OBJECTIVES: Stably expressed transforming growth factor -beta 1(TGF-β1)MCs were obtained and the effects of centellaasiatica (CA) granule on the expressions of Smad 2/3, Smad 7 and collagen Ⅳ and the level of Smad 2/3 phosphorylation were observed. METHODS: Lipofectin method was used to transfect TGF-β1 vector into MC, and the stably expressed TGF-β1 cell lines were selected by G418. The cells were divided into three groups. Control group:normal MC + RPMI 1640 + 10% normal rat serum; TGF-β1 group:stably expressed TGF-β1 MC + RPMI 1640 + 10% normal rat serum; CA group:stably expressed TGF-β1 MC + RPMI 1640 + 10% rat serum containing high CA. The experiments were repeated for five times. The contents of TGF-β1 and collagen Ⅳ in the culture medium were detected with ELISA, the expressions of mRNA and protein of TGF-β1, Smad 2/3, Smad 7 and the level of Smad 2/3 phosphorylation were detected by using real time quantitative polymerase chain reaction and Western blot. RESULTS: The contents of TGF-β1 and collagen Ⅳ in the culture medium of stably-expressed TGF-β1 MC were increased significantly, and the CA could reverse the effects of TGF-β1. The expressions of mRNA and protein of TGF-β1, Smad 2/3 and the level of Smad 2/3 phosphorylation were increased significantly in TGF-β1 transfected MC, and CA could dramatically reduce the expressions of mRNA and protein of TGF-β1, Smad 2/3 and the level of Smad 2/3 phosphorylation. The high expression of TGF-β1 decreased the expression of Smad 7 mRNA and protein, and the CA could antagonize the effect of mRNA expression. CONCLUSIONS The MCs stably-expressed TGF-β1 can activate the TGF-β1/Smad signal pathway and increase the expression of collagen Ⅳ. CA can decrease the occurrence of diabetic nephropathy(DN) by reducing the production of collagen Ⅳ through inhibiting the TGF-β1/Smad signal pathway.
Animals ; Cells, Cultured ; Centella ; chemistry ; Collagen Type IV ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Mesangial Cells ; drug effects ; metabolism ; Rats ; Signal Transduction ; Smad Proteins ; metabolism ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVE: To investigate the effects of centella asiatica (CA) granule on the expression of transform growth factor-β(TGF-β) and related down-stream signals in rats with early diabetic nephropathy(DN) and to clarify the molecular mechanisms of CA molecular mechanism of on preventing and curing early diabetic kidney disease DN by studying the effects of centella asiatica on TGF-β expression and related down-stream signals. METHODS: Sixty male SD rats were divided into control group(=10) and DN model group(=50). The model rats were made a right nephrectomy. One week later, diabetic nephropathy was induced by intraperitoneal injection of streptocozin(30 mg/kg) for three consecutive days. High blood glucose level of Tail vein (fasting glucose ≥ 16.7 mmol/L) and high urinary protein level(total protein level in DN group was more than twice higher than the control group) were measured to confirm early DN in rats. In the sham operation group, the right renal capsule was damaged and the corresponding amount of saline was injected. The model rats were administrated by the means of intragastric administration. The DN model group were divided into DN group, DN+fosinopril group(1.6 mg/kg·d), DN+high CA group(16.8 mg/kg·d), DN+medium CA group(11.2 mg/kg·d) and DN+low CA group(5.6 mg/kg·d), and each group was intragastric administration one time every morning last for 16 weeks. The expressions of mRNA and protein of TGF-β, TβR1, TβR2, Smad2/3, Smad7 and the level of Smad2/3 phosphorylation were detected by using real time quantitative polymerase chain reaction and Western blot. RESULTS: The expressions of mRNA and protein of TGF-β, TβR1, TβR2, Smad2/3 and the level of Smad2/3 phosphorylation were significantly increased, the expressions of mRNA and protein of Smad7 were dramatically decreased. The fosinopril and high dosage CA could reverse the effects of DN. CONCLUSIONS CA plays an important role in preventing and curing DN through regulating the TGF-β/Smad signaling pathways.
Animals ; Centella ; chemistry ; Diabetes Mellitus, Experimental ; Diabetic Nephropathies ; chemically induced ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; physiopathology ; Male ; Rats ; Rats, Sprague-Dawley ; Receptor, Transforming Growth Factor-beta Type I ; metabolism ; Receptor, Transforming Growth Factor-beta Type II ; metabolism ; Signal Transduction ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

Transforming growth factor-β (TGF-β) is a driving force of renal fibrosis, which may lead to chronic kidney diseases and even end stage renal diseases. By activating canonical and non-canonical signaling pathways, TGF-β promotes the synthesis of extracellular matrix while preventing their degradation. In the injured kidney, TGF-β induces apoptosis, proliferation and fibrotic response of renal cells including epithelial cells, endothelial cells, podocytes, fibroblasts, pericytes and macrophages, and it also promotes transdifferentiation, activation and proliferation of myofibroblasts. Additionally, TGF-β exerts profibrotic effects by interplaying with other signaling pathways like BMP-7, Wnt/β-catenin and MAP kinase. Smad3 is the central pathological gene in renal fibrosis, and epigenetic regulation of TGF-β/Smad3 is a hot topic in kidney field. Although direct targeting TGF-β may cause side effects including tumorigenesis and immune diseases, the therapeutic strategies targeting the balance of downstream Smad3 and Smad7 may prevent or delay the progression of fibrotic kidney disease.
Epigenesis, Genetic ; Fibrosis ; Humans ; Kidney Diseases ; pathology ; Signal Transduction ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo explore the role of Treg cells in the pathogenesis of idiopathic thrombocytopenic purpura (ITP).

METHODSThe ITP mouse model was established, the Treg cell ratio in peripheral blood and spleen was detected by flow cytometry, the CD4+ CD25+ T cells were sorted by immunomagnetic beads, the Treg cell associated transcription factors (Foxp3, Smad7, STAT5 and Akt-1) and cytokines (IL-10, TGF-β) in CD4+ CD25+ T cells were enriched from spleen mononuclear cells, and the mRNA expression of Treg cell was measured by real-time PCR.

RESULTSThe ratio of Tregs in peripheral blood and spleen decreased significantly in ITP mouse, as compared with the controls (P<0.01). In addition, the mRNA expression of IL-10, TGF-β and Foxp3 decreased significantly in spleen CD4+ CD25+ T cells (P<0.05). Expression of Smad 7 mRNA was higher than that of controls.

CONCLUSIONThe alteration in Treg frequency and function may be responsible for the immune dysfunction in ITP disease. It is also speculated that the lower mRNA expression of Foxp3 and higher mRNA expression of Smad 7 may inhibit the proliferation and differentiation of Treg cells.

Animals ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Interleukin-10 ; metabolism ; Mice ; Purpura, Thrombocytopenic, Idiopathic ; immunology ; pathology ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Smad7 Protein ; metabolism ; Spleen ; cytology ; T-Lymphocytes, Regulatory ; cytology ; Transforming Growth Factor beta ; metabolism

Transforming growth factor (TGF)-β signaling plays an important role in the pathogenesis of psoriasis. CD109, a novel TGF-β co-receptor, which inhibits TGF-β signaling by enhancing Smad7-dependent degradation of TGF-β type I receptor (TGF-β RI), is abnormally expressed in psoriasis. To date, the expression of Smad7 and the correlation between CD109 and Smad7 expression in psoriasis have not been fully elucidated. This study was designed to investigate the expression and the correlation of CD109 and TGF-β signaling associated proteins in psoriasis and their roles in the pathogenesis of psoriasis. Thirty-two psoriasis specimens were subjected to immunohistochemical staining for CD109, Smad7, TGF-β RI and Ki67. Ten normal skin (NS) specimens served as controls. The positive expression rate (% positive cells) of Smad7 and Ki67 in psoriasis was significantly higher than in NS (62.6%±19.9% vs. 17.2%±4.4%, and 50.7%±14.3% vs. 19.5%±3.2%, respectively, P<0.001), and the expression levels of CD109 and TGF-β RI were reduced significantly in psoriasis as compared with NS (8.1%±6.7% vs. 35.8%±6.7% and 27.3%±3.4% vs. 3.0%±3.4%, respectively, P<0.001). There were significantly negative correlations between CD109 and Smad7 (r=-0.831, P<0.01). These findings indicated that CD109 might play a certain role in the pathogenesis of psoriasis. Lower expression of CD109 and TGF-β RI was highly correlated with higher expression of Smad7 and Ki67, suggesting that CD109 may induce the pathogenesis of psoriasis through Smad7-mediated degradation of TGF-β RI, and lead to the termination of TGF-β signaling.
Adolescent ; Adult ; Antigens, CD ; genetics ; metabolism ; Case-Control Studies ; Down-Regulation ; Female ; GPI-Linked Proteins ; genetics ; metabolism ; Humans ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; metabolism ; Psoriasis ; metabolism ; pathology ; Signal Transduction ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta ; metabolism ; Up-Regulation

OBJECTIVETo explore the effects of Wenyang Huazhuo Tongluo Recipe (WYHZTLR) containing serum on transforming growth factor β1 (TGF-β1)/Smad signaling pathway of skin fibroblasts in systemic sclerosis (SSc).

METHODSTotally 36 SSc patients were randomly assigned to Chinese medicine (CM) group, Western medicine (WM) group, and integrative medicine (IM) group according to random digit table, 12 in each group. Patients in the CM group took WYHZTLR decoction (one dose per day). Patients in the WM group took penicillamine tablet (0. 125 g each time, bid) and Prednisone Acetate Tablet (PAT 20 mg, qd). Patients in the IM group took penicillamine, PAT, and WYHZTLR decoction (in the same dosage of corresponding drugs as aforesaid). All patients were treated for one month to get drug containing serum. Besides, 10 untreated SSc patients' serum was taken as the control group. Healthy subjects' skin fibroblasts were originated from healthy skin tissue of the upper arms of 2 female patients undergoing plastic surgery. Corresponding serum of each group was added in the culture system of SSc patients' and healthy subjects' dermal fibroblasts respectively. Expression levels of TGF-β1 receptor type I (TGF-β1 RI), TGF-β1 receptor II (TGF-β1 RII), p-Smad2/3 and Smad7 protein were examined by Western blot. Expression levels of collagen type I and collagen type III (Col-I, Col-III) mRNA were examined by reverse transcription PCR. Contents of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in the supernatant of SSc, skin fibroblasts were examined by ELISA.

RESULTSCompared with the control group, expression levels of TGF-β1 R I and p-Smad2/3 protein significantly decreased, but expression levels of Smad7 protein significantly increased in skin fibroblasts of SSc patients and healthy subjects of WM, CM, and IM groups (P <0.05, P <0. 01). Meanwhile, the expression level of TGF-β1 RII decreased in the IM group (P <0. 05, P <0. 01). Compared with the WM group, expression levels of TGF-β1 RI and p-Smad2/ 3 protein significantly decreased, but that of Srnad7 protein significantly increased in IM groups (P <0. 01). mRNA expression levels of Col-I and Col-II in SSc skin fibroblasts significantly decreased more in WM, CM, and IM groups than in the control group (P <0. 05, P <0. 01). Besides, the expression level of Col-III mRNA was significantly lower in the IM group than in the WM group (P <0.01). Compared with the control group, serum levels of MMP-9 and MMP-9/TIMP-1 ratios increased more obviously in WM, CM, and IM groups (P <0.05, P <0.01). But expression levels of TIMP-1 decreased significantly in CM and IM groups (P <0.01). Expression levels of MMP-9 and MMP-9/TIMP-1 ratios increased more in the IM group than in the WM group (P <0. 01). Expression levels of TIMP-1 decreased more in CM and IM groups than in the WM group (P <0.01).

CONCLUSIONWYHZTLR containing serum could reduce expression levels of Col-I and Col-III possibly through regulating key signal molecules, such as TGF-β1 RI, p-Smad2/3, and Smad7 in TGF-β1/Smad signaling pathway of SSc skin fibroblasts, and inhibiting transduction of TGF-β1/Smad signaling pathway.

Collagen Type I ; Collagen Type III ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Fibroblasts ; Humans ; Matrix Metalloproteinase 9 ; Scleroderma, Systemic ; drug therapy ; metabolism ; Signal Transduction ; drug effects ; Smad2 Protein ; Smad7 Protein ; Transforming Growth Factor beta1 ; metabolism

Oral mucositis, a severe oral ulceration, is a common toxic effect of radio- or chemoradio-therapy and a limiting factor to using the maximum dose of radiation for effective cancer treatment. Among cancer patients, at least 40% and up to 70%, of individuals treated with standard chemotherapy regimens or upper-body radiation, develop oral mucositis. To date, there is no FDA approved drug to treat oral mucositis in cancer patients. The key challenges for oral mucositis treatment are to repair and protect ulcerated oral mucosa without promoting cancer cell growth. Oral mucositis is the result of complex, multifaceted pathobiology, involving a series of signaling pathways and a chain of interactions between the epithelium and submucosa. Among those pathways and interactions, the activation of nuclear factor-kappa B (NF-κB) is critical to the inflammation process of oral mucositis. We recently found that activation of TGFβ (transforming growth factor β) signaling is associated with the development of oral mucositis. Smad7, the negative regulator of TGFβ signaling, inhibits both NF-κB and TGFβ activation and thus plays a pivotal role in the prevention and treatment of oral mucositis by attenuating growth inhibition, apoptosis, and inflammation while promoting epithelial migration. The major objective of this review is to evaluate the known functions of Smad7, with a particular focus on its molecular mechanisms and its function in blocking multiple pathological processes in oral mucositis.
Animals ; Humans ; Mouth Diseases ; metabolism ; pathology ; prevention & control ; Smad7 Protein ; metabolism ; Stomatitis ; metabolism ; pathology ; prevention & control

The deubiquitinating enzyme ubiquitin specific peptidase 15 (USP15) is regarded as a regulator of TGFβ signaling pathway. This process depends on Smad7, the inhibitory factor of the TGFβ signal, and type I TGFβ receptor (TβR-I), one of the receptors of TGFβ. The expression level of USP15 seems to play vital roles in the pathogenesis of many neoplasms, but so far there has been no report about USP15 in psoriasis. In this study, immunohistochemical staining of USP15, TβR-I and Smad7 was performed in 30 paraffin-embedded psoriasis specimens and 10 normal specimens to investigate the expression of USP15, TβR-I and Smad7 in psoriasis and to explore the relevance among them. And USP15 small interfering RNA (USP15 siRNA) was used to transfect Hacat cells to detect the mRNA expression of TβR-I and Smad7. Of 30 cases of psoriasis in active stage, 28, 24 and 26 cases were positive for USP15, TβR-I and Smad7 staining, respectively. The positive rates of USP15 and Smad7 were significantly higher in psoriasis specimens than in normal skin specimens (44.1%±26.0% vs. 6.1%±6.6%, 47.2%±27.1% vs. 6.6%±7.1%), and positive rate of TβR-I (20.3%±22.2%) in psoriasis was lower than that in normal skin specimens (46.7%±18.2%). There was a significant positive correlation between USP15 and Smad7 expression, and significant negative correlations between USP15 and TβR-expression, an I d between TβR- and Smad7 expression I in psoriasis. After transfection of USP15 siRNA in Hacat cells, the expression of TβR-mRNA was up I -regulated and that of Smad7 was down-regulated. It is concluded that USP15 may play a role in the pathogenesis of psoriasis through regulating the TβR-I/Smad7 pathway and there may be other cell signaling pathways interacting with USP15 to take part in the development of psoriasis.
Adult ; Cell Line ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; Keratinocytes ; cytology ; metabolism ; Male ; Middle Aged ; Protein-Serine-Threonine Kinases ; biosynthesis ; genetics ; Psoriasis ; genetics ; metabolism ; RNA Interference ; Receptors, Transforming Growth Factor beta ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; genetics ; Skin ; metabolism ; Smad7 Protein ; biosynthesis ; genetics ; Ubiquitin-Specific Proteases ; biosynthesis ; genetics ; Young Adult

MicroRNAs (miRNAs) have recently been recognized to have a role in human orthopedic disorders. The objective of our study was to explore the expression profile and biological function of miRNA-17-5p (miR-17-5p), which is well known to be related to cancer cell proliferation and invasion, in osteoblastic differentiation and in cell proliferation. The expression levels of miR-17-5p in the femoral head mesenchymal stem cells of 20 patients with non-traumatic osteonecrosis (ON) and 10 patients with osteoarthritis (OA) were examined by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, the interaction between miR-17-5p and SMAD7 was observed. We found that in non-traumatic ON samples the level of mature miR-17-5p was significantly lower than that of OA samples (P=0.0002). By targeting SMAD7, miR-17-5p promoted nuclear translocation of beta-catenin, enhanced expression of COL1A1 and finally facilitated the proliferation and differentiation of HMSC-bm cells. We also demonstrated that restoring expression of SMAD7 in HMSC-bm cells partially reversed the function of miR-17-5p. Together, our data suggested a theory that dysfunction of a network containing miR-17-5p, SMAD7 and beta-catenin could contribute to ON pathogenesis. The present study prompts the potential clinical value of miR-17-5p in non-traumatic ON.
Adult ; Base Sequence ; Bone Morphogenetic Protein 2/metabolism ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Female ; *Gene Expression Regulation ; Humans ; Male ; MicroRNAs/genetics/*metabolism ; Middle Aged ; Osteoarthritis/genetics/metabolism/pathology ; Osteoblasts/*cytology/metabolism/*pathology ; Osteogenesis ; Osteonecrosis/*genetics/metabolism/pathology ; Signal Transduction ; Smad7 Protein/*genetics/metabolism ; beta Catenin/metabolism

OBJECTIVETo establish a rabbit model of silicotic pulmonary fibrosis and to investigate the effect of cordyceps sinensis in this model.

METHODSThirty healthy male white rabbits were randomly divided into control group, silicosis model group, and intervention group. The rabbits in silicosis model group and intervention group received endotracheal perfusion of silicon dioxide suspension (120 mg/kg), and the control group was treated with the same volume of saline. All the rabbits were sacrificed 30 days later. The lung coefficient was calculated by comparing the lung weight and body weight; the right lung tissue was stained with hematoxylin-eosin (HE). The content of hydroxyproline in lung tissue was measured by alkaline hydrolysis. The mRNA levels of transforming growth factor beta 1 (TGF-β₁) and mothers against decapentaplegic homolog 7 (Smad7) in rabbit lung sections were determined by real-time PCR.

RESULTSNo abnormalities were observed by HE staining in the lung tissues of control group, while fibrosis and silicotic nodules were discovered in the silicosis model group and intervention group. The lung coefficient and the content of hydroxyproline in lung tissue were significantly higher in the silicosis model group than in the control group and intervention group (P < 0.05 or P < 0.01). Compared with the control group, the silicosis model group and intervention group had significantly increased TGF-β₁ mRNA levels but significantly reduced Smad7 mRNA levels (P < 0.02). Compared with the silicosis model group, the intervention group had a significantly reduced TGF-β₁ mRNA level but a significantly increased Smad7 mRNA level (P < 0.05).

CONCLUSIONCordyceps sinensis is able to reduce the expression of TGF-β₁ mRNA and increase the expression of Smad7 mRNA in lung tissues of rabbits with silicotic pulmonary fibrosis, and thus postpone the progression of fibrosis.

Animals ; Cordyceps ; chemistry ; Disease Models, Animal ; Lung ; metabolism ; Male ; Pulmonary Fibrosis ; drug therapy ; metabolism ; Rabbits ; Silicosis ; drug therapy ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism