Effects of Shenyan 1 Prescription on renal fibrosis improvement in rats with unilateral ureteral obstruction based on TGF-β1/Smad3 signaling pathway / 国际中医中药杂志

Objective:To investigate the protective effects and mechanism of Shenyan 1 Prescription on renal fibrosis of unilateral ureteral obstruction (UUO) rats through TGF- β 1/Smad homologous 3 (Smad3) pathway regulating ferroptosis.Methods:Totally 48 male SD rats were divided into four groups: sham-operation group, UUO model group, and Shenyan 1 Prescription low-(10 drug/kg) , and high-dosage (20 crude drug/kg) groups according to random number table method, with 12 rats in each group. The UUO model was induced by the method of unilateral ureteral obstruction except for those sham-operation group. After modeling, rats received corresponding drugs or normal saline by gavage for 4 weeks, once per day. After 4 weeks, the body mass and the left kidney weight were measured. The 24 h urine protein and the levels of serum albumin (ALB), alanine aminotransferase (ALT), serum creatinine (SCr) and blood urea nitrogen (BUN) were detected by biochemical analysis method; the ROS level in renal tissue was measured using a chemical fluorescence assay kit, and the SOD and MDA levels in left renal tissue of rats were measured using ELISA method; the morphology of renal tissue and the specific blue staining of hemosiderin were observed using HE and Prussian blue staining methods, respectively; the expressions of transforming growth factor-β1 (TGF-β1), Smad3, glutathione peroxidase 4 (GPX4), and solute carrier family 1 member 5 (SLC1A5) were detected by Western blot.Results:Compared with the model group, the 24 h urinary protein excretion in Shenyan 1 Prescription high-dosage group decreased ( P<0.05), the serum ALB level increased ( P<0.05), the ALT level decreased ( P<0.05), and the expression of SLC1A5 in renal tissue decreased ( P<0.05); the left kidney weight/body decreased in Shenyan 1 Prescription low- and high-dosage groups ( P<0.05); the levels of serum ROS and MDA decreased ( P<0.05), and the activity of SOD significantly increased ( P<0.05); the expressions of TGF-β1 and Smad3 in renal tissue decreased ( P<0.05), and the expression of GPX4 increased ( P<0.05), and the renal pathological injury and ion deposition were improved. Conclusion:Shenyan 1 Prescription has a protective effect on the structure and function of renal tissues in UUO rats through regulating ferroptosis via inhibition of the TGF-β1/ Smad3 pathway to inhibit renal fibrosis of UUO rats.

The mechanism of miR-10b targeting TGFBR1/SMAD3 pathway on chondrocyte proliferation and hypertrophy in idiopathic short stature / 天津医药

Objective To investigate the effect and mechanism of microRNA-10b(miR-10b)on idiopathic short stature(ISS).Methods A total of 54 children with ISS and 54 healthy children were collected.The serum expression of miR-10b was detected by RT-qPCR,and the relationship between serum miR-10b expression and clinical data of children with ISS was analyzed.miR-10b inhibitor,si-TGFBR1 and their negative control transfection C28/I2 cells were used.CCK-8 experimental detection was used to detect C28/I2 cell proliferation.Western blot assay was used to detect gnome related transcription factor 2(RUNX2),collagen type X alpha 1 chain(COL10A1),transforming growth factor beta receptor 1(TGFBR1),SMAD3 and pSMAD3 protein expression.The target of miR-10b was screened in StarBase database,and the targeting relationship between miR-10b and TGFBR1 was verified by dual luciferase reporter gene assay.Results The serum expression of miR-10b was higher in the ISS group than that of the healthy control group,and the higher the miR-10b expression,the more obvious the decrease of child height,IGF-1 and alkaline phosphatase(P＜0.05).Compared with the NC group,the cell proliferation ability and RUNX2,COL10A1,TGFBR1,and pSMAD3 protein expression were up-regulated in the miR-10b inhibitor group(P＜0.05).StarBase database suggested that miR-10b had a binding site of TGFBR1,and dual luciferase reporter gene assay confirmed that TGFBR1 interacted with miR-10b(P＜0.05).Compared with the si-NC group,the expression of TGFBR1 was down-regulated and the cell proliferation ability was decreased in the si-TGFBR1 group(P＜0.05).Conclusion miR-10b inhibits chondrocyte proliferation and hypertrophy in idiopathic short stature by targeting TGFBR1/SMAD3 pathway.

Uncovering pharmacological mechanisms of Phellinus linteus on focal segmental glomeruloscleosis rats through tandem mass tag-based quantitative proteomic analysis, network pharmacology analysis and experimental validation.

OBJECTIVE: To explore the underlying molecular mechanism of (). METHODS: We used a tandem mass tag-based quantitative proteomic method to determine the differentially expressed proteins. Network pharmacology analysis was used to analysis the main components of and construct the compound-target network. Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) were used to validate the analyses results. RESULTS: The expression levels of thrombospondin-1 (TSP-1) and transforming growth factor (TGF)-ß1/Smad3 signaling pathway proteins were significantly upregulated in focal segmental glomeruloscleosis (FSGS) rats. The reduced the expression levels of TSP-1 and TGF-ß1 signaling pathway proteins. Network pharmacology analysis revealed that protocatechualdehyde was the main active component. Subsequent and experiments validated the results of proteomic and network pharmacology analyses. CONCLUSIONS: Our results suggested that may inhibit renal sclerosis by inhibiting TSP-1-activated TGF-ß1 signaling and may have potential applications in the treatment of FSGS.

Effects of exosomes of mesenchymal stem cells on cholesterol-induced hepatic fibrogenesis.

Objectives: Free cholesterol in the diet can cause liver fibrosis by accumulating in Hepatic stellate cells (HSCs). The rate of mortality of this disease is high worldwide and there is no definite remedy for it, but might be treated by anti-fibrotic therapies. MSCs-derived exosomes are known as the new mechanism of cell-to-cell communication, showing that exosomes can be used as a new treatment. In this study, we investigated the ability of exosomes of WJ-MSCs as a new remedy to reduce cholesterol-induced liver fibrosis in the LX2 cell line. Materials and Methods: MSCs were isolated from Wharton's jelly of the umbilical cord and the exosomes were extracted. The LX2 cell line was cultured in DMEM medium with 10% FBS, then cells were treated with 75 and 100 µM concentrations of cholesterol for 24 hr. The mRNA expression of TGF-ß, αSMA, and collagen1α genes, and the level of Smad3 protein were measured to assess liver fibrosis. Results: Cholesterol increased the expression of TGF-ß, αand -SMA, and collagen1α genes by increasing the phosphorylation of the Smad3 protein. Treatment with Exosomes significantly reduced the expression of TGF-ß, α-SMA, and collagen1α genes (fibrosis genes). Treatment with exosomes prevented the activation of HSCs by inhibiting the phosphorylation of the Smad3 protein. Conclusion: The exosomes of WJ-MSCs can inhibit the TGFß/Smad3 signaling pathway preventing further activation of HSCs and progression of liver fibrosis. So, the exosomes of WJ-MSCs s could be introduced as a treatment for liver failure.

Paracrine effect of the stromal vascular fraction containing M2 macrophages on human chondrocytes through the Smad2/3 signaling pathway.

The adipose-derived stromal vascular fraction (SVF) is composed of a heterogeneous mix of adipose-derived stem cells (ADSCs), macrophages, pericytes, fibroblasts, blood, and other cells. Previous studies have found that the paracrine effects of SVF cells may be therapeutic, but their role in osteoarthritis treatment remains unclear. This study aimed to investigate the therapeutic effect of SVF cells on chondrocytes. Chondrocytes were seeded on culture plates alone (control) or cocultured with SVF or ADSCs on cell culture inserts. After 48 h of coculture, chondrocyte collagen II, tissue inhibitors of metalloproteinases-3 (TIMP-3), and matrix metalloproteinases-13 (MMP-13) messenger RNA (mRNA) expression levels were evaluated using reverse-transcription polymerase chain reaction, and the transforming growth factor-ß (TGF-ß) levels in the supernatant were measured using ELISA. Immunohistochemical staining and flow cytometry were used to evaluate the macrophages in the SVF. These macrophages were characterized according to phenotype using the F4/80, CD86, and CD163 markers. To determine whether the Smad2/3 signaling pathways were involved, the chondrocytes were pre-treated with a Smad2/3 phosphorylation inhibitor and stimulated with the SVF, and then Smad2/3 phosphorylation levels were analyzed using western blot. The mRNA expression levels of various paracrine factors and chondrocyte pellet size were also assessed. Collagen II and TIMP-3 expression were higher in the SVF group than in the ADSC group and controls, while MMP-13 expression was the highest in the ADSC group and the lowest in the controls. TGF-ß levels in the SVF group were also elevated. Immunohistochemical staining and flow cytometry revealed that the macrophages in the SVF were of the anti-inflammatory phenotype. Western blot analysis showed that the SVF increased Smad2/3 phosphorylation, while Smad2/3 inhibitors decreased phosphorylation. Smad2/3 inhibitors also reduced the expression of various other paracrine factors and decreased chondrocyte pellet size. These findings suggested that the paracrine effect of heterogeneous cells, such as anti-inflammatory macrophages, in the SVF partly supports chondrocyte regeneration through TGF-ß-induced Smad2/3 phosphorylation.

Role of CD73 in endogenous protective mechanism of hepatic ischemia-reperfusion injury in mice and the relationship with TGF-β 1/Smad3 signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of ecto-5′-nucleotidase (CD73) in endogenous protective mechanism of hepatic ischemia-reperfusion (I/R) injury in mice and the relationship with transforming growth factor beta1 (TGF-β 1)/Smad3 signaling pathway. Methods:Twenty-four SPF healthy male C57BL/6 mice, aged 8-10 weeks, weighing 20-23 g, were divided into 3 groups ( n=8 each) by the random number table method: sham operation group (S group), hepatic I/R group (IR group) and hepatic I/R plus CD73 specific inhibitor group (APCP group). The hepatic hilum was only exposed but not occluded in group S. The hepatic portal was occluded for 30 min followed by reperfusion to develop the model of hepatic I/R in anesthetized animals in group IR.CD73-specific inhibitor APCP 40 mg/kg was intraperitoneally injected, and 10 min later hepatic I/R was performed.Orbital venous blood samples were collected at 6 h of reperfusion for determination of serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations.Then the mice were sacrificed, and liver tissues were obtained for determination of the expression of CD73, TGF-β 1 and phosphorylated Smad3 (p-Smad3) (by Western blot), contents of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay), and content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) (with a visible spectrophotometer) and for microscopic examination of the pathological changes of liver tissues (with a light microscope). Results:Compared with group S, the concentrations of AST and ALT in serum and contents of IL-1β, TNF-α and MDA in liver tissues were significantly increased, the activity of SOD was decreased, and the expression of CD73, TGF-β 1 and p-Smad3 was up-regulated in IR and APCP groups ( P<0.05). Compared with group IR, the concentrations of AST and ALT in serum and contents of IL-1β, TNF-α and MDA in liver tissues were significantly increased, the activity of SOD was decreased, and the expression of CD73, TGF-β 1 and p-Smad3 in liver tissues was down-regulated in group APCP ( P<0.05). The pathological changes of liver tissues were accentuated in group APCP as compared with group IR. Conclusions:CD73 is involved in the process of endogenous protective mechanism of hepatic I/R injury in mice, which may be related to the regulation of TGF-β 1/Smad3 signaling pathway.

Latency-Associated Transcript-Derived MicroRNAs in Herpes Simplex Virus Type 1 Target SMAD3 and SMAD4 in TGF-ß/Smad Signaling Pathway.

Background: During its latent infection, hepatic stellate cell (HSV-1) produces only a micro RNA (miRNA) precursor called latency-associated transcript (LAT), which encodes six distinct miRNAs. Recent studies have suggested that some of these miRNAs could target cellular mRNAs. One of the key cell signaling pathways that can be affected by HSV-1 is the TGF-ß/Smad pathway. Herein, we investigated the potential role of the LAT as well as three LAT-derived miRNAs in targeting SMAD3 and SMAD4, as two main mediators in TGF-ß/Smad. Methods: The selection of LAT-derived miRNAs was based on the search results obtained from an online miRNA prediction tool. HEK293T cells were transfected with each miRNA-expressing lentivector and with the construct-expressing LAT. To survey the effect of LAT on the expression of pro-fibrotic markers, we transfected LX-2 cells with LAT construct. The impact of viral miRNA overexpression on SMADs and fibrotic markers was measured by quantitative PCR and luciferase assays. Results: Among the LAT-derived miRNAs, miR-H2, miR-H3, and miR-H4 were selected for the study. Our results demonstrated that while miR-H2 binds to both SMAD mRNAs, miR-H3 and miR-H4 inhibit only the expression of the SMAD4 and SMAD3, respectively. Transfection of the LX-2 with LAT also decreased pro-fibrotic genes expression. Conclusion: Our findings display that LAT negatively regulates TGF-ß/Smad through targeting SMAD3 and SMAD4 by its miRNAs. These viral miRNAs can also contribute to the development of therapeutic interventions in diseases for which prevention or treatment can be achieved through targeting TGF-ß pathway.

SUV39H1-Mediated DNMT1 is Involved in the Epigenetic Regulation of Smad3 in Cervical Cancer.

BACKGROUND: SMAD3 is a pivotal intracellular mediator for participating in the activation of multiple immune signal pathways. OBJECTIVE: The epigenetic regulation mechanism of the positive immune factor SMAD3 in cervical cancer remains unknown. Therefore, the epigenetic regulation on SMAD3 is investigated in this study. METHODS: The methylation status of SMAD3 was detected by Methylation-Specific PCR (MS-PCR) and Quantitative Methylation-Specific PCR (MS-qPCR) in cervical cancer tissues and cell lines. The underlying molecular mechanisms of SUV39H1-DNMT1-SMAD3 regulation were elucidated using cervical cancer cell lines containing siRNA or/and over-expression systems. The regulation of DNMT1 by SUV39H1 was confirmed using Chromatin Immunoprecipitation-qPCR (ChIP-qPCR). The statistical methods used for comparing samples between groups were paired t-tests and one-way ANOVAs. RESULTS: H3K9me3 protein regulated by SUV39H1 directly interacts with the DNMT1 promoter region to regulate its expression in cervical cancer cells, resulting in the reduced expression of the downstream target gene DNMT1. In addition, DNMT1 mediates the epigenetic modulation of the SMAD3 gene by directly binding to its promoter region. The depletion of DNMT1 effectively restores the expression of SMAD3 in vitro. Moreover, in an in vivo assay, the expression profile of SUV39H1-DNMT1 was found to correlate with SMAD3 expression in accordance with the expression at the cellular level. Notably, the promoter region of SMAD3 was hypermethylated in cervical cancer tissues, and this hypermethylation inhibited the subsequent gene expression. CONCLUSION: These results indicate that SUV39H1-DNMT1 is a crucial SMAD3 regulatory axis in cervical cancer. SUV39H1-DNMT1 axis may provide a potential therapeutic target for the treatment of cervical cancer.

[Mechanism of transcriptional regulation of Meox1 by transforming growth factor ß (1) and its effect on cell migration of adult human dermal fibroblasts].

Objective: To explore the transcriptional regulation mechanism of transforming growth factor ß(1) (TGF-ß(1)) on Meox1 and its effect on cell migration of adult human dermal fibroblasts (HDF-a). Methods: (1) HDF-a cells were cultured in RPMI 1640 complete medium (hereinafter referred to as routinely cultured). The cells were divided into TGF-ß(1) stimulation group and blank control group. The cells in TGF-ß(1) stimulation group were stimulated with 10 µL TGF-ß(1) in the mass concentration of 1 mg/µL, while the cells in blank control group were stimulated with the equal volume of phosphate buffer solution. After 72 hours in culture, partial cells in both groups were collected for transcriptome sequencing. The genes with differential expression ratio greater than or equal to 2 and P<0.01 between the two groups were selected to perform enrichment analysis and analysis of metabolic pathways of the Kyoto Gene and Genome Encyclopedia with, and the expression value of Meox1 per million transcripts (TPM) was recorded (n=3). Partial cells from the two groups were used to detect the Meox1 mRNA expression by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) (n=3). (2) Cultured HDF-a cells in the logarithmic growth phase (the same growth phase of cells below) were divided into empty plasmid group, Smad2 overexpression (OE) group, Smad3 OE group, and Smad4 OE group, which were transfected respectively with 2 µg empty pcDNA3.1 plasmid and pcDNA3.1 plasmids separately carrying Smad2, Smad3, and Smad4 for 6 hours, and then were routinely cultured for 48 hours. The Meox1 mRNA expression in the transfected cells of each group was detected by real-time fluorescent quantitative RT-PCR (n=3). (3) HDF-a cells were routinely cultured and grouped the same as in experiment (1). After 72 hours in culture, the enrichment of Smad2, Smad3, and Smad4 protein on the Meox1 promoter in the cells of each group was detected by chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) (n=3). (4) HDF-a cells were routinely cultured and divided into negative interference group, small interference RNA (siRNA)-Smad2 group, siRNA-Smad3 group, siRNA-Smad4 group, empty plasmid group, Smad2 OE group, Smad3 OE group, and Smad4 OE group, which were transfected respectively with 50 µmol/L random siRNA, siRNA-Smad2, siRNA-Smad3, siRNA-Smad4, 2 µg empty pcDNA3.1 plasmid and pcDNA3.1 plasmids separately carrying Smad2, Smad3, and Smad4 for 6 hours and then routinely cultured for 48 hours. The enrichment of Smad2, Smad3, and Smad4 protein on the Meox1 promoter in the cells of corresponding group was detected by ChIP-qPCR (n=3). (5) Two batches of HDF-a cells were cultured and divided into negative interference group, siRNA-Meox1 group, empty plasmid group, and Meox1 OE group, which were transfected respectively with 50 µmol/L random siRNA, siRNA-Meox1, 2 µg empty pcDNA3.1 plasmid and pcDNA3.1 plasmid carrying Meox1 for 6 hours and then routinely cultured for 24 hours. One batch of cells were subjected to scratch test with the scratch width being observed 24 hours after scratching and compared with the initial width for scratch wound healing; the other batch of cells were subjected to Transwell assay, in which the migrated cells were counted after being routinely cultured for 24 hours (n=3). (6) From January 2018 to June 2019, 3 hypertrophic scar patients (2 males and 1 female, aged 35-56 years) were admitted to the First Affiliated Hospital of Army Medical University (the Third Military Medical University) 8-12 months after burns. The scar tissue and normal skin tissue along the scar margin resected during surgery were taken, and immunohistochemical staining was performed to observe the distribution of Meox1 protein expression. Data were statistically analyzed with one-way analysis of variance and independent sample t test. Results: (1) After 72 hours in culture, a total of 843 genes were obviously differentially expressed between the two groups, being related to tissue repair, cell migration, inflammatory cell chemotaxis induction process and potential signaling pathways such as tumor necrosis factor, interleukin 17, extracellular matrix receptor. The TPM value of Meox1 in the cells of blank control group was 45.9±1.9, which was significantly lower than 163.1±29.5 of TGF-ß(1) stimulation group (t=6.88, P<0.01) with RNA-sequencing. After 72 hours in culture, the Meox1 mRNA expression levels in the cells of blank control group was 1.00±0.21, which was significantly lower than 11.00±3.61 of TGF-ß(1) stimulation group (t=4.79, P<0.01). (2) After 48 hours in culture, the Meox1 mRNA expression levels in the cells of Smad2 OE group, Smad3 OE group, and Smad4 OE group were 198.70±11.02, 35.47±4.30, 20.27±2.50, respectively, which were significantly higher than 1.03±0.19 of empty plasmid group (t=31.07, 13.80, 13.12, P<0.01). (3) After 72 hours in culture, the enrichment of Smad2, Smad3, and Smad4 protein on the promoter of Meox1 in the cells of TGF-ß(1) stimulation group was significantly higher than that of blank control group respectively (t=12.99, 41.47, 29.10, P<0.01). (4) After 48 hours in culture, the enrichment of Smad2 protein on the promoter of Meox1 in the cells of negative interference group was (0.200 000±0.030 000)%, significantly higher than (0.000 770±0.000 013)% of siRNA-Smad2 group (t=11.67, P<0.01); the enrichment of Smad2 protein on the promoter of Meox1 in the cells of empty plasmid group was (0.200 000±0.040 000)%, significantly lower than (0.700 000±0.090 000)% of Smad2 OE group (t=8.85, P<0.01). The enrichment of Smad3 protein on the promoter of Meox1 in the cells of negative interference group was (0.500 0±0.041 3)%, significantly higher than (0.006 0±0.001 3)% of siRNA-Smad3 group (t=17.79, P<0.01); the enrichment of Smad3 protein on the promoter of Meox1 in the cells of empty plasmid group was (0.470 0±0.080 0)%, which was significantly lower than (1.100 0±0.070 0)% of Smad3 OE group (t=9.93, P<0.01). The enrichment of Smad4 protein on the promoter of Meox1 in the cells of negative interference group was similar to that of siRNA-Smad4 group (t=2.11, P>0.05); the enrichment of Smad4 protein on the promoter of Meox1 in the cells of empty plasmid group was similar to that of Smad4 OE group (t=0.60, P>0.05). (5) Twenty-four hours after scratching, the scratch healing width of cells in siRNA-Meox1 group was narrower than that of negative interference group, while that of Meox1 OE group was wider than that of empty plasmid group. After 24 hours in culture, the number of migration cells in negative interference group was significantly higher than that in siRNA-Meox1 group (t=9.12, P<0.01), and that in empty plasmid group was significantly lower than that in Meox1 OE group (t=8.99, P<0.01). (6) The expression of Meox1 protein in the scar tissue was significantly higher than that in normal skin of patients with hypertrophic scars. Conclusions: TGF-ß(1) transcriptionally regulates Meox1 expression via Smad2/3 in HDF-a cells, thus promoting cell migration.

Danggui Buxue Tang ameliorates bleomycin-induced pulmonary fibrosis in rats through inhibiting transforming growth factor-ß1/Smad3/ plasminogen activator inhibitor-1 signaling pathway.

OBJECTIVE: To investigate the effect of Danggui Buxue Tang (DBT), a decoction from Traditional Chinese Medicine, on bleomycin-induced pulmonary fibrosis in rats, and to propose the possible underlying mechanism. METHODS: Forty male Sprague-Dawley rats were randomly divided into sham group, model group, prednisone group and DBT group. Pulmonary fibrosis rat model was established by intratracheal injection with bleomycin. Body weight and lung index were monitored. Histopathologic examination and collagen deposition were determined using Hematoxylin and eosin (HE) and Masson's trichrome staining. Immunohistochemistry staining was applied to observe the expression of alpha-smooth muscle actin (α-SMA). mRNA expression of α-SMA, collagen Ⅰ and collagen Ⅲ were measured by real-time fluorescence quantitative PCR (RT-qPCR). Inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and IL-1ß in serum were detected by Enzyme-linked immunosorbent assay. Alkali hydrolysis method was conducted to investigate the content of hydroxyproline (HYP). Transforming growth factor-ß1 (TGF-ß1), Smad3 and plasminogen activator inhibitor-1 (PAI-1) protein level were examined by Western blot assay. RESULTS: DBT significantly reduced the severity of bleomycin-induced pulmonary fibrosis and inflammation as indicated by minimizing the lost of weight, and by lowering the levels of lung index, inflammation score, Ashcroft score, collagen volume fraction (%), HYP, α-SMA, collagen Ⅰ, collagen Ⅲ, TNF-α, IL-6, IL-1ß, TGF-ß1, Smad3 and PAI-1, consistent with the effect of prednisone. CONCLUSION: Our findings suggest that DBT is able to ameliorate the pulmonary fibrosis, the possible mechanism may involve inhibition of pulmonary inflammation and collagen deposition, possibly via suppressing TGF-ß1/Smad3/PAI-1 signaling pathway.

MiR-182 inhibits kidney fibrosis by regulating transforming growth factor ß1/Smad3 pathway in autosomal dominant polycystic kidney disease.

The aim of the present study was to investigate the molecular mechanism of miR-182 in kidney fibrosis in polycystic kidney disease (PKD). We measured the expression of miR-182 in kidney tissue of autosomal dominant PKD. Additionally, we investigated the relationship between miR-182 and fibrotic protein by transfecting miR-182 mimics and miR-182 inhibitor into polycystic kidney cyst-lined epithelial cells, respectively. Furthermore, we observed the interaction between transforming growth factor ß1 (TGF-ß1) and miR-182 and fibrinogen factors of cyst-lined epithelial cells after TGF-ß1 intervention, and measured the expression of Smad2 and Smad3 protein. Results are presented as follows: (a) MiR-182 was positively correlated with fibrosis of cyst-lined epithelial cells; (b) TGF-ß1 could induce fibrosis of cyst-lined epithelial cells; (c) the expression of miR-182 had a remarkably impact on the fibrosis induced by TGF-ß1, but had little effect on the expression of TGF-ß1; (d) the expression of Smad3 protein in TGF-ß1 induce-cyst-lined epithelial cells was increased. TGF-ß1 and miR-182 promoting the fibrosis of polycystic kidney cyst-lined epithelial cells may be mediated by the TGF-ß1/Smad3 signaling pathway, of which Smad3 was an important regulator.

Effect of Yanghe decoction on the expression of Smad3 and NF-κB in breast cancer MDA-MB-231 cells / 国际中医中药杂志

Objective To investigate the effect of Yanghe decoction serum on the proliferation of breast cancer stem cells MDA-MB-231 and the expression of Smad3 and NF-κB. Methods According to the random number table method, 20 female SD rats were divided into blank control group and Yanghe decoction high, medium and low dose group. Drug serum were given gastric gavage of Yanghe decoction 28, 14, 7 g/kg, and control group were given gastric gavage of same volume saline. Each group of rats was given orally for 3 days to prepare 10% Yanghe decoction serum and blank serum. MDA-MB-231 cells were divided into blank control group and Yanghe decoction low, medium and high dose group according to random number table method. Low, medium and high dose Yanghe decoction groups were cultured with medium containing 10% Yanghe decoction high, medium and low dose drug serum, and the blank control group was cultured with medium containing 10% control serum. After drug intervention, the effects of each group on the proliferation of MDA-MB-231 cells at 24 h, 48 h, and 72 h were detected by MTT assay. The expression of Smad3 and NF-κB protein in each group was detected by Western blot. The expression of Smad3 and NF-κB mRNA in each group were detected by real-time fluorescence quantitative PCR. Results Compared to the control group, Yanghe decoction low, medium and high dose group of cells for 24 h (1.143 ± 0.093, 0.953 ± 0.069, 0.874 ± 0.041 vs. 1.239 ± 0.160), 48 h (1.223 ± 0.129, 0.989 ± 0.093, 0.864 ± 0.105 vs. 1.389 ± 0.095), 72 h (1.092 ± 0.147, 0.881 ± 0.095, 0.719 ± 0.086 vs. 1.353 ± 0.150) proliferation activity were significantly lower (P<0.05 or P<0.01). After 48 h of drug intervention, the expression of Smad3 protein (0.974 ± 0.098, 0.844 ± 0.084, 0.789 ± 0.105 vs. 1.214 ± 0.012), NF-κB p50 protein (0.994 ± 0.047, 0.911 ± 0.015, 0.765 ± 0.084 vs. 1.147 ± 0.103) in the low, medium and high dose Yanghe decoction group were significantly lower than those in the control group (P<0.05 or P<0.01). The expression of Smad3 mRNA (1.223 ± 0.129, 0.989 ± 0.093, 0.864 ± 0.105 vs.1.389 ± 0.095), NF-κB mRNA (1.092 ± 0.147, 0.881 ± 0.095, 0.719 ± 0.086 vs. 1.353 ± 0.150) significantly decreased in the Yanghe decoction group (P<0.05 or P<0.01). Conclusions Yanghe decoction can inhibit the proliferation of breast cancer stem cell MDA-MB-231, and lower the expression of Smad3 and NF-κB.

Effect of Yanghe decoction on the expression of Smad3 and NF-κB in breast cancer MDA-MB-231 cells / 国际中医中药杂志

Objective@#To investigate the effect of Yanghe decoction serum on the proliferation of breast cancer stem cells MDA-MB-231 and the expression of Smad3 and NF-κB.@*Methods@#According to the random number table method, 20 female SD rats were divided into blank control group and Yanghe decoction high, medium and low dose group. Drug serum were given gastric gavage of Yanghe decoction 28, 14, 7 g/kg, and control group were given gastric gavage of same volume saline. Each group of rats was given orally for 3 days to prepare 10% Yanghe decoction serum and blank serum. MDA-MB-231 cells were divided into blank control group and Yanghe decoction low, medium and high dose group according to random number table method. Low, medium and high dose Yanghe decoction groups were cultured with medium containing 10% Yanghe decoction high, medium and low dose drug serum, and the blank control group was cultured with medium containing 10% control serum. After drug intervention, the effects of each group on the proliferation of MDA-MB-231 cells at 24 h, 48 h, and 72 h were detected by MTT assay. The expression of Smad3 and NF-κB protein in each group was detected by Western blot. The expression of Smad3 and NF-κB mRNA in each group were detected by real-time fluorescence quantitative PCR.@*Results@#Compared to the control group, Yanghe decoction low, medium and high dose group of cells for 24 h (1.143 ± 0.093, 0.953 ± 0.069, 0.874 ± 0.041 vs. 1.239 ± 0.160), 48 h (1.223 ± 0.129, 0.989 ± 0.093, 0.864 ± 0.105 vs. 1.389 ± 0.095), 72 h (1.092 ± 0.147, 0.881 ± 0.095, 0.719 ± 0.086 vs. 1.353 ± 0.150) proliferation activity were significantly lower (P<0.05 or P<0.01). After 48 h of drug intervention, the expression of Smad3 protein (0.974 ± 0.098, 0.844 ± 0.084, 0.789 ± 0.105 vs. 1.214 ± 0.012), NF- κB p50 protein (0.994 ± 0.047, 0.911 ± 0.015, 0.765 ± 0.084 vs. 1.147 ± 0.103) in the low, medium and high dose Yanghe decoction group were significantly lower than those in the control group (P<0.05 or P<0.01). The expression of Smad3 mRNA (1.223 ± 0.129, 0.989 ± 0.093, 0.864 ± 0.105 vs.1.389 ± 0.095), NF-κB mRNA (1.092 ± 0.147, 0.881 ± 0.095, 0.719 ± 0.086 vs. 1.353 ± 0.150) significantly decreased in the Yanghe decoction group (P<0.05 or P<0.01).@*Conclusions@#Yanghe decoction can inhibit the proliferation of breast cancer stem cell MDA-MB-231, and lower the expression of Smad3 and NF-κB.

[Effects of human erythropoietin on transforming growth factor ß1/Smad3 signal transduction pathway in acute wounds of rats].

Objective: To explore the effects of human erythropoietin (hEPO) on healing related transforming growth factor ß1 (TGF-ß1)/Smad3 signal transduction pathway in acute wounds of rats. Methods: Seventy-two healthy Sprague Dawley rats were divided into normal saline control group, low dose group, middle dose group, and high dose group according to the random number table, with 18 rats in each group, after round acute wounds with diameter of 2.5 cm were inflicted on the back of rats. Rats in the 4 groups had debridement routinely. Wounds of rats in normal saline control group were covered by gauzes infiltrated with 1 mL normal saline, while wounds of rats in low dose group, middle dose group, and high dose group were respectively covered by gauze infiltrated with 1 mL hEPO in doses of 50, 100, and 150 U every day, and then the wounds were bandaged with 6 layers of dry gauze. Dressing change was performed once every day. On treatment day (TD) 3, 7, and 14, 6 rats from each group were taken for general observation and calculation of wound healing rate. Then the wound tissue samples were harvested after the rats were sacrificed for observation of expressions of CD31 and transforming growth factor ß1 (TGF-ß1) with immunohistochemical method. Protein expression of phosphorylated Smad3 of the wound tissue of 3 rats were detected by Western blotting. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) On TD 3, obvious exudation and scab were observed in the wounds of rats in the 4 groups. On TD 7, the wounds of rats in low dose group, middle dose group, and high dose group were reduced compared with those in normal saline control group. On TD 14, all wounds of rats in the 4 groups were healed. On TD 7, the wound healing rates of rats in middle dose group and high dose group were significantly higher than the rate in normal saline control group (P<0.01). At the other time points, the wound healing rates of rats in the 4 groups were close (P>0.05). (2) CD31 mainly expressed in blood vessels. Except for those in low dose group on TD 3 and 7 (P>0.05), the expressions of CD31 in wound tissue of rats in low dose group on TD 14 and in middle dose group and high dose group on TD 3, 7, and 14 were significantly higher than those in normal saline control group (P<0.01). Except for those on TD 3 (P>0.05), the expressions of CD31 in wound tissue of rats in middle dose group and high dose group on TD 7 and 14 were significantly higher than those in low dose group (P<0.01). Except for that on TD 3 (P>0.05), the expressions of CD31 in wound tissue of rats in high dose group on TD 7 and 14 were significantly higher than those in middle dose group (P<0.01). (3) Except for that in low dose group on TD 3 (1.9±0.7, P>0.05), the expressions of TGF-ß1 in wound tissue of rats in low dose group on TD 7 and 14 (3.3±1.0, 3.7±0.7), and in middle dose group and high dose group on TD 3, 7, and 14 (3.3±1.0, 3.6±1.0, 3.9±0.9, 3.4±0.7, 3.8±0.8, 4.2±0.4) were significantly higher than those in normal saline control group (1.7±0.5, 2.7±1.0, 3.0±0.9, P<0.01). Except for those on TD 7 (P>0.05), the expressions of TGF-ß1 in wound tissue of rats in middle dose group and high dose group on TD 3 and 14 were significantly higher than those in low dose group (P<0.01). Except for that on TD 14 (P<0.01), the expressions of TGF-ß1 in wound tissue of rats in high dose group on TD 3 and 7 were close to those in middle dose group (P>0.05). (4) Except for those in low dose group on TD 3 and 14 and in middle dose group and high dose group on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in the 3 groups at the other time points were significantly higher than those in normal saline control group (P<0.01). Except for those on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in middle dose group and high dose group on TD 3 and 7 were significantly higher than those in low dose group (P<0.01). Except for that on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in high dose group on TD 3 and 7 were significantly lower than those in middle dose group (P<0.01). Conclusions: Exogenous hEPO can increase the expressions of CD31, TGF-ß1, and phosphorylated Smad3 in acute wounds of rats, promote angiogenesis of wounds, and activate TGF-ß1/Smad3 signal transduction pathway to promote wound healing.

Hypoxia-Inducible Factor 1α Regulates the Transforming Growth Factor ß1/SMAD Family Member 3 Pathway to Promote Breast Cancer Progression.

PURPOSE: The transforming growth factor ß1 (TGF-ß1)/SMAD family member 3 (SMAD3) pathway, and hypoxia-inducible factor 1α (HIF-1α) are two key players in various types of malignancies including breast cancer. The TGF-ß1/SMAD3 pathway can interact with HIF-1α in some diseases; however, their interaction in breast cancer is still unknown. Therefore, our study aimed to investigate the interactions between the TGF-ß1/SMAD3 pathway and HIF-1α in breast cancer. METHODS: Expression of HIF-1α in serum of breast cancer patients and healthy controls was detected by quantitative reverse transcription polymerase chain reaction, and the diagnostic value of HIF-1α for breast cancer was evaluated by receiver operating characteristic curve analysis. Breast cancer cell lines overexpressing SMAD3 and HIF-1α were established. Cell apoptosis and proliferation following different treatments were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and cell counting kit-8, respectively. Expression of related proteins was detected by western blot. RESULTS: Serum levels of HIF-1α were higher in breast cancer patients than in normal controls. Both SMAD3 and HIF-1α overexpression inhibited cell apoptosis and promoted cell proliferation. Treatment with inhibitors of HIF-1α and SMAD3 promoted apoptosis in breast cancer cells and inhibited their proliferation. Overexpression of HIF-1α promoted the expression of TGF-ß1 and SMAD3, while SMAD3 overexpression did not significantly affect expression of HIF-1α or TGF-ß1. CONCLUSION: HIF-1α serves as an upstream regulator of the TGF-ß1/SMAD3 pathway and promotes the growth of breast cancer.

Expression of TGF-beta receptor 1 and Smads in the tissues of primary spontaneous pneumothorax.

BACKGROUND: Primary spontaneous pneumothorax (PSP) is a common disease which is often caused by the rupture of bullae in the lungs. The underlying pathogenesis of PSP remains unclear. Some molecules may be involved in the development of PSP potentially. The aim of this study was to investigate the expression of TGF-beta receptor 1 (TßR1), Smad2, Smad3 and Smad4 in the resected bullae of patients with PSP. METHODS: From May 2015 to May 2016, 34 patients with PSP underwent video-assisted thoracoscopic surgery (VATS) bullectomy. Immunohistochemistry was performed to identify the expression of TßR1, Smad2, Smad3 and Smad4 in the resected pulmonary bullae tissues. The levels of these cytokines were calculated by immunoreactivity scoring system (IRS). Ten patients without pneumothorax associated disease were selected as the control group. RESULTS: The analysis showed that the expression levels of TßR1, Smad2 and Smad4 were significantly higher in bullae tissues of patients with PSP than that in normal lung tissues (P=0.012, 0.031, 0.000 respectively). There was no significant difference between the expression level of Smad3 in bullae tissue of PSP patients and that in normal lung tissues of the control group (P=0.140). However, the absolute quantity of Smad3 expression in PSP bullae tissues was (4.2529±1.7193), scored by the IRS, which is higher than that in the control lung tissues (3.2600±2.2132). Also, the expression of TßR1, Smad2, Smad3 and Smad4 were not showed correlation with the clinical characteristics of PSP patients, such as age, sex, body mass index (BMI), recurrence and side of pneumothorax. CONCLUSIONS: TßR1, Smad2 and Smad4 highly expressed in bullae tissues of PSP patients. Our findings suggested that TßR1, Smad2 and Smad4 may be related to the development of PSP bullae.

Hypoxia-Inducible Factor 1α Regulates the Transforming Growth Factor β1/SMAD Family Member 3 Pathway to Promote Breast Cancer Progression / 한국유방암학회지

PURPOSE: The transforming growth factor β1 (TGF-β1)/SMAD family member 3 (SMAD3) pathway, and hypoxia-inducible factor 1α (HIF-1α) are two key players in various types of malignancies including breast cancer. The TGF-β1/SMAD3 pathway can interact with HIF-1α in some diseases; however, their interaction in breast cancer is still unknown. Therefore, our study aimed to investigate the interactions between the TGF-β1/SMAD3 pathway and HIF-1α in breast cancer. METHODS: Expression of HIF-1α in serum of breast cancer patients and healthy controls was detected by quantitative reverse transcription polymerase chain reaction, and the diagnostic value of HIF-1α for breast cancer was evaluated by receiver operating characteristic curve analysis. Breast cancer cell lines overexpressing SMAD3 and HIF-1α were established. Cell apoptosis and proliferation following different treatments were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and cell counting kit-8, respectively. Expression of related proteins was detected by western blot. RESULTS: Serum levels of HIF-1α were higher in breast cancer patients than in normal controls. Both SMAD3 and HIF-1α overexpression inhibited cell apoptosis and promoted cell proliferation. Treatment with inhibitors of HIF-1α and SMAD3 promoted apoptosis in breast cancer cells and inhibited their proliferation. Overexpression of HIF-1α promoted the expression of TGF-β1 and SMAD3, while SMAD3 overexpression did not significantly affect expression of HIF-1α or TGF-β1. CONCLUSION: HIF-1α serves as an upstream regulator of the TGF-β1/SMAD3 pathway and promotes the growth of breast cancer.

Effects of human erythropoietin on transforming growth factor β1/Smad3 signal transduction pathway in acute wounds of rats / 中华烧伤杂志

Objective@#To explore the effects of human erythropoietin (hEPO) on healing related transforming growth factor β1 (TGF-β1)/Smad3 signal transduction pathway in acute wounds of rats.@*Methods@#Seventy-two healthy Sprague Dawley rats were divided into normal saline control group, low dose group, middle dose group, and high dose group according to the random number table, with 18 rats in each group, after round acute wounds with diameter of 2.5 cm were inflicted on the back of rats. Rats in the 4 groups had debridement routinely. Wounds of rats in normal saline control group were covered by gauzes infiltrated with 1 mL normal saline, while wounds of rats in low dose group, middle dose group, and high dose group were respectively covered by gauze infiltrated with 1 mL hEPO in doses of 50, 100, and 150 U every day, and then the wounds were bandaged with 6 layers of dry gauze. Dressing change was performed once every day. On treatment day (TD) 3, 7, and 14, 6 rats from each group were taken for general observation and calculation of wound healing rate. Then the wound tissue samples were harvested after the rats were sacrificed for observation of expressions of CD31 and transforming growth factor β1 (TGF-β1) with immunohistochemical method. Protein expression of phosphorylated Smad3 of the wound tissue of 3 rats were detected by Western blotting. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction.@*Results@#(1) On TD 3, obvious exudation and scab were observed in the wounds of rats in the 4 groups. On TD 7, the wounds of rats in low dose group, middle dose group, and high dose group were reduced compared with those in normal saline control group. On TD 14, all wounds of rats in the 4 groups were healed. On TD 7, the wound healing rates of rats in middle dose group and high dose group were significantly higher than the rate in normal saline control group (P<0.01). At the other time points, the wound healing rates of rats in the 4 groups were close (P>0.05). (2) CD31 mainly expressed in blood vessels. Except for those in low dose group on TD 3 and 7 (P>0.05), the expressions of CD31 in wound tissue of rats in low dose group on TD 14 and in middle dose group and high dose group on TD 3, 7, and 14 were significantly higher than those in normal saline control group (P<0.01). Except for those on TD 3 (P>0.05), the expressions of CD31 in wound tissue of rats in middle dose group and high dose group on TD 7 and 14 were significantly higher than those in low dose group (P<0.01). Except for that on TD 3 (P>0.05), the expressions of CD31 in wound tissue of rats in high dose group on TD 7 and 14 were significantly higher than those in middle dose group (P<0.01). (3) Except for that in low dose group on TD 3 (1.9±0.7, P>0.05), the expressions of TGF-β1 in wound tissue of rats in low dose group on TD 7 and 14 (3.3±1.0, 3.7±0.7), and in middle dose group and high dose group on TD 3, 7, and 14 (3.3±1.0, 3.6±1.0, 3.9±0.9, 3.4±0.7, 3.8±0.8, 4.2±0.4) were significantly higher than those in normal saline control group (1.7±0.5, 2.7±1.0, 3.0±0.9, P<0.01). Except for those on TD 7 (P>0.05), the expressions of TGF-β1 in wound tissue of rats in middle dose group and high dose group on TD 3 and 14 were significantly higher than those in low dose group (P<0.01). Except for that on TD 14 (P<0.01), the expressions of TGF-β1 in wound tissue of rats in high dose group on TD 3 and 7 were close to those in middle dose group (P>0.05). (4) Except for those in low dose group on TD 3 and 14 and in middle dose group and high dose group on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in the 3 groups at the other time points were significantly higher than those in normal saline control group (P<0.01). Except for those on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in middle dose group and high dose group on TD 3 and 7 were significantly higher than those in low dose group (P<0.01). Except for that on TD 14 (P>0.05), the protein expressions of phosphorylated Smad3 in wound tissue of rats in high dose group on TD 3 and 7 were significantly lower than those in middle dose group (P<0.01).@*Conclusions@#Exogenous hEPO can increase the expressions of CD31, TGF-β1, and phosphorylated Smad3 in acute wounds of rats, promote angiogenesis of wounds, and activate TGF-β1/Smad3 signal transduction pathway to promote wound healing.

Treadmill exercise promotes the expression of TGF-β1 and Smad3 protein, inhibiting cell apoptosis after cerebral ischemia / 中华物理医学与康复杂志

Objective To observe the effect of treadmill exercise on the expression of transforming growth factor-β1 (TGF-β1) and its receptor Smad3 protein as well as on cell apoptosis in the ischemic boundary zone,so as to explore how exercise promotes the recovery of neurological function after cerebral ischemia.Methods Thirty adult male Sprague-Dawley rats were randomly divided into a sham group (n=6),a model group (n=12) and an exercise group (n=12).A modified Longa's method was used to establish an animal model of cerebral ischemia by occluding the right middle cerebral artery in the rats of the model and exercise groups.Those of the sham group were subjected to the same surgical procedure except that no thread was inserted.After 24h the exercise group began treadmill training,while the other two groups were left on the treadmill without training.Modified neurological severity scores (mNSSs) were used to quantify the rats' neurological functioning on the 3rd,7th and 14th day after the surgery.The ischemic boundary zone tissue was then dissected to detect the expression of TGF-31 and Smad3 protein using western blotting.Cell apoptosis was detected by TUNEL staining.Results The average mNSS scores of the exercise group on the 7th and the 14th day were significantly lower than those of the model group at the same time points.The average expression level of TGF-β1 and Smad3 protein in the exercise group was significantly higher than in the model group.The number of TUNEL-positive cells in the exercise group was significantly lower than in the model group on the 14th day.Conclusions Treadmill exercise can improve the recovery of neurological function after cerebral ischemia.It may be partly due to upregulating the expression of TGF-β1 and Smad3 protein,which inhibit cell apoptosis in the ischemic boundary zone.

A small-molecule inhibitor of SMAD3 attenuates resistance to anti-HER2 drugs in HER2-positive breast cancer cells.

PURPOSE: Resistance against anti-HER2 drugs in HER2-positive breast cancer is a major obstacle to the improving prognosis. Transforming growth factor ß (TGFß) is a cytokine involved in the acquisition of more malignant phenotypes through epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. The aim of this study was to investigate the effects of TGFß and its downstream SMAD pathway on resistance to anti-HER2 drugs. METHODS: HER2-positive breast cancer cell lines were stimulated with TGFß for 14 days. Then, the sensitivity to trastuzumab and lapatinib and the expression levels of various EMT and CSC markers were examined. The correlation of nuclear SMAD3 expression in untreated breast tumor tissues with trastuzumab efficacy in neoadjuvant settings was examined. The effect of a small-molecule inhibitor of SMAD3 (SIS3) on resistance to anti-HER2 drugs was explored. RESULTS: We found that continuous activation of the TGFß-SMAD3 pathway induced resistance to anti-HER2 drugs and CSC traits in HER2-positive breast cancer cells. The induction of drug resistance by TGFß required strong activation of SMAD3. In fact, activated SMAD3 regulated multiple genes that harbor SMAD-binding elements and are involved in trastuzumab resistance. Nuclear SMAD3 expression in tumor tissue was inversely correlated with sensitivity to neoadjuvant treatment with trastuzumab. SIS3 not only prevented the acquisition of resistance to anti-HER2 drugs but also restored trastuzumab sensitivity in trastuzumab-resistant cells. CONCLUSIONS: This study indicates that the TGFß-SMAD3 pathway plays an important role in the induction and maintenance of resistance to anti-HER2 drugs. Thus, SMAD3 is a potential therapeutic target that can inhibit resistance and restore sensitivity to anti-HER2 drugs.