Epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis and provides novel strategies for cancer therapy. Hypaconitine (HpA), a diester-diterpenoid alkaloid isolated from the root of the Aconitum species, exhibits anti-inflammatory, analgesic, and especially, cardiotoxic activities. Here, we reported the anti-metastatic potentials of HpA in transforming growth factor-β1 (TGF-β1)-induced EMT in lung cancer A549 cells. The cytotoxic effect of HpA was determined by MTT assay. A549 cells were treated with TGF-β1 with or without HpA co-treatment, and the morphological alterations were observed with a microscopy. The expression of E-cadherin, N-cadherin, and NF-κB was determined by both Western blotting and immunofluorescence analyses. The adhesion, migration, and invasion were detected with Matrigel, wound-healing, and transwell assays, respectively. The expression of Snail was determined by Western blotting. The expression of NF-κB p65, IκBα, and p-IκBα in nuclear and cytosolic extracts was assessed by Western blotting. The results showed that low concentration of HpA (<16 μmol·L) had no obvious cytotoxicity to A549 cells. Morphologically, TGF-β1 treatment induced spindle-shaped alteration in the cells. The upregulation of N-cadherin, NF-κB, and Snail and the downregulation of E-cadherin were detected after TGF-β1 treatment. The adhesion, migration and invasion abilities were also increased by TGF-β1. Besides, TGF-β1 induced expression of Snail in a time-dependent manner. Furthermore, TGF-β1 induced nuclear translocation of NF-κB p65. All these alterations were dramatically inhibited by HpA co-treatment. In addition, the NF-κB inhibitor PDTC showed similar inhibitory effect. In conclusion, these results showed that HpA inhibited TGF-β1-induced EMT in A549 cells, which was possibly mediated by the inactivation of the NF-κB signaling pathway, providing an evidence for anti-cancer effect of HpA.
A549 Cells ; Aconitine ; analogs & derivatives ; pharmacology ; Active Transport, Cell Nucleus ; drug effects ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cadherins ; analysis ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Dose-Response Relationship, Drug ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Neoplasm Invasiveness ; Transforming Growth Factor beta1 ; antagonists & inhibitors ; physiology

To investigate the role of glucose transporter 1 (GLUT1) and sodium-glucose cotransporter 1 (SGLT1) in high glucose dialysate-induced peritoneal fibrosis.Thirty six male SD rats were randomly divided into 6 groups (6 in each):normal control group, sham operation group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorizin group (PD+Z group), PD+phloretin+phlorizin group (PD+T+Z group). Rat model of uraemia was established using 5/6 nephrotomy, and 2.5% dextrose peritoneal dialysis solution was used in peritoneal dialysis. Peritoneal equilibration test was performed 24 h after dialysis to evaluate transport function of peritoneum in rats; HE staining was used to observe the morphology of peritoneal tissue; and immunohistochemistry was used to detect the expression of GLUT1, SGLT1, TGF-β1 and connective tissue growth factor (CTGF) in peritoneum. Human peritoneal microvascular endothelial cells (HPECs) were divided into 5 groups:normal control group, peritoneal dialysis group (PD group), PD+phloretin group (PD+T group), PD+phlorezin group (PD+Z group), and PD+phloretin+phlorezin group (PD+T+Z group). Real time PCR and Western blotting were used to detect mRNA and protein expressions of GLUT1, SGLT1, TGF-β1, CTGF in peritoneal membrane and HPECs., compared with sham operation group, rats in PD group had thickened peritoneum, higher ultrafiltration volume, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-β1 were significantly increased (all<0.05); compared with PD group, thickened peritoneum was attenuated, and the mRNA and protein expressions of GLUT1, SGLT1, CTGF, TGF-β1 were significantly decreased in PD+T, PD+Z and PD+T+Z groups (all<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in peritoneum were positively correlated with the expressions of TGF-β1 and CTGF (all<0.05)., the mRNA and protein expressions of GLUT1, SGLT1, TGF-β1, CTGF were significantly increased in HPECs of peritoneal dialysis group (all<0.05), and those in PD+T, PD+Z, and PD+T+Z groups were decreased (all<0.05). Pearson's correlation analysis showed that the expressions of GLUT1, SGLT1 in HPECs were positively correlated with the expressions of TGF-β1 and CTGF (all<0.05).High glucose peritoneal dialysis fluid may promote peritoneal fibrosis by upregulating the expressions of GLUT1 and SGLT1.
Animals ; Cells, Cultured ; Connective Tissue Growth Factor ; analysis ; drug effects ; Dialysis Solutions ; adverse effects ; chemistry ; pharmacology ; Gene Expression Regulation ; drug effects ; Glucose ; adverse effects ; pharmacology ; Glucose Transporter Type 1 ; analysis ; drug effects ; physiology ; Hemodiafiltration ; adverse effects ; methods ; Humans ; Male ; Peritoneal Dialysis ; adverse effects ; methods ; Peritoneal Fibrosis ; chemically induced ; genetics ; physiopathology ; Peritoneum ; chemistry ; drug effects ; pathology ; Phloretin ; Phlorhizin ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Sodium-Glucose Transporter 1 ; analysis ; drug effects ; physiology ; Transforming Growth Factor beta1 ; analysis ; drug effects ; Uremia ; chemically induced

α-smooth muscle actin (α-SMA) and tenascin-C are stress-induced phenotypic features of myofibroblasts. The expression levels of these two proteins closely correlate with the extracellular mechanical microenvironment. We investigated how the expression of α-SMA and tenascin-C was altered in the periodontal ligament (PDL) under orthodontic loading to indirectly reveal the intrinsic mechanical microenvironment in the PDL. In this study, we demonstrated the synergistic effects of transforming growth factor-β1 (TGF-β1) and mechanical tensile or compressive stress on myofibroblast differentiation from human periodontal ligament cells (hPDLCs). The hPDLCs under higher tensile or compressive stress significantly increased their levels of α-SMA and tenascin-C compared with those under lower tensile or compressive stress. A similar trend was observed in the tension and compression areas of the PDL under continuous light or heavy orthodontic load in rats. During the time-course analysis of expression, we observed that an increase in α-SMA levels was matched by an increase in tenascin-C levels in the PDL under orthodontic load in vivo. The time-dependent variation of α-SMA and tenascin-C expression in the PDL may indicate the time-dependent variation of intrinsic stress under constant extrinsic loading.
Actins ; analysis ; drug effects ; Adult ; Animals ; Biomechanical Phenomena ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Cellular Microenvironment ; physiology ; Humans ; Male ; Myofibroblasts ; physiology ; Orthodontic Wires ; Periodontal Ligament ; chemistry ; cytology ; Pressure ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical ; Tenascin ; analysis ; drug effects ; Time Factors ; Tooth Movement Techniques ; instrumentation ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the clinical significance of transforming growth factor-beta 1 (TGF-β1) in children with primary IgA nephropathy (IgAN).

METHODSThirty children who were diagnosed with primary IgAN by renal biopsy between May 2008 and October 2012 were included in the study. Thirty healthy children were used as the control group. Urinary and blood TGF-β1 levels were measured using enzyme-linked immunosorbent assay, and the protein expression of TGF-β1 in the renal tissue was measured by immunohistochemistry. The correlation between TGF-β1 levels in blood, urine, and renal tissue and their relationship with clinical indices were analyzed.

RESULTSChildren with primary IgAN had significantly higher urinary and blood TGF-β1 levels than the control group (P<0.01). Urinary TGF-β1 level was positively correlated with the pathological grade of renal tissue (r=0.557, P=0.001), and a significant positive correlation was also found between the TGF-β1 expression in the renal tissue and the pathological grade of renal tissue (r=0.682, P<0.01). There was no correlation between TGF-β1 levels in blood and renal tissue (r=0.038, P=0.844).

CONCLUSIONSUrinary TGF-β1 level is significantly positively correlated with the pathological severity of disease in children with primary IgAN. Clinical measurement of urinary TGF-β1 may be of great practical value in predicting the progression and prognosis of chronic nephropathy.

Adolescent ; Child ; Female ; Glomerulonephritis, IGA ; metabolism ; pathology ; Humans ; Kidney ; chemistry ; pathology ; Male ; Transforming Growth Factor beta1 ; analysis ; physiology ; urine

OBJECTIVETo explore the effects and mechanisms of multi-glycoside of Tripterygium wilfordii (GTW) on improving glomerular inflammatory lesion in rats with diabetic nephropathy (DN).

METHODDN model was induced by unilateral nephrectomy and intraperitoneal injection of STZ (35 mg x kg(-1)) twice. The rats were randomly divided into 3 groups, the sham-operated group (Sham group, n = 5), the vehicle-given group (Vehicle group, n = 5 ) and GTW-treated group (GTW group, n = 5). After the model was successfully established, the rats in GTW group were daily oral administrated with GTW suspension (50 mg x kg(-1) x d(-1)), meanwhile, the rats in Vehicle group were daily oral administrated with distilled water (2 mL) for 8 weeks. From the beginning of the administration, all rats were killed 8 weeks later. Blood and renal tissues were collected,and then UAlb, renal function, glomerular morphology characteristics and glomerular macrophages (ED1 + cells) infiltration, as well as the protein expressions of inflammatory cytokines including tumor necrosis factor(TNF)-α and interleukin(IL)-lβ, and the key molecules in p38MAPK signaling pathway including p38 mitogenactivated protein kinase (MAPK), phosphorylated p38 (p-p38MAPK) and transforming growth factor(TGF)-β1 were investigated respectively.

RESULTGTW not only ameliorated the general state of health and body weight,but also attenuated UAlb, glomerulosclerosis, the infiltration of glomerular ED1 + cells and the protein expressions of TNF-α, IL-1β, p-p38MAPK and TGF-β1 in the kidney in DN model rats.

CONCLUSIONBy means of DN model rats, we demonstrated that GTW has the protective effect on renal inflammatory damage in vivo via inhibiting inflammatory cells infiltration and inflammatory cytokines expression. Furthermore, GTW could improve renal inflammatory lesion through down-regulating the expressions of the key signaling molecules in p38MAPK pathway such as p-p38MAPK and TGF-β1 ,and inhibiting the activation of p38MAPK signaling in the kidney.

Animals ; Diabetic Nephropathies ; drug therapy ; Disease Models, Animal ; Glomerulonephritis ; drug therapy ; Glycosides ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; analysis ; Tripterygium ; chemistry ; p38 Mitogen-Activated Protein Kinases ; physiology

BACKGROUNDPomegranate (punica granatum) belongs to the family Punicaceae, and its peel has been used as a traditional Chinese medicine because of its efficacy in restraining intestine, promoting hemostasis, and killing parasites. Pomegranate peel has been reported to possess wound-healing properties which are mainly attributed to its polyphenol extracts. The purpose of this study was to investigate the effect of pomegranate peel polyphenols (PPP) gel on cutaneous wound healing in diabetic rats.

METHODSAlloxan-induced diabetic rats were given incisional wounds on each side of the mid-back and then treated daily with PPP gel (polyphenol mass fraction = 30%) post-wounding. Rats were sacrificed on days 4, 7, 14, and 21 post-wounding to assess the rates of wound closure, histological characteristics; and to detect the contents of hydroxyproline, production of nitric oxide (NO), and activities of NO synthase (NOS), as well as the expressions of transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) in wound tissue.

RESULTSWound closure was significantly shortened when PPP gel was applied to the wounds of diabetic rats. Histological examination showed the ability of PPP gel to increase fibroblast infiltration, collagen regeneration, vascularization, and epithelialization in the wound area of diabetic rats. In addition, PPP gel-treated diabetic rats showed increased contents of hydroxyproline, production of NO, and activities of NOS and increased expressions of TGF-β1, VEGF, and EGF in wound tissues.

CONCLUSIONPPP gel may be a beneficial method for treating wound disorders associated with diabetes.

Alloxan ; Animals ; Diabetes Mellitus, Experimental ; pathology ; physiopathology ; Female ; Gels ; Hydroxyproline ; analysis ; Male ; Nitric Oxide ; biosynthesis ; Polyphenols ; pharmacology ; Punicaceae ; Rats ; Rats, Wistar ; Transforming Growth Factor beta1 ; physiology ; Vascular Endothelial Growth Factor A ; physiology ; Wound Healing ; drug effects

BACKGROUNDCell transplantation has great potential for promoting endothelial repair and reducing the complications of percutaneous coronary intervention (PCI). The aim of this study was to investigate the effect of transplantation of human umbilical cord blood endothelial progenitor cells (EPCs) on injured arteries.

METHODSUmbilical cord blood mononuclear cells were obtained from post-partum lying-in women, and EPCs were isolated, cultured, expanded and identified by immunofluorescence. The carotid arterial endothelium of New Zealand white rabbits was injured by dilatation with a 3F balloon, and the EPCs were injected into the lumen of the injured artery in the transplanted group (n = 16), while an equal volume of phosphated buffered saline (PBS) was injected into the control group after balloon injury (n = 16). The animals were sacrificed after either 2 or 4 weeks, and the grafted cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibodies. Arterial cross sections were analyzed by pathology, immunohistochemistry and morphometry to evaluate the reparative effects of EPCs. Proliferating cell nuclear antigen (PCNA) and transforming growth factor (TGF)-β1 mRNA expression were detected by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSFluorescence-labeled EPCs were found in the neointima. The neointimal area and the neointimal/medial area ratio were significantly lower in the transplanted group than in the control group (P < 0.05). von Willebrand factor (vWF) immunohistostaining showed more VWF-positive cells in the transplanted animals than in the controls (8.75 ± 2.92 vs. 4.50 ± 1.77, P < 0.05). Compared with the control group, the transplanted group had lower expression of PCNA mRNA (0.67 ± 0.11 vs. 1.25 ± 0.40, P < 0.01) and higher expression of TGF-β1 mRNA (1.10 ± 0.21 vs. 0.82 ± 0.07, P < 0.05).

CONCLUSIONSEPCs derived from human umbilical cord blood were successfully transplanted into injured vessels. The transplanted EPCs inhibited neointimal hyperplasia and promoted vascular re-endothelialization.

Animals ; Carotid Artery Injuries ; immunology ; pathology ; therapy ; Cell Differentiation ; Cells, Cultured ; Cytokines ; genetics ; Endothelial Cells ; cytology ; physiology ; Fetal Blood ; cytology ; Humans ; Hyperplasia ; Male ; Neointima ; pathology ; Proliferating Cell Nuclear Antigen ; genetics ; RNA, Messenger ; analysis ; Rabbits ; Stem Cell Transplantation ; Transforming Growth Factor beta1 ; genetics

OBJECTIVETo investigate the changes in the levels of interleukin-17 (IL-17) and transforming growth factor beta 1 (TGF-β1) in serum and bronchoalveolar lavage fluid (BALF) and their clinical significance among children with asthma.

METHODSFifty-six children with asthma were divided into moderate or severe asthma (n=37) and mild asthma groups (n=19) and 18 children without asthma were selected as the control group. Cells in BALF were counted under a microscope. The levels of IL-17 and TGF-β1 in serum and BALF were measured using ELISA.

RESULTSwere no significant differences in total cell count and percentage of macrophages between the two asthma groups and the control group (P>0.05). The percentages of neutrophils, eosinophils and epithelial cells in BALF were significantly higher in the two asthma groups than in the control group (P<0.05). The two asthma groups had significantly higher levels of IL-17 and TGF-β1 in serum and BALF than the control group (P<0.05), and the moderate or severe asthma group had significantly higher levels of IL-17 and TGF-β1 in serum and BALF than the mild asthma group (P<0.05). Levels of IL-17 and TGF-β1 in serum were significantly positively correlated with those in BALF (r=0.935 and 0.943, P<0.05 for both). In children with asthma, serum IL-17 level was significantly positively correlated with the percentage of neutrophils, eosinophils and epithelial cells in BALF (r=0.802, 0.799, and 0.674, P<0.05 for all), and a significant positive correlation was also seen between serum levels of IL-17 and TGF-β1 (r=0.878, P<0.05).

CONCLUSIONSLevels of IL-17 and TGF-β1 in serum and BALF are elevated in children with asthma. IL-17 and TGF-β1 may be involved in the occurrence and development of asthma, and they play important roles in asthma attack and aggravation.

Asthma ; immunology ; pathology ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Interleukin-17 ; analysis ; blood ; physiology ; Male ; Transforming Growth Factor beta1 ; analysis ; blood ; physiology

OBJECTIVETo study the effect of 1,25-(OH)2D3 supplementation during gestation and lactation on TGF-β1 and Smad3 expression in lungs of rat offspring with asthma.

METHODSThirty-two female Wistar rats were randomly divided into four groups: low-, medium- and high-dose 1,25-(OH)2D3 supplementation and control groups (n=8 each). From the 7th day of gestation, the three 1,25-(OH)2D3 supplementation groups were administered with 2,10 and 20 μg/mL of 1,25-(OH)2D3 respectively every other day until weaning (rat offspring: 21 days old). The control group received normal saline instead. Then, bronchial asthma was induced in rat offspring from the 4 groups. The protein and mRNA expression of TGF-β1 and Smad3 in the lung tissue was measured by immunochemistry and RT-PCR.

RESULTSEosinophil cell infiltration and airway inflammation decreased in rat offspring from the low- and medium-dose 1,25-(OH)2D3 groups, but increased in rat offspring of the high-dose 1,25-(OH)2D3 group compared with the control group. Immunohistochemistry of lung tissues showed that the expression of TGF-β1 protein and pSmad3 decreased in rat offspring from the low- and medium-dose 1,25-(OH)2D3 groups (P<0.05), but increased significantly in rat offspring from the high-dose 1,25-(OH)2D3 group compared with the control group (P<0.05). PCR showed that the expression of TGF-β1 and Smad3 mRNA in the lung tissue decreased in rat offspring from the low- and medium-dose 1,25-(OH)2D3 groups (P<0.05), but increased significantly in rat offspring from the high-dose 1,25-(OH)2D3 group compared with the control group (P<0.05).

CONCLUSIONS1,25-(OH)2D3 supplementation plays a role in regulating the immune system in asthmatic rats. Its mechanism may be associated with regulation of the expression of TGF-β/Smad signal pathway-related proteins through the vitamin D receptor signal pathway.

Animals ; Asthma ; metabolism ; Cholecalciferol ; administration & dosage ; analogs & derivatives ; Dietary Supplements ; Female ; Lactation ; metabolism ; Lung ; metabolism ; pathology ; Male ; Pregnancy ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Signal Transduction ; Smad3 Protein ; genetics ; physiology ; Transforming Growth Factor beta1 ; genetics ; physiology

BACKGROUNDMetformin has become a cornerstone in the treatment of patients with type-2 diabetes. Accumulated evidence suggests that metformin supports direct cardiovascular effects. The present study aimed to investigate if metformin has beneficial effects on primary cardiomyocytes damaged by H2O2, and reveal the potential mechanism of action of metformin.

METHODSCardiomyocytes were incubated in the presence of 100 µmol/L H2O2 for 12 hours. Cardiomyocytes were pretreated with metformin at different concentrations and time and with aminoimidazole carboxamide ribonucleotide (AICAR) (500 µmol/L), an adenosine monophophate (AMP)-activated protein kinase (AMPK) agonist for 60 minutes before the addition of H2O2. Other cells were preincubated with compound C (an AMPK antagonist, 20 µmol/L) for 4 hours. The viability and apoptosis of cells were analyzed. AMPK, endothelial nitric oxide synthase (eNOS), and transforming growth factor (TGF)-β1 were analyzed using immunblotting.

RESULTSMetformin had antagonistic effects on the influences of H2O2 on cell viability and attenuated oxidative stress-induced apoptosis. Metformin also increased phosphorylation of AMPK and eNOS, and reduced the expression of TGF-β1, basic fibroblast growth factor (bFGF), and tumor necrosis factor (TNF)-α.

CONCLUSIONSMetformin has beneficial effects on cardiomyocytes, and this effect involves activation of the AMPK-eNOS pathway. Metformin may be potentially beneficial for the treatment of heart disease.

AMP-Activated Protein Kinases ; physiology ; Aminoimidazole Carboxamide ; analogs & derivatives ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Hypoglycemic Agents ; pharmacology ; Metformin ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Ribonucleotides ; pharmacology ; Transforming Growth Factor beta1 ; genetics ; Tumor Necrosis Factor-alpha ; genetics