OBJECTIVETo explore the effect of Bushen Tiaojing Recipe (BTR) on the expressions of drosophila mothers against decapentaplegic protein (Smadl), Smad5, Smad8, and Smad4 on human mural granulosa cells.

METHODSSixty-six patients undergoing in vitro fertilization-embryo transfer (IVF-ET) were randomly assigned to two groups in the ratio of 1:2, the treatment group and the control group. Twenty-three patients in the treatment group were treated with BTR and GnRHa/FSH/hCG, while forty-three patients in the control group were treated with GnRHa/FSH/hCG. The mRNA expressions of Smad1, Smad5, Smad8, and Smad4 on mural granulosa cells of the mature follicle were detected by real-time PCR on the ovum retrieval day. The expressions of Smad1, Smad5, Smad8, and Smad4 at the protein level were observed using cell immunofluorescence method.

RESULTSThe mRNA and protein expressions of Smadl in the granulosa cells were significantly higher in the treatment group than in the control group (P <0.05). There was no statistical difference in the mRNA and protein expressions of Smad5, Smad8, and Smad4 between the two groups.

CONCLUSIONThe mechanisms of BTR for improving the pregnancy rate and the ovarian functions might be correlated with up-regulating mRNA and protein expressions of Smadl of human mural granulosa cells.

Adult ; Drugs, Chinese Herbal ; pharmacology ; Female ; Granulosa Cells ; drug effects ; metabolism ; Humans ; Ovarian Follicle ; cytology ; Signal Transduction ; Smad1 Protein ; metabolism ; Smad4 Protein ; metabolism ; Smad5 Protein ; metabolism ; Smad8 Protein ; metabolism

Zfyve16 (a.k.a. endofin or endosome-associated FYVE-domain protein), a member of the FYVE-domain protein family, is involved in endosomal trafficking and in TGF-β, BMP, and EGFR signaling. The FYVE protein SARA regulates the TGF-β signaling pathway by recruiting Smad2/3 and accelerating their phosphorylation, thereby altering their susceptibility to TGF-β-mediated T cell suppression. Zfyve16 binds to Smad4 and their binding affects the formation of Smad2/3-Smad4 complex in TGF-β signaling. However, the in vivo function of Zfyve16 remains unknown. In this study, we generated a Zfyve16 knockout mouse strain (Zfyve16) and examined its hematopoietic phenotypes and hematopoietic reconstruction ability. The proportion of Tcells in the peripheral blood of Zfyve16 mice increases compared with that in wild-type mice. This finding is consistent with the role of Zfyve16 in facilitating TGF-β signaling. Unpredictably, B cell proliferation is inhibited in Zfyve16 mice. The proliferation potential of Zfyve16 B-lymphoid cells also significantly decreases in vitro. These results suggest that Zfyve16 inhibits the proliferation of T cells, possibly through the TGF-β signaling, but upregulates the proliferation of B-lymphoid cells.
Animals ; B-Lymphocytes ; metabolism ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Movement ; Cell Proliferation ; genetics ; Intracellular Signaling Peptides and Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Mice ; Mice, Knockout ; Serine Endopeptidases ; genetics ; metabolism ; Signal Transduction ; Smad Proteins, Receptor-Regulated ; metabolism ; Transforming Growth Factor beta ; metabolism ; Up-Regulation

OBJECTIVESTo study the expression of Smad1 and Smad5 in the testis of infertile rats with adenine-modeled kidney-yang deficiency and the pathological mechanism of infertility with kidney-yang deficiency, attempting to obtain experimental evidence for the prevention and treatment of male infertility.

METHODSForty-eight 60 d male SD rats were divided randomly into 6 groups with 8 in each: 7 d, 14 d and 21 d kidney-yang deficiency groups, and 7 d, 14 d and 21 d control groups. The experimental rats had been fed with adenine (300 mg/kg) and the expression levels of Smad1 and Smad5 were measured with immunohistochemical SABC method at the 7th, 14th and 21st day.

RESULTSSmad1 immunoreactivity was mainly located in the spermatogonia, spermatocytes and spermatids, and the reactive substance distributed in cytoplasm with negative nuclei. Sertoli cells and Leydig cells were negative. Compared with the control, the expression level of Smad1 was decreased significantly at the 21st day (P < 0.05), but with no significant difference at the 7th and 14th day (P > 0.05). Smad5 immunoreactivity was mainly located in the spermatogonia and spermatocytes, and the reactive substance distributed in cytoplasm with negative nuclei. Compared with the control, the expression level of Smad5 was not significantly different at the 7th day (P > 0.05). The expression of Smad5 was negative at the 14th and the 21st day.

CONCLUSIONThe weaker expression of Smad1 and no expression of Smad5 may be one of the pathological mechanisms of infertility with adenine-modeled kidney-yang deficiency.

Animals ; Infertility, Male ; metabolism ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Smad1 Protein ; biosynthesis ; Smad5 Protein ; biosynthesis ; Testis ; metabolism ; Yang Deficiency ; metabolism ; pathology

AIMTo study the expression and regulation of Smad1, Smad2 and Smad4 proteins (intracellular signaling molecules of transforming growth factor-b family) in rat testis during postnatal development.

METHODSThe whole testes were collected from SD rats aged 3, 7, 14, 28 and 90 (adult) days. The cellular localization and developmental changes were examined by immunohistochemistry ABC method with the glucose oxidase-DAB-nickel enhancement technique. Quantitative analysis of the immunostaining was made by the image analysis system. The Smads proteins coexistence in the adult rat testis was tested by the double immune staining for CD14-Smad4 and Smad2-Smad4. The protein expression of Smad during rat testicular development was examined by means of Western blots.

RESULTSSmad1, Smad2 and Smad4 were present throughout testicular development. The immunostaining of Smad1 and Smad2 were present in spermatogenic cells. A positive immunoreactivity was located at the cytoplasm, but the nucleus was negative. Smad1 was immunolocalized at the d14, d28 and adult testes, while Smad2, at the d7, d14, d28 and adult testis. There was positive immunoreaction in the Sertoli cells and Leydig cells as well. The immunolocalization of Smad4 was exclusively at the cytoplasm of Leydig cells and the nuclei were negative throughout the testicular development. No expression was detected in the germ cells. The results of image and statistical analysis showed that generally the expression of Smad1, Smad2 and Smad4 in the testis tended to increase gradually with the growth of the rat.

CONCLUSIONThe present data provide direct evidences for the molecular mechanism of TGF-bgr action in rat testes during postnatal development and spermatogenesis.

Animals ; Blotting, Western ; DNA-Binding Proteins ; analysis ; biosynthesis ; Immunohistochemistry ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology ; Smad Proteins ; Smad1 Protein ; Smad2 Protein ; Smad4 Protein ; Testis ; chemistry ; growth & development ; physiology ; Trans-Activators ; analysis ; biosynthesis

OBJECTIVETo observe the effect of single herb pilose antler (PA) on the expression of Smad2 and Smad3 in the cartilage of osteoarthritis (OA) rats.

METHODSOne hundred 3-month old female healthy SD rats, (200 +/- 20) g, were recruited and routinely fed for 1 week. They were randomly divided into 5 groups, i.e., the low dose PA group, the high dose PA group, the normal saline control group, the model group, and the normal control group, 20 in each group. The model was prepared using classic Hulth method except the normal control group. After 6-week modeling, the model was confirmed successful by pathologic observation. PA at 0.021 g/100 g and 0.084 g/1 00 g was given by gastrogavage to rats in the low dose PA group and the high dose PA group respectively. Normal saline was administered to those in the normal saline control group. No treatment was given to rats in the normal control group and the model group. Bilateral knee cartilages were harvested at week 2,4, and 6. mRNA and protein expressions of Smad2 and Smad3 were detected by immunohistochemical assay, fluorescent quantitative PCR, and Western blot.

RESULTSOA model was successfully prepared by pathological observation. Results of immunohistochemical assay showed that Smad2 and Smad3 expressed extensively in the cartilage, and located inside the chondrocyte membrane. Compared with the model group, mRNA expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 2, 4, and 6, showing statistical difference (P < 0.05). Compared with the same group at week 4 after gastrogavage, mRNA expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.05). Compared with the model group, protein expression of Smad2 and Smad3 obviously increased in the chondrocytes of the low dose PA group and the high dose PA group at week 2 and 4, showing statistical difference (P < 0.01). Compared with the same group at week 2 after gastrogavage, protein expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 4, showing statistical difference (P < 0.01). Compared with the same group at week 4 after gastrogavage, protein expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.01).

CONCLUSIONS(1) The pilose antler could repair cartilages by regulating mRNA and protein expressions of Smad2 and Smad3. (2) Up-regulating mRNA and protein expressions of Smad2 and Smad3 might be one of important mechanisms for the pathogenesis of OA.

Animals ; Antlers ; chemistry ; Cartilage ; cytology ; metabolism ; Chondrocytes ; drug effects ; metabolism ; Female ; Medicine, Chinese Traditional ; Osteoarthritis ; drug therapy ; metabolism ; Rats ; Rats, Sprague-Dawley ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism

OBJECTIVETo observe Smads protein expression in lung tissue of quartz exposed mice and to explore its association with pulmonary fibrosis in silicosis.

METHODSThe experimental mice were divided into control and quartz groups. 0.2 g/kg weight of quartz was injected intratracheally in quartz group. Samples were collected at the 1st, 3rd, 5th, 7th, 14th and 28th day after injection. Immunohistochemical methods with quantitative image analysis were used to assay the protein expression of transforming growth factor beta(1) (TGF-beta(1)), Smad 2/3, Smad 4, and Smad 7 protein levels. Protein expression level is presented by positive unit (PU).

RESULTSSmad 2/3 protein expression increased from day 3, reaching its peak level in day 14 [(42.2 +/- 2.4) PU], and decreased gradually. The elevation of Smad 4 protein level began from day 5, and the highest degree came into day 14 [(40.0 +/- 1.8) PU], decreased thereafter. The expression of Smad 7 presented a decreasing tendency at the beginning and reaching the lowest level in day 14 [(33.5 +/- 3.3) PU]. It seemed to elevate in day 28, but was still lower than the controls. There were positive correlation between Smad 2/3, Smad 4 and TGF-beta(1) (r = 0.91, r = 0.71, respectively, P < 0.05) and also between Smad 2/3 and hydroxyproline contents of lung tissue (r = 0.85, P < 0.05) except Smad 7.

CONCLUSIONSmad protein may have certain association with pulmonary fibrosis in silicosis.

Animals ; DNA-Binding Proteins ; immunology ; metabolism ; Lung ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Quartz ; toxicity ; Smad2 Protein ; Smad3 Protein ; Smad4 Protein ; Smad7 Protein ; Trans-Activators ; immunology ; metabolism ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo investigate the expression of TGF-betaR I, Smad2, Smad3 and Smad7 in keloids, normal scars and normal skins. Discuss the significance of these proteins in the course of keloid.

METHODSImmunohistochemistry method was used to detect the expression intensity and distribution of these proteins in above 3 kinds of different tissues in 44 cases. Statistics was used to analyze the data.

RESULTSThe expression of TGF-betaR I were much stronger in keloid than in the other two tissues. The expression of Smad7 were lower in keloids. The increase expression of Smad2,3 were not obvious, but they were found to accumulate in the nucleus.

CONCLUSIONSThe results indicate that over-expression of TGF-betaR I, low-expression of Smad7 and accumulation of Smad2,3 may be one of the etiological factors of keloids. This research may provide a new idea to prevent and treat keloids or other fibrosis diseases in the future.

Adolescent ; Adult ; Female ; Humans ; Keloid ; metabolism ; Male ; Middle Aged ; Protein-Serine-Threonine Kinases ; metabolism ; Receptors, Transforming Growth Factor beta ; metabolism ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Young Adult

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

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 mechanism of losartan treating glomerulosclerosis and to observe the effect of losartan on the expressions of TGF-beta1, p-Smad2/3, and Smad7 in the renal tissues of 5/6 nephrectomized rats. METHODS: Male Wistar rats were randomly divided into a sham-operated group, a 5/6 nephrectomized model group, and a losartan treated group. The rats in the model group and the losartan treated group were performed 5/6 nephrectomy by the method with 2 procedures. Twelve weeks after of the operation, all rats were killed. The 24-hour urinary protein, serum creatinine, and urea nitrogen were detected. Pathological changes of the renal tissues were observed by HE and Masson staining, and the expressions of TGF-beta1, p-Smad2/3, and Smad7 were detected by immunohistochemical staining. RESULTS: The 24-hour urinary protein, serum creatinine, urea nitrogen, and the relative area of collagen in the renal tissues of the rats in the model group significantly increased (P<0.01), and losartan could reduce these indexes. The expressions of TGF-beta1 and p-Smad2/3 were just at a low level in the renal tissues of the rats in the sham-operated group, and were strongly positive in the model group; but losartan could decrease the expressions of TGF-beta1 and p-Smad2/3 (P<0.01). The expression of Smad7 in the model group was fewer than that in the sham-operated group (P<0.01), but losartan could improve the expression of Smad7 (P<0.01). CONCLUSION Losartan may implement its anti-glomerulosclerosis by affecting TGF-beta1, p-Smad2/3, and Smad7 of TGF-beta/Smads pathway of the renal tissues of 5/6 nephrectomized rats.
Animals ; Kidney ; drug effects ; metabolism ; Losartan ; pharmacology ; Male ; Nephrectomy ; methods ; Rats ; Signal Transduction ; drug effects ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism