to a random number table, including DM, XST50 and XST100 groups. XST treatment groups were daily i.p. injected with 50 or 100 mg/kg XST for 60 days, respectively. The control and DM groups were given i.p. injection with saline. Blood glucose level and body weight were recorded every week. Histological changes in the retina tissues were observed with hematoxylin-eosin staining. Apoptosis and inflammation related factors, including cleaved caspase-3, glial fifibrillary acidic protein (GFAP), tumor necrosis factor-α (TNF-α) and intercellular cell adhesion molecule-1 (ICAM-1) were detected by Western blot or real-time polymerase chain reaction. Then, the levels of advanced glycation end product (AGE) and its receptor (RAGE) were investigated. Tight junctions proteins (Zonula occludens-1 (ZO-1), Occludin and Claudin-5) of blood-retinal barrier were detected by Western blot. The levels of retinal fifibrosis, transforming growth factor-β1 (TGF-β1)-Smad2/3 signaling pathway were evaluated at last.@*RESULTS@#There was no signifificant difference in the body weight and blood glucose level between XST and DM groups (P>0.05). Compared with the DM group, XST treatment signifificantly increased the retinal thickness of rats (P<0.05 or P<0.01), and suppressed cleaved caspase-3 expression (P<0.01). XST increased the protein expressions of ZO-1, Occludin and Claudin-5 and decreased the mRNA expressions of matrix metalloproteinase 2 (MMP-2) and MMP-9 (P<0.05 or P<0.01). Moreover, XST signifificantly reduced the productions of AGE and RAGE proteins in the retina of rats (P<0.05 or P<0.01), suppressed the over-expression of TNF-α, and decreased the elevated level of ICAM-1 in retina of rats (P<0.05 or P<0.01). XST signifificantly reduced the levels of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), TGF-β1 and phosphorylation of Smad2/3 protein in rats (P<0.05 or P<0.01).@*CONCLUSIONS@#XST had protective effects on DR with possible mechanisms of inhibiting the inflammation and apoptosis, up-regulating the expression of tight junction proteins, suppressing the productions of AGE and RAGE proteins, and blocking the TGF-β/Smad2/3 signaling pathway. XST treatment might play a role for the future therapeutic strategy against DR.
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Objective: To further explore the role of TGF-β1/Smads signaling pathway in the tissue remodeling process of cultured nasal polyps in vitro. Methods: Fifteen cases of chronic rhinosinusitis with nasal polyps (CRSwNP) and 15 cases of chronic rhinosinusitis without nasal polyps (CRSsNP) were screened out, and the control group was 10 patients with deviation of nasal septum. The location and expression of proteins in tissues were analyzed by immunohistochemistry. The expression and distribution of collagen were detected by using the method of Masson trichrome staining. The levels of mRNA and protein expression were detected by quantitative realtime polymerase chain reaction (qRT-PCR) and Western blotting respectively. Nasal polyps were treated ex vivo by TGF-β1 (n=15). The levels of mRNA and protein expression in culture pellets and collagen expression in culture supernatant were analyzed by qRT-PCR, Western blotting and ELISA respectively. Results: The TGF-β1, Smad2, Smad3 mRNA and protein expression levels were significantly decreased in the CRSwNP group compared with the CRSsNP group (all P<0.05). TGF-β1 and pSmad2/3 protein level showed positive correlation in CRS (r=0.991, P<0.01), so was TGF-β1 and Smad2, Smad3 mRNA levels (r=0.581, r=0.658, both P<0.01). TGF-β1 had positive correlation with collagen expression in CRS (r=0.982, P<0.01). Compared with the controls ex vivo, the levels of Smad2, Smad3, pSmad2/3 and collagen were markedly increased after TGF-β1 treatment. Conclusion: TGF-β1/Smads signaling pathway may play an important role in tissue remodeling of CRSwNP, and cause collagen reduction and edematous mucous membrane in nasal polyps.
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Myocardial fibrosis leads to cardiac remodeling and heart failure after myocardial infarction .The mechanism of ven-tricular remodeling after myocardial infarction is complicated , which results from multi-factor interactions .TGF-β1-dependent Smads signal pathway is a definite mechanism which plays an important role in the process of myocardial fibrosis .Myocardial infarction activates the TGF-β1, which links the downstream Smads signaling to play a sustained biological effects via the ac-cumulation of activated cardiac fibroblasts and excess deposition of extracellular matrix , and the differentiation from myocardial fibroblasts to myofibroblasts .Therefore, how to block or control TGF-β1/Smads signaling pathway is an important research di-rection for the treatment of myocardial fibrosis after myocardial infarction.This paper reviews the current studies of the treat-ment of myocardial fibrosis after myocardial infarction based on the TGF-β1/Smads signaling pathway .
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Objective: To investigate the inhibitory effects of ginsenoside Re on intimal hyperplasia in balloon-injuried rats and further explore the role of TGF-β1/Smads signaling pathway in this protection. Methods: Fifty SD rats were randomly divided into five groups, including Sham operation group, model group, ginsenoside Re low, medium, and high-dose groups. The injured model of carotid artery intima was established by 2F balloon catheters in each group except the sham operation group. One day after model was established, animals were daily ig administered with distilled water in model group, Sham operation group, and ginsenoside Re (12.5 mg/kg, 25 mg/kg and 50 mg/kg) groups. Two weeks later, animals were sacrificed and the injured artery was taken for HE staining. The histopathological changes were observed and the lumen area, intima area, and media area as well as the ratio of intimal area/media area were detected. The expression of transforming growth factors β1 (TGF-β1), SMAD family member 2 (Smad 2) and Smad family member 3 (Smad 3) were measured by real time RT-PCR and immunohistochemistry. Results: Compared with the Sham operation group, the vessel cavity in the model group was narrower (P < 0.01); Compared with the model group, the medium and high dose of ginsenoside Re obviously alleviated vascular intimal hyperplasia (P < 0.05). Compared with the Sham operation group, the mRNA and protein expressions levels of TGF-β1, Smad 2, and Smad 3 in model group were higher (P < 0.01), which were obviously decreased in the medium and high-dose ginsenoside Re (P < 0.05). Conclusion: Ginsenoside Re could alleviate the vascular neointimal hyperplasia through suppressing the TGF-β1/Smads signaling pathway.
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Objective To investigate the effects of all-trans retinioc acid (ATRA)on proliferation of rat hepatic stellate cells (HSC-T6)and expressions of collagen Ⅰ,matrix metalloproteinase-2 (MMP-2),tissue inhibitor of metalloproteinases-1 (TIMP-1 )and signal protein Smad2/3 in TGF-β1-simulated HSC-T6 so as to explore the impact and molecular mechanisms of ATRA on liver fibrosis in vitro .Methods Cultured HSC-T6s were treated with different concentrations of ATRA (0.1,1,10 μmol/L)for fixed time (12,24,48 hours).After intervention time,cell proliferation was evaluated by MTT.Meanwhile,HSC-T6s stimulated by TGF-β1 (5 ng/mL)were treated with different concentrations of ATRA for 24 h.The mRNA expressions of COL1α2,MMP-2 and TIMP-1 were quantified by RT-PCR;the expression of Smad 2/3 protein was determined by cell immunochemistry.Results The proliferation of hepatic stellate cells was inhibited by ATRA in a dose-dependent manner (P < 0.05 ).After induced by TGF-β1,the mRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein were increased significantly compared with control group (P <0.05).However,ATRA could obviously reduce themRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein in HSC-T6 induced by TGF-β1 (P < 0.05 ).Conclusion ATRA can inhibit the proliferation of HSC-T6s and reduce the mRNA expressions of COL1α2,MMP-2 and TIMP-1 in HSC-T6 which were induced by TGF-β1.The anti-hepatic fibrosis function of ATRA may be related to its inhibition on the expression of Smad 2/3 protein in HSC-T6 to influence TGF-β1/Smad signaling pathway.
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AIM To explore the effects of Bushen Huoxue Recipe (Fluoritum,Psoraleae Fructus,Cuscutae Semen,etc.) on the protein expressions of transforming growth factor (TGF)-β1,TGF-βRⅡ and Smad2/3 in follicle wall granulosa cells in mice with autoimmune premature ovarian failure (POF).METHODS Balb/c female mice were subcutaneously injected with mouse zona pellucida 3 at multiple points to establish autoimmune POF model.POF mice were divided into model group,positive group (progynova),low-,middle-and high-dose of Bushen Huoxue Recipe groups.After intervention for 30 days,ovarian tissue was stained by hematoxylin-eosin (HE),and the protein expressions of TGF-β1,TGF-βRⅡ and Smad2/3 in follicle wall granulosa cells and ovarian tissue were detected by immunohistochemistry and Western blot,respectively.RESULTS Compared with the model group,the number of mature follicles in the Bushen Huoxue Recipe groups and the positive group was obviously increased,together with the decreased number of atretic follicles.The protein expressions of TGF-β1,TGF-βRⅡ and Smad2/3 in follicle wall granulosa cells and ovarian tissue in the Bushen Huoxue Recipe groups and the positive group were much higher than those in the model group (P ＜ 0.05).CONCLUSION Bushen Huoxue Recipe can improve ovarian function by up-regulating the protein expressions of TGF-β1,TGF-βRⅡ and Smad2/3 in granulosa cells.
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Objective To investigate the effects of all-trans retinioc acid (ATRA)on proliferation of rat hepatic stellate cells (HSC-T6)and expressions of collagen Ⅰ,matrix metalloproteinase-2 (MMP-2),tissue inhibitor of metalloproteinases-1 (TIMP-1 )and signal protein Smad2/3 in TGF-β1-simulated HSC-T6 so as to explore the impact and molecular mechanisms of ATRA on liver fibrosis in vitro .Methods Cultured HSC-T6s were treated with different concentrations of ATRA (0.1,1,10 μmol/L)for fixed time (12,24,48 hours).After intervention time,cell proliferation was evaluated by MTT.Meanwhile,HSC-T6s stimulated by TGF-β1 (5 ng/mL)were treated with different concentrations of ATRA for 24 h.The mRNA expressions of COL1α2,MMP-2 and TIMP-1 were quantified by RT-PCR;the expression of Smad 2/3 protein was determined by cell immunochemistry.Results The proliferation of hepatic stellate cells was inhibited by ATRA in a dose-dependent manner (P < 0.05 ).After induced by TGF-β1,the mRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein were increased significantly compared with control group (P <0.05).However,ATRA could obviously reduce themRNA expressions of COL1α2,MMP-2 and TIMP-1 and the expression of Smad 2/3 protein in HSC-T6 induced by TGF-β1 (P < 0.05 ).Conclusion ATRA can inhibit the proliferation of HSC-T6s and reduce the mRNA expressions of COL1α2,MMP-2 and TIMP-1 in HSC-T6 which were induced by TGF-β1.The anti-hepatic fibrosis function of ATRA may be related to its inhibition on the expression of Smad 2/3 protein in HSC-T6 to influence TGF-β1/Smad signaling pathway.
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AIM To explore the effects of Bushen Huoxue Recipe (Fluoritum,Psoraleae Fructus,Cuscutae Semen,etc.) on the protein expressions of transforming growth factor (TGF)-β1,TGF-βRⅡ and Smad2/3 in follicle wall granulosa cells in mice with autoimmune premature ovarian failure (POF).METHODS Balb/c female mice were subcutaneously injected with mouse zona pellucida 3 at multiple points to establish autoimmune POF model.POF mice were divided into model group,positive group (progynova),low-,middle-and high-dose of Bushen Huoxue Recipe groups.After intervention for 30 days,ovarian tissue was stained by hematoxylin-eosin (HE),and the protein expressions of TGF-β1,TGF-βRⅡ and Smad2/3 in follicle wall granulosa cells and ovarian tissue were detected by immunohistochemistry and Western blot,respectively.RESULTS Compared with the model group,the number of mature follicles in the Bushen Huoxue Recipe groups and the positive group was obviously increased,together with the decreased number of atretic follicles.The protein expressions of TGF-β1,TGF-βRⅡ and Smad2/3 in follicle wall granulosa cells and ovarian tissue in the Bushen Huoxue Recipe groups and the positive group were much higher than those in the model group (P ＜ 0.05).CONCLUSION Bushen Huoxue Recipe can improve ovarian function by up-regulating the protein expressions of TGF-β1,TGF-βRⅡ and Smad2/3 in granulosa cells.
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Objective To screen for selective transforming growth factor β(TGF-β)inhibitors from the compound library, and analyze their structure-activity relationship. Methods The inhibiting activities of 170 compounds to TGF-βpathway were evaluat-ed by the SMAD3 luciferase reporter system;the positive hits were examined for their selectivity towards activin receptor like kinase (ALK)4、ALK5 or ALK7 by a molecule based screening system composed of SMAD3,ATP and the purified kinase domain for ALK4, ALK5 or ALK7;the EGFP-SMAD2 fusion protein redistribution assay was used to confirm the inhibiting effects of positive hits. The structure-activity relationship was analyzed by comparing the docking module of SB431542 with ALK5 kinase domain. Results Fif-teen compounds were found capable of inhibiting luciferase expression downstream of SMAD3 with≥25%inhibitory rate;several of them showed different selectivity towards ALK4,ALK5 and ALK7. Compound 63 selectively inhibited the activity of ALK4 and ALK7 with IC500.234 and 0.370μmol/L,respectively,while compound 64 showed inhibiting activity towards all three kinases with the IC50 values 10,6 and 85 nmol/L for ALK4、ALK5 and ALK7,respectively. In addition,compounds 63 and 64 further inhibited the TGF-β1 induced EGFP-SMAD2 nuclear translocation,with the IC50 values of 0.45 and 6.30μmol/L,respectively. The MTT anti-proliferative assay indicated that compounds 63 and 64 exerted these activities at non-toxic concentrations. The analysis of structure-activity rela-tionship indicated that the compounds sharing a core structure,the 1,2,4-triarylimizazole or 1,3,5-triarylpyrazoline,with the 3,4 methyoenedioxyphenyl,6-methylpyridine and 4-aminocarboxyl substitution groups tended to exhibit better activities. Conclusion The two potent TGF-βpathway inhibitors,63 and 64 are identified through this screening project,of which,63 selectively inhibited the ALK4 and ALK7 activity,while 64 showed inhibiting activity towards all three tested types of ALKs.
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Objective To study the effect of paeonol (PAE) and PNS on the expression of transforming growth factor (TGF)- beta 1/ Smad2/3 pathway in rats with acute myocardial infarction (AMI), and the possible molecular mechanism thereof. Methods Model of AMI was made using left anterior descending coronary branch ligation. According to the inter?vention methods rats were divided into model group, PAE group (8 mg·kg-1), PNS group (40 mg·kg-1), PAE (4 mg·kg-1)+PNS (20 mg·kg-1) low dose group, PAE (8 mg·kg-1)+PNS (40 mg·kg-1) high dose group and captopril positive control group (10 mg · kg-1). Rats without ligation were used as Sham operation group. Left ventricular systolic blood pressure (LVSP), left ventricular diastolic pressure (LVEDP) and the maximum rise and fall rate (/dtmax DP) were detected after 28-day treat?ment. HE staining was used to observe changes of myocardial tissue. The protein expression levels of TGF-β1 and Smad2/3 were detected by Western blot assay. Results There were significant differences in parameters used for detecting treatment group and model group, formula group and single drug group, formula high dose group and formula low dose group (P <0.01). The model group showed pathological changes. All treatment groups showed different degrees of pathological improve?ment. There was the most significant improvement in formulae group and captopril group. Compared with the model group, TGF-β1 and Smad2/3 protein expressions were decreased in treatment group. The expression levels of TGF-β1 and Smad2/3 were significantly decreased in formula group than those of PAE group and PNS group, and lower levels in formula high dose group than those of formula low dose group (P<0.05). Conclusion Paeonol and PNS can inhibit the expressions of TGF-β/Smad 2/3 protein in rats with AMI, by blocking TGF-β/Smad pathway.
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BACKGROUND: Imbalance among TGF-beta/Smad pathway, MMP-1, and TIMP-1 expressions results in sclerotic skin disease, such as scleroderma, hypertrophic scar, and keloids. Curcumin, a phytochemical extracted from the rhizomes of Curcuma longa, showed an anti-fibrotic effect in an animal study, such as a pulmonary and cholangioductal fibrosis animal model. However, the expressions of type I collagen, MMP-1, Smad2/3, and TIMP-1 in curcumin treated human skin fibroblasts is largely unknown. OBJECTIVE: The purpose of this study was to investigate the expressions of type I collagen, MMP-1, Smad2/3, and TIMP-1 proteins in curcumin-treated human skin fibroblasts. METHODS: Human skin fibroblasts were treated by various concentrations of curcumin (1~40 uM). The expressions of type I collagen, MMP-1, Smad2/3, and TIMP-1 proteins were analyzed by Western blot analysis. In addition, activities of type I collagen promoter were analyzed by the CAT assay. RESULTS: The expression of type I collagen decreased but the expression of MMP-1 and TIMP-1 increased by curcumin treatment in a dose and time dependent manner in Western blot analysis. Type I collagen promoter activities were decreased by curcumin treatment in the CAT assay. Smad2/3 expression decreased by curcumin treatment but TGF-beta1 induced Smad2/3 activation was not decreased by curcumin treatment following Western blot analysis. CONCLUSION: Decrease of type I collagen expression through the inhibition of Smad2/3 and increase of the expression of MMP-1, which is the degradating enzyme of type I collagen, in human skin fibroblasts by curcumin treatment offer expectation of curcumin as antifibrotic agent.
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Animais , Gatos , Humanos , Western Blotting , Cicatriz Hipertrófica , Colágeno Tipo I , Curcuma , Curcumina , Regulação para Baixo , Fibroblastos , Fibrose , Queloide , Modelos Animais , Proteínas , Rizoma , Pele , Dermatopatias , Inibidor Tecidual de Metaloproteinase-1 , Fator de Crescimento Transformador beta1RESUMO
Objective To investigate the role of transforming growth factor-β1 (TGF-β1), Smad2/3 and Smad7 expressions in carotid artery remodeling in renovascular hypertensive rats, and also the therapeutic effect of Enalapril and Amlodipine. Methods The renovascular hypertensive rat (RHR) models with two-kidney and one-clip were established, including model group (n = 6), sham-operated group (n = 6), Enalapril group (10 mg/kg per day, n = 6), Amlodipine group (5 mg/kg per day, n = 6) and combination group (Amlodipine 2.5 mg/kg per day + Enalapril 5mg/kg per day, n = 6). The medication were continuous administrated for six weeks. Carotid artery morphological and structural changes in the media were observed by HE staining, Masson staining and immuno histochemical staining. Media thickness (MT), MT and lumen diameter ratio (MT/LD), and the expression levels of media α-smooth muscle actin (α-actin), proliferating cell nuclear antigen (PCNA), TGF-β1, phosphorylated Smad2/3 (p-Smad2/3) and Smad7 in carotid arteries were measured. Results The media of carotid arteries in RHR model group was significantly thickened, the volume of smooth muscle cell was increased, and the array was in disorder; MT, MT/LD, the proliferation index of smooth muscle cell and collagen fiber area percentage of carotid arteries in the model group were significantly higher than those in the sham-operated group (P < 0.01). Compared to sham-operated group, the model group had significantly higher expressions of TGF-β1 and p-Smad2/3 (P < 0.05) and lower Smad7 expression. Both Enalapril and Amlodipine improved smooth muscle hypertrophy and collagen deposition, reduced RHR carotid MT, MT/LD, proliferation index of smooth muscle cell, collagen fiber area percentage and the expressions of TGF-β1 and p-Smad2/3 (P < 0.05), increased Smad7 expression (P < 0.05). Moreover, the combination treatment of Enalapril and Amlodipine had significantly better effects than single Amlodipine group (P < 0.05), but not single Enalapril group. Conclusions TGF-β1/Smads pathway may participate in the mechanism of carotid artery remodeling in RHR; the role of Amlodipine and Enalapril in inversing carotid artery remodeling may be related to the change of TGF-β1/Smads pathway, the combination treatment of Amlodipine and Enalapril had better effects than single administration of Amlodipine.
RESUMO
OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.
OBJETIVO: Investigar a expressão de proteínas SMAD em tecidos de tiroide humana desde que a inativação dos componentes da sinalização de TGF-β/activina é relatada em diversos tipos de câncer. SMAD 2 e SMAD3 fosforilados (pSMAD2/3) associados com SMAD4 induzem a transmissão do sinal gerado por TGF-β e activina, enquanto SMAD7 inibe essa sinalização intracelular. Embora TGF-β e activina exerçam efeitos antiproliferativos nas células foliculares da tiroide, tumores de tiroide expressam altos níveis dessas proteínas. MATERIAIS E MÉTODOS: A expressão proteica de SMADs foi avaliada em bócio multinodular, adenoma folicular, carcinomas papilífero e folicular por imuno-histoquímica. RESULTADOS: A expressão de pSMAD2/3, SMAD4 e SMAD7 foi observada tanto em tumores benignos como malignos da tiroide. Embora pSMAD2/3, SMAD4 e SMAD7 exibissem alta positividade citoplasmática em carcinomas, a positividade nuclear de pSMAD2/3 não foi diferente entre lesões benignas e malignas da tiroide. CONCLUSÕES: O achado da expressão de SMADs em células tiroidianas e a presença das proteínas pSMAD2/3 e SMAD4 no núcleo de células tumorais indicam propagação da sinalização TGF-β/activina. Contudo, a alta expressão de SMAD7 inibitório, principalmente em tumores malignos, poderia contribuir para atenuação da sinalização antiproliferativa de SMADs em carcinomas de tiroide.
Assuntos
Humanos , Ativinas/fisiologia , Proteínas Smad Reguladas por Receptor/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/fisiologia , Adenoma/metabolismo , Carcinoma Papilar, Variante Folicular/metabolismo , Bócio Nodular/metabolismo , Transdução de Sinais/fisiologia , /análise , /análise , /análise , /análiseRESUMO
PURPOSE: Identifying the signal cross-talk between integrin signaling cascade and TGF-beta 1 signaling cascade in articular chondrocytes. MATERIALS AND METHODS: To analyze integrin or TGF-beta 1 mediated signaling pathways from extracellular stimuli, type II collagen was coated on the cell culture plate and TGF-beta 1 was added to cell culture media. Chondrocytes were cultured in the conditioned media with each or both stimuli. Altered activation of signaling proteins detected with western blot technique. RESULTS: More rapid attachment of cells was observed in the type II collagen coated group than non-coated group. The phosphorylated SMAD 2 and 3 were expressed in the type II collagen coated group and synergistically up-regulated phosphorylation in the co-treated group. The phosphorylated FAK at tyrosine 925 was activated by TGF-beta 1 treatment and synergistically up-regulated by both stimuli. But there was no meaningfully changed phosphorylation of extracellular signal regulated protein kinase (ERK) 1/2 and p38, as known downstream molecules of FAK cascade. CONCLUSION: This result means that SMAD 2, SMAD 3 and tyrosine 925 of FAK are involved in this signal cross-talking in articular chondrocytes.