Animals ; Epithelial-Mesenchymal Transition ; Lung/metabolism* ; Male ; Pulmonary Disease, Chronic Obstructive ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad2 Protein/metabolism* ; Syndecan-1 ; Transforming Growth Factor beta1/metabolism*

To explore the influence for combination of nourishing yin and tonifying yang sequential therapy (NYTYST) with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve (DOR) based on TGF-β1/Smads signaling pathway. 
 Methods: A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group (n=8 in each group). The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy (HRT) and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor β1 receptor (TGF-β1R) in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot.
 Results: Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group (P<0.01); the levels of TGF-β1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly (P<0.01). Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group (P<0.05 or P<0.01). The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group (P<0.05). Compared with the Western medicine group, the levels of TGF-β1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group (P<0.05 or P<0.01). 
 Conclusion: NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-β1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.
Animals ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Gene Expression Regulation ; drug effects ; Infertility ; therapy ; Medicine, Chinese Traditional ; Ovarian Reserve ; drug effects ; Rats ; Signal Transduction ; drug effects ; Smad2 Protein ; genetics ; metabolism ; Smad3 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

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

OBJECTIVETo investigate the effect of tumor necrosis factor-α-induced protein 8 like-2 (TIPE2) on apoptosis of CD4T lymphocytes in a murine model of severe burn injury.

METHODSA total of 140 male mice were randomly allocated into 6 groups. Small RNA interference technique was used to construct a siTIPE2-overexpressing lentivirus, and severe burn injury models were established in the mice. CD4T cells were purified from spleen of the mice, and the expressions of TIPE2, Smad2/Smad3, P-Smad2/P-Smad3 and Bcl-2/Bimprotein in CD4Tregs were detected. The changes in mitochondrial membrane potential and cytochrome C in CD4T cells were detected, and the activities of caspase-3, caspase-8, and caspase-9 were analyzed.

RESULTSDown-regulation of TIPE2 promoted the apoptosis of CD4T lymphocytes in siTIPE2-burn group, in which the protein expressions of P-smad2/P-Smad3 decreased, Bcl-2 expression increased and Bim expression decreased significantly as compared with the other groups (P<0.01 or 0.05). The mitochondrial membrane potential and cytochrome C expression in CD4T cells were down-regulated in siTIPE2-burn group (P<0.05) with a lowered caspase-3 activity compared with TIPE2-burn group (P<0.01) and decreased caspase-8 and caspase-9 compared with the other groups (P<0.05). The apoptosis rate was the highest in TIPE2-burn group, whose Smad2/Smad3 was higher than that in the sham group (P<0.05) and the expression of P-smad2/P-Smad3 significantly increased compared with the other groups (P<0.05). In TIPE2-burn group, the mitochondrial membrane potential in CD4T cells was decreased (P<0.01), the expression of cytochrome C increased markedly (P<0.01), and the activities of caspase-3, caspase-8, and caspase-9 were all obviously higher than those in the other groups (P<0.05).

CONCLUSIONAs an important immunoregulatory molecule, TIPE2 can promote the apoptosis of CD4T lymphocyte in mice with sever burn injury.

Animals ; Apoptosis ; Burns ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Down-Regulation ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Mice ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Spleen

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

OBJECTIVETo elucidate the role of transforming growth factor-beta1(TGF-β1) in epithelial-mesenchymal transition of mesothelial cells and peritoneal metastasis of gastric cancer.

METHODSHMrSV5 cells, a human peritoneal mesothelial cell line, were incubated with TGF-β1, and their morphological changes were observed by phase contrast microscopy. Expressions of α-smooth muscle actin (α-SMA), vimentin, cytokeratin, E-cadherin, phosphorylated-Smad2 and Smad2 were examined by Western blotting. After fibroblastic-like mesothelial cells were co-incubate with HSC-39 cells(gastric cancer cell line), the adhesion and invasion potential of HSC-39 were evaluated by adhesion and invasion assay in vitro.

RESULTSFew mesothelial cells converted to spindle fibroblast-like morphology for 24 h, and remarkable phenotypic changes were observed at 72 h of TGF-β1 activation. TGF-β1 could induce α-SMA and vimentin expression, and down-regulate cytokeratin and E-cadherin expression in mesothelial cells (P<0.05). TGF-β1 induced phosphorylation of Smad2 within 15 min of stimulation, reached a maximum at 30 min after treatment and remained high level during the experiment without affecting total Smad2 expression(P>0.05). The percentage of HSC-39 gastric cancer cells adhered were significantly increased as compared to the control. When the mesothelial cells were treated by TGF-β1 for 72 h, the increased adhesion percentage was(146±17)%(P<0.05). After fibroblastic-like mesothelial cells co-incubated with HSC-39 cells for 48 h, more cancer cells [(61.1±11.4) cells/view field] invaded the coated membrane as compared to the control group [(31.9±8.1) cells/view field] (P<0.05).

CONCLUSIONTGF-β1 can induce the transition of mesothelial cells into myofibroblasts and Smad2 signal pathway may play a role in this transition, which is associated with increased adhesion and invasiveness of gastric cancer cells, and provides favorable environment for the dissemination of gastric cancer.

Cadherins ; Cell Line, Tumor ; Epithelial Cells ; Epithelial-Mesenchymal Transition ; Epithelium ; Fibroblasts ; Humans ; Peritoneal Neoplasms ; Signal Transduction ; Smad2 Protein ; Stomach Neoplasms ; Transforming Growth Factor beta1 ; Vimentin

OBJECTIVETo explore the mechanism of serotonin-promoted osteoblast differentiation.

METHODSExpression levels of collagen I and lysyl oxidase (LOX) in osteoblast were measured by RT-PCR after treated by (50, 100, 200 and 400 ng/L) serotonin. LOX siRNA effect was measured by Western blot, and protein levels of collagen I were determined by ELISA after treated by serotonin. Expression levels of Smad2 and Smad3 in osteoblasts were also measured by RT-PCR after treated by serotonin.Moreover, expression levels of LOX were measured by RT-PCR after Smad3 was knockout.

RESULTSSerotonin promoted collagen I and LOX expression. The expression level of collagen I was significantly decreased by LOX siRNA. Furthermore, serotonin up-regulated the expression of Smad2 and Smad3 in osteoblasts, and the expression level of LOX was inhibited by Smad3 siRNA.

CONCLUSIONSerotonin promoted collagen I expression by activating Smads signaling pathway and up-regulating the LOX expression.

Blotting, Western ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; metabolism ; Humans ; Osteoblasts ; drug effects ; metabolism ; Protein-Lysine 6-Oxidase ; metabolism ; RNA, Small Interfering ; Serotonin ; pharmacology ; Signal Transduction ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Up-Regulation

Previous studies showed that three methods for regulating and invigorating lung and kidney (lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, and Qi supplementing and kidney nourishing) could regulate inflammatory signaling pathways of chronic obstructive pulmonary disease (COPD) in rats, so as to alleviate inflammation. In the present study, R-value comprehensive evaluation method was used to evaluate the comprehensive effect of three methods for regulating and invigorating lung and kidney on inflammatory signaling pathways. Rats were randomly divided into control, model, lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing and aminophylline groups. The COPD rat models were established by cigarette smoking combined with bacterial infection, and orally administered with drugs between the 9th and 20th week. Afterwards, efforts were made to observe the long-term effects between the drug withdrawal and the 32rd week and detect indicators in two batches in the 20th week and 32th week. Specifically, (1) Linking JAK/STAT signaling pathway: JAK2 mRNA, and protein expressions of STAT-1, STAT-3, STAT-5, JAK-2; (2) NF-kappaB signaling pathway: Smad2 mRNA and protein expressions of I-kappaB, NF-kappaB, TGF-beta1; (3) PPARgamma and antioxidant signaling pathway: SOD, PGE mRNA, PPARgamma protein. According to the results, 5 indicators in JAK/STAT pathway, 4 indicators in NF-kappaB pathway, and 3 indicators in PPARgamma pathway were significantly rectified by three methods for regulating and invigorating lung and kidney in between the 20th week and 32nd week. Between the 20th and 32nd week, the recipes for rectifying JAK/STAT pathway with intensity from high to low were recipes for lung invigorating and spleen strengthening, Qi supplementing and kidney nourishing, lung invigorating and kidney tonifying, aminophylline, particularly those for lung invigorating and spleen strengthening; The recipes for rectifying NF-kappaB pathway with intensity from high to low were recipes for lung invigorating and spleen strengthening, lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing and aminophylline, particularly the first three types of drugs. The recipes for rectifying PPARgamma and antioxidant signaling pathway with intensity from high to low were recipes for lung invigorating and kidney tonifying, Qi supplementing and kidney nourishing, lung invigorating and spleen strengthening and aminophylline. Therefore, three methods for regulating and invigorating lung and kidney showed better long-term effects in regulating COPD lung inflammation signaling pathways. Specifically, recipe for lung invigorating and spleen strengthening showed a better effect in JAK/STAT and NF-kappaB pathways, while recipe for lung invigorating and kidney tonifying and Qi supplementing and kidney nourishing showed better effects in PPARgamma and antioxidant signaling pathways. In conclusion, R-value comprehensive evaluation method can evaluate the comprehensive effect of medicines and define the ranking of multiple drugs and their main targets.
Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Kidney ; drug effects ; physiopathology ; Lung ; drug effects ; immunology ; metabolism ; physiopathology ; NF-kappa B ; immunology ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; immunology ; metabolism ; physiopathology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effects of total flavonoids from astragalus complanatus (FAC) on paraquat poisoning-induced pulmonary fibrosis in rats.

METHODSThe rats were divided into six groups randomly: control group, paraquat group, prednisolone group and FAC low-dose, middle-dose, high-dose group. Pulmonary fibrosis model was replicated by intratracheal injection of paraquat. In the mext day,the rats were treated by intragastric administration once a day. After 28 days, the rats were sacrificed. The lung index and the levels of HYP and T-AOC were measured, and the pathologic changes of the lung tissue were obtained by HE staining. The levels of TGF-β, Smad2, α-SMA protein were analyzed by Western blot.

RESULTSFAC improved the activity of T-AOC in serum and reduced pulmonary index and the content of HYP as well (P<0.05 or P<0.01), the alveolitis and fibrosis extent were attenuated. The expression of Smad2 significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.31±0.11, 0.45±0.12 and 0.30±0.05) as compared with that of the PQ group (0.85±0.34) (P<0.05). The expression of α-SMA significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.31±0.11, 0.35±0.07 and 0.32±0.10) as compared with that of the PQ group (0.45±0.08) (P<0.05). The expression of TGF-β significantly decreased in groups of FAC low-dose, middle-dose and high-dose (0.35±0.04, 0.27±0.05 and 0.18±0.04)as compared with that of the PQ group (0.63±0.11) (P<0.05).

CONCLUSIONFAC can alleviate PQ-induced pulmonary fibrosis in rats through inhibiting TGF-β/Smad signaling pathway.

Actins ; metabolism ; Animals ; Astragalus Plant ; chemistry ; Flavonoids ; pharmacology ; Lung ; pathology ; Paraquat ; poisoning ; Phytochemicals ; pharmacology ; Pulmonary Fibrosis ; chemically induced ; drug therapy ; Rats ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism