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

Glucosyltransferases ; metabolism ; HEK293 Cells ; Humans ; Proteasome Endopeptidase Complex ; metabolism ; Protein Stability ; Proteolysis ; Smad3 Protein ; chemistry ; metabolism ; Ubiquitin ; metabolism

OBJECTIVE: To observe the effect of traditional Chinese medicine for intervention of phlegm and blood stasis in regulating TGF-β1/Smad3 signaling and relieving nephropathy in diabetic rats. METHODS: SD rats were divided into blank group (NC), diabetic model group (MC group), intervention of phlegm and blood stasis (RPDBS) group, phlegm-removing (RP) group and blood-removing (DBS) group. Diabetic models were established in all the rats except for those in the blank group. After 4 weeks of feeding, the rats in RPDBS group, RP group and DBS group were given corresponding drug intervention for 8 weeks. HE staining was used to observe the changes in renal histopathology. Western blotting and real-time fluorescence quantitative PCR were used to detect the expression levels of transforming growth factor-β1 (TGF-β1) and Smad3. RESULTS: The structure and arrangement of the glomeruli and renal tubules improved significantly in the treatment groups in comparison with those in the MC group. The expression levels of TGF-β1, Smad3 and p-Smad3 were significantly downregulated at both the protein and mRNA levels in the treatment groups ( < 0.05), and the down-regulation was more obvious in RPDBS group than in RP group and DBS group ( < 0.05). CONCLUSIONS Intervention of phlegm and blood stasis may inhibit the activation of TGF-β1/Smad3 signaling pathway and delay diabetic nephropathy and fibrosis to protect the renal function in diabetic rats.
Animals ; Diabetes Mellitus, Experimental ; Diabetic Nephropathies ; Kidney ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad3 Protein ; Transforming Growth Factor beta1

OBJECTIVE: To clarify the molecular signaling mechanism underlying the inhibitory effect of metformin on transforming growth factor-β1 (TGF-β1)-stimulated collagen I production in rat biliary fibroblasts. METHODS: Primary biliary fibroblasts were isolated under aseptic condition from 50 Sprague-Dawley rats (half male and half female), and microscopic observation identified no obvious difference in the morphology or viability of the cells from rats with different sexes or body weight. The cells were treated with TGF-β1 (10 ng/mL), Smad3 siRNA+TGF-β1, CTGF siRNA+TGF-β1, metformin (10 mmol/L)+ TGF-β1, or Compound C (10 μmol/L)+metformin+TGF-β1. The expressions of CTGF and collagen I in the treated cells were determined using ELISA kit or Western blotting; the phorsphorylated and total Smad3 and AMPK expressions were detected using immunoblotting. RESULTS: TGF-β1 time- and dose-dependently induced collagen I production in rat biliary fibroblasts. The activated AMPK by metformin dose-dependently inhibited TGF-β1-induced collagen I production. Pre-incubation of cells with the AMPK inhibitor Compound C restored the inhibitory effect of AMPK on TGF-β1-induced collagen I secretion ( < 0.01). Activation of AMPK by metformin significantly reduced TGF-β1-induced collagen I production by suppressing Smad3-driven CTGF expression ( < 0.01), and the application of Compound C reversed such changes in the fibroblasts ( < 0.01). CONCLUSIONS Metformin inhibits TGF-β1-stimulated collagen I production by activating AMPK and inhibiting Smad3- driven CTGF expression in rat biliary fibroblasts.
Animals ; Cells, Cultured ; Collagen ; Female ; Fibroblasts ; Male ; Metformin ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad3 Protein ; Transforming Growth Factor beta1

To investigate the amelioration effect of saponins extracted from Panax japonicas (SPJ) on myocardial fibrosis in natural aging rats and its mechanisms, male SD rats aged 18 months were randomly divided into 3 groups (aging model group, low-dose SPJ group and high-dose SPJ group), with 10 rats in each group. SPJ groups were given SPJ at different doses (10, 60 mg·kg⁻¹·d⁻¹) consecutively for 6 months, meanwhile, aging model group was treated with the equal volume of saline for 6 months until 24 months old. Another 10 rats aged 6 month were used as young control group. The changes of myocardial morphological were observed by haematoxylin-eosin (HE) staining. Masson staining was used to observe the changes of collagen deposition in rat hearts. RT-PCR was used to detect the mRNA expression levels of myofibroblast marker α-SMA, collagen-related protein COL1α2, COL3α1 and matrix metalloproteinase MMP2, MMP9. Western blot was used to test the changes of the protein expressions of TGF-β1, p-Smad3, IL-1β and TNF-α in heart tissues. SPJ can effectively improve the arrangement of myocardial fibers, decrease inflammatory infiltration and reduce collagen deposition in aging rats. SPJ can effectively down-regulate the mRNA expression levels of COL1α2, COL3α1, α-SMA, MMP9, MMP2 and inhibit the protein expressions of TGF-β1, p-Smad3, TNF-α, IL-1β in the natural aging heart tissues. SPJ can effectively alleviate myocardial fibrosis in natural aging rats, and its mechanisms was related to the inhibition of the protein expressions of TGF-β1, p-Smad3 and the reduction of myocardial inflammation in rat hearts.
Animals ; Fibrosis ; Male ; Panax ; Rats ; Rats, Sprague-Dawley ; Saponins ; Signal Transduction ; Smad3 Protein ; Transforming Growth Factor beta1

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 investigate the relationship between expression of Smad3, Smad7 and ventricular remodeling in rats after myocardial infarction.

METHODSMyocardial infarction was induced by left anterior descending coronary artery ligation in rats (n = 11) and sham-operated rats were used as control (n = 10). The rats were sacrificed 8 weeks later. Heart weight/body weight (HW/BW), mean blood pressure, left ventricular end diastolic pressure (LVEDP), collagen content in the un-infarcted area were examined. The mRNA levels of transforming growth factor (TGF)beta(1), Smad 3, Smad7 were determined by RT-PCR.

RESULTCompared with controls, the level of HW/BW, LVEDP and collagen content were significant increased. The mRNA expression of TGFbeta(1) and Smad3 was significantly increased in areas of myocardial infarction, border of the infarction, interventricular septum and right ventricle. The expression of Smad7 mRNA in these areas was decreased.

CONCLUSIONThese results indicated that TGFbeta(1)-Smads signaling was correlated to the ventricular remodeling after myocardial infarction. Smad3 might promote the process while Smad7 inhibit the process.

Animals ; Male ; Myocardial Infarction ; metabolism ; physiopathology ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta ; metabolism ; Ventricular Remodeling

Objective: To investigate the clinicopathological features, immunophenotypes and molecular genetics of EWSR1-SMAD3 positive fibroblastic tumor (ESFT) with an emphasis on differential diagnosis. Methods: The clinicopathological data, immunohistochemical profiles and molecular profiles of 3 ESFT cases diagnosed at the Department of Pathology, Fudan University Shanghai Cancer Center from 2018 to 2021were analyzed. The related literature was also reviewed. Results: There were two males and one female. The patients were 24, 12 and 36 years old, respectively. All three tumors occurred in the subcutis of the foot with the disease duration of 6 months to 2 years. The tumors were presented with a slowly growing mass or nodule, accompanied with pain in 1 patient. The tumors ranged in size from 0.1 to 1.6 cm (mean, 1.0 cm). Microscopically, the tumors were located in the subcutaneous tissue with a nodular or plexiform growth pattern. They were composed of cellular fascicles of bland spindle cells with elongated nuclei and fine chromatin. One of the tumors infiltrated into adjacent adipose tissue. There was no nuclear atypia or mitotic activities. All three tumors showed prominent stromal hyalinization with zonal pattern present in one case. Focal punctate calcification was noted in two cases. The immunohistochemical studies showed that tumor cells were diffusely positive for ERG and negative for CD31 and CD34, with Ki-67 index less than 2%. Fluorescence in situ hybridization on the two tested cases identified EWSR1 gene rearrangement. The next generation sequencing analysis demonstrated EWSR1-SMAD3 fusion in all three cases. During the follow up, one patient developed local recurrence 24 months after the surgery. Conclusions: ESFT is a benign fibroblastic neoplasm and has a predilection for the foot, characterized by ERG immunoreactivity and EWSR1-SMAD3 fusion. Local recurrence might occur when incompletely excised. Familiarity with its clinicopathological features is helpful in distinguishing it from other spindle cell neoplasms that tend to occur at acral sites.
Adult ; Child ; Female ; Humans ; Male ; Biomarkers, Tumor/analysis* ; China ; In Situ Hybridization, Fluorescence ; Neoplasms, Fibrous Tissue/pathology* ; RNA-Binding Protein EWS/genetics* ; Smad3 Protein/genetics* ; Soft Tissue Neoplasms/surgery*

Objective To investigate the effect of SMAD family member 3(SMAD3) silenced by small interfering RNA (siRNA) on macrophage polarization and transforming growth factor β1 (TGF-β1)/ SMAD family signaling pathway in rheumatoid arthritis (RA). Methods RA macrophages co-cultured with rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were used as a cell model. TGF-β1 was used to stimulate macrophages, and SMAD3-specific siRNA (si-SMAD3) and negative control siRNA (si-NC) were transfected into human RA macrophages co-cultured in Transwell^TM chamber. The expression of SMAD3 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of TGF-β1, SMAD3 and SMAD7 protein was detected by Western blot analysis. The contents of TGF-β1 and IL-23 in cell culture supernatant were determined by ELISA. Cell proliferation was detected by CCK-8 assay. Transwell^TM chamber was used to measure cell migration. Results Compared with the model group and the si-NC group, the expression of TGF-β1, SMAD3 mRNA and protein in RA macrophages decreased significantly after silencing SMAD3. In addition, the secretion of IL-23 decreased significantly, and the cell proliferation activity and cell migration were inhibited, with high expression of SMAD7. Conclusion Knockdown of SMAD3 can promote M2 polarization and SMAD7 expression in RA macrophages.
Humans ; Arthritis, Rheumatoid/genetics* ; Interleukin-23 ; Macrophages ; RNA, Messenger ; RNA, Small Interfering/genetics* ; Smad7 Protein/genetics* ; Transforming Growth Factor beta1/genetics* ; Smad3 Protein/genetics* ; Gene Silencing

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