ABSTRACT
Objective:To explore the relationship between the expression of angiopoietin 1 (ANGPT1) and Smadhomolog 9 (Smad9) genes in cancer tissues and tumor metastasis, invasion behavior and prognosis in patients with lung adenocarcinoma.Methods:Sixty patients with lung adenocarcinoma in Chengwu Hospital Affiliated to Shandong First Medical University from October 2018 to December 2019 were selected as the research objects. The expressions of ANGPT1 and Smad9 mRNA in cancer tissues and adjacent tissues were compared, as well as the expressions of ANGPT1 and Smad9 mRNA in cancer tissues of patients with different tumor metastasis and invasion behaviors. The relationship between ANGPT1 and Smad9 mRNA expression and tumor metastasis and invasion behavior of lung adenocarcinoma were analyzed, and the 1-year survival rate of patients with lung adenocarcinoma was calculated. The 1-year survival rate of patients with different ANGPT1 and SMAD9 mRNA expression levels were compared.Results:The relative expression of ANGPT1 mRNA and Smad9 mRNA in cancer tissues were lower than those in adjacent tissues: 2.45 ± 0.26 vs. 11.18 ± 0.93, 4.23 ± 0.31 vs. 7.58 ± 0.65, the differences were statistically significant ( P<0.05). There were significant differences in the relative gene expression of ANGPT1 and Smad9 mRNA in different clinical stages, tumor diameter, degree of differentiation, lymph node metastasis and pleural invasion ( P<0.05). Spearman correlation analysis showed that the expression of ANGPT1 and Smad9 mRNA were negatively correlated with clinical stage, tumor diameter, lymph node metastasis and pleural invasion ( r = - 0.517, - 0.539, - 0.606, - 0.679, P<0.05), and positively correlated with the degree of differentiation ( r = 0.628, P<0.05). The 1-year survival rate of 58 patients was 72.41%. Kaplan-Meier curve analysis showed that the 1-year survival rate of patients with low expression of ANGPT1 and Smad9 mRNA in cancer tissues were were lower than those in patients with high expression ( P<0.05). Conclusions:Down-regulation of ANGPT1 and Smad9 genes in cancer tissues will accelerate the metastasis and invasion behavior of lung adenocarcinoma. Up-regulating the expression of both genes can be a potential way to improve survival.
ABSTRACT
Objective:To investigate the protective effect and possible mechanism of Tangshenbao on renal damage in diabetic nephropathy (DN) rats.Methods:Totally 36 SPF male SD rats were randomly divided into normal group ( n=6) and model group ( n=30). The DN rat model was prepared by single high-dose intraperitoneal injection of STZ. According to the random number table method, the rats were divided into model group, irbesartan group and Tangshenbao low-, medium- and high-dosage groups, with 6 rats in each group. Drug intervention lasted for 8 weeks. The general condition and body weight of rats in each group were recorded. The blood glucose, kidney index, 24 h urine protein (24 h UTP), SCr and BUN levels were detected. The pathological morphology of renal tissue was observed by PAS staining and transmission electron microscopy. The mRNA and protein expressions of Ets-1, TGF-β1, Smad2 and Smad3 in renal tissue were detected by real-time fluorescence quantitative PCR and Western blot. Results:Compared with model group, the body weight of Tangshenbao low, medium and high dose groups and irbesartan group significantly increased ( P<0.01). The kidney index decreased ( P<0.05 or P<0.01). The contents of 24 hUTP, BUN and SCr significantly decreased ( P<0.05 or P<0.01). Glomerular volume was significantly reduced ( P<0.05 or P<0.01), the mRNA expressions of Ets-1 (1.59 ± 0.06, 1.47 ± 0.04, 1.31 ± 0.03, 1.39 ± 0.03 vs. 1.64 ± 0.04), TGF-β1 (1.65 ± 0.05, 1.59 ± 0.03, 1.38 ± 0.05, 1.49 ± 0.04 vs. 1.77 ± 0.08), Smad2 (1.48 ± 0.05,1.39 ± 0.05, 1.22 ± 0.03, 1.31 ± 0.04 vs. 1.54 ± 0.05), Smad3 (1.57 ± 0.04, 1.48 ± 0.03, 1.28 ± 0.03, 1.39 ± 0.02 vs. 1.64 ± 0.05) in renal tissue of rats significantly decreased ( P<0.05 or P<0.01), the protein expressions of Ets-1 (1.33 ± 0.32, 1.16 ± 0.38, 0.77 ± 0.06, 0.84 ± 0.06 vs. 1.97 ± 0.43), TGF-β1 ( 1.35 ± 0.14, 1.24 ± 0.22, 0.94 ± 0.13, 1.07 ± 0.06 vs. 1.63 ± 0.20), Smad2 (1.24 ± 0.26, 1.14 ± 0.31, 0.77 ± 0.28, 0.85 ± 0.19 vs. 1.72 ± 0.34) and Smad3 (1.29 ± 0.14, 1.19 ± 0.21, 0.85 ± 0.39, 0.90 ± 0.37 vs. 1.76 ± 0.21) decreased ( P<0.05 or P<0.01). Conclusion:Tangshenbao can improve renal damage in DN rats, and its mechanism may be related to the inhibition of Ets-1 expression and TGF-β1/Smads signaling pathway.
ABSTRACT
Objective:To investigate the effect of miRNA-186-3p (miR-186-3p) on the apoptosis, immigration and invasion of cervical cancer CaSki cells and its relationship with transforming growth factor-β (TGF-β)-Smad signaling pathway.Methods:Plasmids containing miR-186-3p suppressor gene and miR-186-3p mimics were transfected into cervical cancer CaSki cells, namely miR-186-3p-I group and miR-186-3p-M group, respectively. In addition, CaSki cells transfected with empty plasmids were used as control (miR-186-3p-group C). Flow cytometry was used to detect the apoptosis rate of each group, and Transwell method was used to detect the migration and invasion ability of each group. Western blotting was used to detect the expression level of TGF-β-Smad signaling pathway related proteins. The target genes of miR-186-3p were predicted by using Starbase software and verified by using dual luciferase reporter gene assay.Results:The apoptosis rate of miR-186-3p-M group was lower than that of miR-186-3p-I group and miR-186-3p-C group [(7.5±3.2)% vs.(13.9±0.7)%, (12.7±0.6)%, all P < 0.05]. The number of migrating cells in miR-186-3p-M group [(218±25) vs. (168±13), (175±13), both P < 0.001] and the number of invasive cells in miR-186-3p-I group and miR-186-3p-C group were higher than those in miR-186-3p-I group and miR-186-3p-C group [(165±21) vs. (130±11), (142±12), all P < 0.001]. The relative expression levels of TGF-β, Smad2, Smad3, phosphorylated Smad2 (p-Smad2) and phosphorylated Smad3 (p-Smad3) in miR-186-3p-M group were the lowest, and the differences were statistically significant compared with the other two groups (all P < 0.001). Starbase software was used to predict the complementary binding of miR-186-3p to XIST RNA. Dual luciferase reporter assay showed that the relative luciferase activity of cells co-transfected with XIST wild-type vector and miR-186-3p mimic plasmid was lower than that of cells co-transfected with XIST wild-type vector and miR-186-3p empty plasmid ( P < 0.001). Conclusion:miR-186-3p may directly bind to XIST RNA and down-regulate the activity of TGF-β-Smad signaling pathway, thereby enhancing the immigration, apoptosis and invasion activities of cervical cancer CaSki cells.
ABSTRACT
Objective To investigate the effect of Yangxue Rougan pills on a rat model of liver fibrosis induced by multiple factors and the mechanism of action of Yangxue Rougan pills in the treatment of liver fibrosis. Methods A total of 50 male rats were randomly divided into blank control group, multi-factor model group, Fuzheng Huayu capsule group, and high-, middle-, and low-dose Yangxue Rougan pill groups. The rats in the blank control group were given normal water and feed, and those in the other groups were given modified high-fat low-protein diet and 5% alcohol, as well as subcutaneous injection of olive oil solution containing 40% carbon tetrachloride and intraperitoneal injection of pig serum 0.5 mL per rat, twice a week for 12 consecutive weeks. Since week 7, the rats in the high-, middle-, and low-dose Yangxue Rougan pill groups were given Yangxue Rougan pills at a dose of 9.5, 4.75, and 2.38 g/kg, respectively, those in the Fuzheng Huayu capsule group were given Fuzheng Huayu capsules at a dose of 0.75 g/kg, and those in the blank control group and the multi-factor model group were given an equal volume of distilled water by gavage every day for 6 consecutive weeks. The rats were treated at week 12. HE staining and Masson staining were used to observe the degree of liver fibrosis in rats, and PCR and Western blot were used to measure the expression of TGF-β1, Smad3, and Smad7 in the liver. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the Dunnett's t -test was used for further comparison between two groups. Results Compared with the blank control group, the multi-factor model group had a severely damaged lobular structure and a significantly higher degree of liver fibrosis, with the formation of pseudolobules with different sizes; compared with the multi-factor model group, the Yangxue Rougan pill groups had a significant improvement in the degree of liver fibrosis, with the most significant therapeutic effect in the high- and middle-dose Yangxue Rougan pill groups. Compared with the blank control group, the multi-factor model group had significant increases in the expression of TGF-β1 and Smad3 and a significant reduction in the expression of Smad7 in liver tissue (all P < 0.05); compared with the multi-factor model group, the Yangxue Rougan pill groups had a significant reduction in the expression of TGF-β1 and a significant increase in the expression of Smad7 (all P < 0.05); compared with the multi-factor model group, the high- and middle-dose Yangxue Rougan pill groups had a significant reduction in the expression of Smad3 (both P < 0.05). Conclusion Yangxue Rougan pills can significantly inhibit liver fibrosis in rats by downregulating the expression of TGF-β1 and Smad3 and upregulating the expression of Smad7, and therefore, the TGF-β1/Smad signaling pathway is one of the mechanisms of action of Yangxue Rougan pills in improving liver fibrosis.
ABSTRACT
Objective To evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on transforming growth factor-β1 (TGF-β1)/Smad signaling pathway during acute lung injury (ALI) in rats.Methods Healthy clean-grade adult male Sprague-Dawley rats,aged 4-5 weeks,weighing 100-120 g,were selected,and BMSCs were prepared and cultured in vitro.Eighty-four healthy clean-grade adult male Sprague-Dawley rats,aged 4 weeks,weighing 170-190 g,were selected and divided into 4 groups (n=21 each) using a random number table method:control group (group C),group ALI,ALI plus BMSCs group (group ALI + BMSCs),and ALI plus phosphate buffer solution (PBS)group (group ALI+PBS).ALI was induced by intraperitoneally injecting 5 mg/kg lipopolysaccharide 0.5 ml in anesthetized rats.In group ALI+BMSCs,1×104 cells/ml BMSCs 0.5 ml (in PBS) was injected via the tail vein after intraperitoneal injection of lipopolysaccharide.PBS 0.5 ml was injected via the tail vein after intraperitoneal injection of lipopolysaccharide in group ALI+PBS.Arterial blood samples were collected at 6,24 and 48 h after injecting BMSCs for blood gas analysis and for determination of wet to dry weight ratio (W/D ratio),pathological changes (using haematoxylin and eosin staining),and expression of TGF-β1,Smad2 and Smad3 in lung tissues (by Western blot).Results Compared with group C,PaO2 was significantly decreased,PaCO2 and W/D ratio were increased,the expression of TGF-β1,Smad2 and Smad3 in lung tissues was up-regulated (P＜0.05),and the pathological changes of lung tissues were accentuated in ALI,ALI+BMSCs and ALI+PBS groups.Compared with group ALI,PaO2 was significantly increased,PaCO2and W/D ratio were decreased,the expression of TGF-β1,Smad2 and Smad3 in lung tissues was down-regulated (P＜0.05),and the pathological changes of lung tissues were significantly reduced in group ALI+BMSCs.Conclusion The mechanism by which BMSCs mitigates ALI may be associated with inhibiting TGF-β1/Smad signaling pathway in rats.
ABSTRACT
Objective@#To study the effect of Neiyi-Tongjingling on the content of TGF-β1 and Smad2/3 protein in ectopic endometrium of rats with endometriosis.@*Methods@#Atotal of 42 rats were randomly divided into the sham operation group, model group, high, medium and low dose groups of traditional Chinese medicine and western medicine group with 7 rats in each group. Except for the sham-operated group, the rats in the other groups established EMs models by means of rat autologous intimal transplantation. The drug was administered at the 5th week after the model was established. The western medicine group was given 0.5 mg/kg gestrinone solution twice a week. The high, medium and low dose groups of traditional Chinese medicine were given 49.50 mg/kg, 24.75 mg/kg and 12.38 mg/kg of Neiyi-Tongjingling liquid, respectively. While the model group and sham operation group were given the same volume of normal saline once per day for 4 weeks. The volume of ectopic lesions in each group was observed. The HE staining was used to observe the pathological changes of intima tissue. Immunohistochemical SP method was used to detect the expression of TGF-β1 and Smad2/3 proteins in intima tissue.@*Results@#The volume of ectopic endometrium (57.91 ± 13.10 mm3, 48.93 ± 8.15 mm3, 76.21 ± 17.14 mm3, 57.88 ± 15.98 mm3 vs. 141.58 ± 54.25 mm3) in the western medicine group and the high, medium and low dose group of Chinese medicine were significantly lower than that in the model group (P<0.05). Compared with the model group, the content of TGF-β1 (0.08 ± 0.00, 0.08 ± 0.01, 0.10 ± 0.00 vs. 0.13 ± 0.03) and Smad2/3 (0.09 ± 0.02, 0.08 ± 0.01, 0.10 ± 0.01 vs. 0.12 ± 0.02) in ectopic endometrium tissue of three Chinese medicine groups decreased significantly (P<0.05).@*Conclusions@#The Neiyi-Tongjingling can treat EMs by inhibiting the growth of ectopic endometrium, reducing the volume of ectopic lesions, and reducing the expression of TGF-β1 and Smad2/3 proteins.
ABSTRACT
BACKGROUND: Transforming growth factor β (TGF-β), 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-β1. METHODS: A549 epithelial cells and CCD-11Lu fibroblasts were incubated and stimulated with or without all-trans RA (ATRA) and TGF-β1 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-β1 and Smad3 were evaluated at 1 and 3 weeks. RESULTS: When A549 cells were pre-stimulated with TGF-β1 prior to RA treatment, RA completely inhibited the p-Smad2/3. However, when A549 cells were pre-treated with RA prior to TGF-β1 stimulation, RA did not completely suppress the p-Smad2/3. When A549 cells were pre-treated with MAPK inhibitor, TGF-β1 failed to phosphorylate Smad2/3. In fibroblasts, p38 MAPK inhibitor suppressed TGF-β1-induced p-Smad2. In a bleomycin-induced lung injury mouse model, RA decreased the expression of TGF-β1 and Smad3 at 1 and 3 weeks. CONCLUSION: RA had inhibitory effects on the phosphorylation of Smad induced by TGF-β1 in vitro, and RA also decreased the expression of TGF-β1 at 1 and 3 weeks in vivo. Furthermore, pre-treatment with a MAPK inhibitor showed a preventative effect on TGF-β1/Smad phosphorylation in epithelial cells. As a result, a combination of RA and MAPK inhibitors may suppress the TGF-β1-induced lung injury and fibrosis.
Subject(s)
Animals , Mice , Apoptosis , Bleomycin , Blotting, Western , Cell Differentiation , Epithelial Cells , Fibroblasts , Fibrosis , In Vitro Techniques , Lung Injury , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Models, Animal , p38 Mitogen-Activated Protein Kinases , Phosphorylation , Protein Kinases , Smad Proteins , Transforming Growth Factor beta , Transforming Growth Factors , TretinoinABSTRACT
Objective@#To investigate the effects of single-hole and double-hole drilling and closed drainage on the expression of Hypoxia inducible factor-1α (HIF-1α), Claudin-5, transforming growth factor-β (TGF-β) and Smad2/3 in the epidermis of patients with chronic subdural hematoma.@*Methods@#100 patients with chronic subdural hematoma were randomly divided into single hole and double hole drainage group according to random number table. Immunohistochemical streptavidin-peroxidase (SP) was used to detect the expression of TGF- β protein, and Western blot was used to detect the expression of HIF-1α, Claudin-5 and Smad2/3 in the epihematoma of the two groups before and after operation.@*Results@#Compared with pre-operation, the expression of HIF-1α, TGF-β and Smad2/3 protein in adventitia of hematoma in both groups decreased after operation, especially in the double-hole group (P<0.05). The expression of Claudin-5 protein in adventitia of hematoma was significantly increased in both groups, especially in the double-hole group, with statistically significant difference (P<0.05).@*Conclusions@#Single-hole and double-hole drilling and sealing drainage is an effective method to treat chronic subdural hematoma. It can reduce the expression of HIF-1α, TGF-β and Smad2/3 protein in the epidermis of hematoma, and significantly increase the expression of Claudin-5, and the effect of double-hole drilling and sealing drainage is more significant.
ABSTRACT
Objective To investigate the effects of single-hole and double-hole drilling and closed drainage on the expression of Hypoxia inducible factor-1α (HIF-1α),Claudin-5,transforming growth factor-β (TGF-β) and Smad2/3 in the epidermis of patients with chronic subdural hematoma.Methods 100 patients with chronic subdural hematoma were randomly divided into single hole and double hole drainage group according to random number table.Immunohistochemical streptavidin-peroxidase (SP) was used to detect the expression of TGF-β protein,and Western blot was used to detect the expression of HIF-1α,Claudin-5 and Smad2/3 in the epihematoma of the two groups before and after operation.Results Compared with pre-operation,the expression of HIF-1 α,TGF-β and Smad2/3 protein in adventitia of hematoma in both groups decreased after operation,especially in the double-hole group (P ＜ 0.05).The expression of Claudin-5 protein in adventitia of hematoma was significantly increased in both groups,especially in the double-hole group,with statistically significant difference (P ＜ 0.05).Conclusions Single-hole and double-hole drilling and sealing drainage is an effective method to treat chronic subdural hematoma.It can reduce the expression of HIF-1α,TGF-β and Smad2/3 protein in the epidermis of hematoma,and significantly increase the expression of Claudin-5,and the effect of double-hole drilling and sealing drainage is more significant.
ABSTRACT
Objective@#To investigate the mechanism of cleft palate in mice induced by excessive all-trans retinoic acid (atRA).@*Methods@#The pregnant mice were randomly divided into atRA-treated group (n=27) and control group (n=27). atRA-treated group was given by gavage a single dose of atRA (100 mg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. The expressions of Smad2, phospho-Smad2 (p-Smad2), Smad4 and Smad7 in mouse embryonic palatal mesenchyme were analyzed by Western blotting.@*Results@#At GD13-GD15, compared with the control, the ratio of BrdU-positive cells in the palatal mesenchyme of atRA-treated fetuses decreased significantly (P<0.05), especially at GD14, atRA inhibition rate was (65.4±1.7)%. Moreover, atRA decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells, whereas the expression of Smad7 was significantly increased at GD14 and GD15.@*Conclusions@#atRA may lead to cleft palate by inhibiting the activation of Smad signaling pathway and affecting the proliferation of palatal mesenchymal cells.
ABSTRACT
BACKGROUND: The transforming growth factor-β/SMAD (TGF-β/SMAD) pathway plays an important role in tissue repair and collagen synthesis. Low-level light therapy (LLLT) is increasingly used to alleviate pain and inflammation and promote wound healing. However, few studies have directly compared the effects of different wavelengths of light-emitting diodes (LEDs) or examined their individual effects at the molecular level. OBJECTIVE: Here we used a mouse model to investigate the effect of blue (410 nm), red (630 nm), and infrared (830 nm) LEDs on wound closure and assessed the underlying changes in a signal transduction pathway. METHODS: A full-thickness wound was created on the dorsal skin of mice using a 6-mm-diameter punch. In part I, the wounds were irradiated using blue, red, and infrared LEDs. In part II, the wounds were irradiated at different time points. Photo documentation, serial skin biopsies, wound measurements, and immunohistochemical staining using TGF-β/SMAD pathway-related molecules were performed. RESULTS: The overall wound closure percentage was highest during the first 10 days when an 830-nm LED was used. The wound closure process was accelerated when the irradiation was initiated immediately after wounding. Irradiation using 830-nm LED upregulated TGF-β and collagen-1 but downregulated SMAD7. CONCLUSION: Our findings show that LLLT using an 830-nm wavelength LED delivered immediately after wound formation may have the best effect on wound healing by upregulating the TGF-β/SMAD signaling pathway.
Subject(s)
Animals , Mice , Biopsy , Collagen , Inflammation , Low-Level Light Therapy , Signal Transduction , Skin , Smad Proteins , Wound Healing , Wounds and InjuriesABSTRACT
Se realizó un compendio de los principales factores etiopatogénicos causantes de la expresividad clínica de la esclerosis sistémica, aún no bien establecida, la asociación genética, herencia, factores ambientales y respuesta inflamatoria, su relación con infecciones endógenas comportándose de manera especial en otras enfermedades reumáticas inflamatorias. El objetivo del artículo fue describir los elementos de la etiopatogenia que distinguen a esta enfermedad del colágeno. Los mecanismos etiopatogénicos basados en anormalidades moleculares, pueden ser correctas en la esclerosis sistémica. Su espectro clínico heterogéneo responde a una etiopatogenia distinta en cada individuo, así como, a eventos complejos que pueden ocurrir en fases iniciales de la enfermedad y no a una patogenia única.
A summary of the main ethiopathogenic factors that cause the clinical expressiveness of the Systemic Sclerosis, still as soon as established, the genetic association, inheritance, environmental factors and inflammatory response, their relationship with endogenous infections behaving from a special way to other inflammatory rheumatic illnesses. The objective of this article was to describe the ethiopathogenic elements that distinguish this illness of the collagen. The ethiopathogenic mechanisms based on molecular abnormalities can be correct in the systemic sclerosis. Its heterogenic clinical spectrum responds to a different etiopathogeny in each individual, as well as to complex events that can happen in initial phases of the illness and not to a unique pathogenic.
ABSTRACT
Hepatic fibrosis is the process in which the human body repairs the liver tissue damaged due to various reasons. Recent studies have found that bone morphogenetic protein-7 (BMP-7) and the downstream Smads can antagonize the effect of transforming growth factor-β1, which induces hepatic fibrosis, through various pathways, including inducing the degradation of extracellular matrix, inhibiting cell apoptosis, reducing the expression of various proinflammatory factors, and promoting the regeneration of hepatocytes. This article investigates the role of BMP-7/Smads signaling pathway in hepatic fibrosis.
ABSTRACT
AIM:To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms .METHODS: CFs were cultured using myocardial tissue with dry method .Hypoxic environment was established for CFs by continuous nitrogen supplement .Type I and type III collagens in supernatants were detected by ELISA.Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents .The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining .RESULTS:Un-der hypoxic condition , TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-de-pendent manner in the CFs .At the concentration of 5μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01).RXR agonist 9-cis-retinoic acid (9-cis-RA;10 -9 ~10 -6 mol/L) decreased TGF-β1 (5μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic con-dition.The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01).Smad2 inhibitor ( 20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF -β1 under hypoxic condition.Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly in-creased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05).CONCLU-SION:Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition .
ABSTRACT
Biliary atresia (BA) is one of the most serious digestive system diseases, which threatens the health of infants. Liver fibrosis is a major cause of death in children with BA. In the process of the pathogenesis of BA, virus infection can in?duce a series of immune and inflammatory reaction, result in a decrease of regulatory T cells (Treg cells) and high expression of CD14, activating a variety of inflammatory pathways and TGF-β/Smad2/3 pro-fibrogenic pathway, which produces a large number of medium damage of liver cells and bile duct cells, releases proinflammatory factor, oxygen metabolism matter and cytokines. These changes further aggravate damage of hepatobiliary system and cause the internal environment imbalance of liver parenchyma cells. The imbalance of internal environment with adaptive degeneration and necrosis in liver parenchyma cells, hepatic macrophages and gathered inflammatory cells leads to the activation of hepatic stellate cells (HSCs). HSCs can be converted into fibroblast cells, and promote the process of liver fibrosis. Immune and inflammatory lesions, pro-fibrogenic pathway are the important factors in contributing to liver fibrosis and cirrhosis of biliary atresia.
ABSTRACT
Objective To observe the effect of transforming growth factor beta 1 (TGF-β1) on the expression of collagen in rat atrial and ventricular fibroblasts, and to investigate its specific molecular mechanisms. Methods Tissue explant attachment was used to culture fibroblasts obtained from the atrium and ventricle of rat heart, and they were identified with SABC immunocytochemical staining, and then the following experiments were carried out. (1) Hydroxyproline digestion was performed to study the effects of TGF-β1, within different concentrations (0, 5, 10ng/ml) and different action time (6, 12, 24, 48h) on the content of hydroxyproline in rat's atrial and ventricular fibroblasts. (2) Rat's atrial and ventricular fibroblasts were stimulated with TGF-β1 in optimal concentration and action time, the expression of α-smooth muscle actin (α-SMA) was determined with Western blotting, and the expressions of typeand III collagen mRNA were evaluated with reverse-transcription PCR. The contents of hydroxyproline in the respective cells were measured with hydroxyproline determination. Western blotting was used to measure the protein expression of Smad2/3, p-Smad2/3 and Smad7. Results (1) TGF-β1 was shown to stimulate the collagen synthesis in rat's atrial and ventricular fibroblasts, and the optimal stimulus was TGF-β1 concentration 5ng/ml with action time of 24h. (2) After being stimulated by optimal stimulation effect of TGF-β1, the expression of typeand III collagen and p-Smad2/3 increased, while that of Smad7 decreased significantly only in atrial fibroblasts (P1 under optimal stimulating conditions. Conclusion TGF-β1 can induce dysbolism of collagen of cardiac fibroblasts with abnormal expression of cytoskeletal protein, which may occur more obviously in rat's atrial fibroblasts than in ventricular fibroblasts, and its mechanism may be related with TGF-β1/SMAD pathway.
ABSTRACT
Objective To investigate the effects of Ruangan granule on transforming growth factor-β1(TGF-β1)/Smads signaling pathway in liver fibrosis in rats. Methods A total of 105 Wistar rats were randomly divided into normal control group, model group and colchicine, Dahuang-Zhechong pill group, high-, medium- and low-dose Ruangan granule groups (n=15 in each group). Liver fibrosis was induced by carbon tetrachloride and a high-cholesterol diet. After modeling, the low-, medium- and high-dose Ruangan granule groups were intragastric administrated Ruangan granule mixed suspension 3.6, 7.2, 14.4 g/(kg?d), respectively;Dahuang-Zhechong pill group was administrated with Dahuang-Zhechong pellets mixed suspension of 0.18 g/(kg?d);the colchicine group was intragastric administrated with colchicine mixed suspension of 0.108 mg/(kg?d);and the normal control group and the model group were intragastric administrated with the equal volume of distilled water. All rats were intragastric administrated for 8 weeks. The expressions of TGF-β1, Smad3 and Smad7 proteins in the liver tissue were detected with immunohistochemical staining method. The expressions of TGF-β1, Smad3, Smad7 mRNAs in the liver tussue were detected by RT-PCR. Results The expressions of TGF-β1 (2.59 ± 0.99 vs. 0.43 ± 0.21) and Smad3 (2.56 ± 0.67 vs. 0.41 ± 0.18) proteins and TGF-β1 mRNA (2.25 ± 0.21 vs. 0.71 ± 0.09) and Smad3 (2.34 ± 0.03 vs. 0.78 ± 0.12) mRNAs in the model group were significantly increased than those in the normal control group (all P<0.01). Compared with the model group, the expressions of TGF-β1 (1.12 ± 0.27 vs. 2.59 ± 0.99) and Smad3 (1.05 ± 0.34 vs. 2.56 ± 0.67) proteins in the high-dose Ruangan granule group decreased significantly, the expression of Smad7 increased significantly (2.33 ± 0.62 vs. 0.36 ± 0.18), and the expressions of TGF-β1 (1.09 ± 0.11 vs. 2.25 ± 0.21) and Smad3 (1.10 ± 0.02 vs. 2.34 ± 0.03) mRNAs decreased significantly, the expression of smad7 mRNA (1.18 ± 0.13 vs. 0.38 ± 0.11) increased significantly (P<0.05). Conclusions Ruangan granule can regulate the TGF-β1/Smads signaling pathway via down-regulation of TGF-β1, Smad3 and up-regulation of Smad7 in liver fibrosis in rats.
ABSTRACT
Objective To investigate the influence of salvianolic acid B on the expressions of TGF-β1/Smad signaling pathway related proteins in human lung fibroblasts,and to explore the mechanism of the inhibitory effect of Sal B on TGF-β1-induced lung fibroblast activation.Methods The human embryonic lung fibroblasts (MRC-5) were cultured in vitro and randomly divided into control group ( the cells were cultured with DMEM without TGF-β1 or Sal B),Sal B group (the cells were cultured with 10μmol·L-1 Sal B),TGF-β1 group (the cells were cultured with 10μg·L-1 TGF-β1),and TGF-β1 (10μg·L-1 )+ Sal B (10μmol·L-1 )group.The protein levels of p-Smad2,p-Smad3,TβRⅠ,and Smad7 in the fibroblasts in various groups were detected by Western blotting method.Results Compared with control group,the expression levels of p-Smad2,p-Smad3,and TβRⅠproteins in TGF-β1 group were significantly increased (P0.05).Compared with TGF-β1 group,the expression levels of p-Smad2,p-Smad3,and TβRⅠ in TGF-β1+ Sal B group were descended (P<0.05),and the expression level of Smad7 was increased (P<0.05).Conclusion Sal B could suppress the TGF-β/Smad signaling pathway in lung fibroblasts and to inhibit the TGF-β1-induced lung fibroblast activation.
ABSTRACT
Objective To explore the regulation function of Icariine on the expression of bone morphogenetic protein signaling pathway Smad1,Smad5,Smad4 and to explore the mechanism of promoting MC3T3-E1 cell proliferation and differentiation.Methods MC3T3-E1 cells were treated by 0,10-9,10-8 and 10-7 mol/L Icariine respectively.After stimulated by Icariine 1 d,2 d and 3 d,MTT method and population diploid time were used to observe the cell proliferation,and the cell alkaline phosphatase (ALP) level was assayed.At 21 days later,the alizarin red staining was proceeded.At 1,2 and 3 days later,the RT-PCR was used to detect the mRNA expression level about Smad1,Smad5 and Smad4,and the Western blot was to detect the Smad1,5 and Smad4 protein.At 2 days later,the RT-PCR was used to detect the mRNA expression level about Runx2,BMP-2 and osteoprotegerin (OPG),and the Western blot was used to analyze osteocalcin (OCN) protein level.Results After simulated by Icariine,the proliferation (MTT test),the ALP activity and mineralization of osteoblasts were increased,the cell population diploid was reduced (P＜0.05).At 1,2 and 3days later,the results of RT-PCR showed that Icariine continued increasing the mRNA level of Smad1,5 and Smad4 in 10-8 mol/L.At 2 days later,Smad1,Smad5 and Smad4 mRNA expression were obviously reduced in 0 mol/L group,and At 3 days later,Smad1,Smad5 and Smad4 mRNA expression were obviously reduced in 10-9 and 10-7 mol/L groups.At 2 days later,BMP-2,Runx2 and OPG mRNA were obviously increased in 10-8 mol/L group.The results of Western blot showed that OCN,Smad1,Smad5 and Smad4 protein were obviously up-regulated in 10-8 mol/L group.Conclusion Icariine occurs the expressions of BMP-2,Runx2,Smad1,Smad5 and Smad4 by stimulating the bone morphology of MC3T3-E1 cells directly,and promotes the osteogenic differentiation in the manner of expression level synchronous rising.
ABSTRACT
Objective To determine the effect of smad anchor for receptor activation (SARA) on renal tubular epithelial to mesenchymal transtion (EMT) induced by high glucose and to investigate the associated mechanism.Methods HK-2 cells were exposed to high glucose (30 mmol/L).HK-2 cells were transfected with the plasmids of wild-type SARA [SARA (WT)] or SARA mutant [SARA with SBD deletion,called SARA (dSBD)] and then was stimulated by high glucose.The gene expression was assayed by real-time PCR and the protein expression was detected by Western blotting.Results During the process of high glucose-induced EMT of HK-2 cells,the gene and protein expression of SARA were down-regulated.The expression of TGF-β1 and Smad3 increased after stimulation of high glucose in HK-2.However,the Smad2 mRNA expression increased while its protein expression was down-regulated in a time-dependent manner.Smad2 and Smad3 were activated by high glucose stimulation and Smad3 kept activation for longer time than Smad2.Compared with high glucose group,over-expression of SARA by transfection of SARA (WT) up-regulated the expression of zona occludens(ZO)1 and down-regulated the expression of vimentin (P＜0.05).However,SARA (dSBD) had no such effects on above expressions.The Smad2 protein expression increased along with the over-expression of SARA.Meanwhile,over-expression of SARA prolonged the activation time of Smad2 and shortened the activation time of Smad3.Conclusions TGF-β1 signaling is activated and SARA expression is down-regulated during the process of high glucose-induced EMT in HK-2 cells.Over-expression of SARA can inhibit the EMT via increase of Smad2 protein expression and longer activation time of Smad2.