OBJECTIVE: To investigate the effects of Ganoderma lucidum polysaccharides (GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. METHODS: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide (MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type I (CICP) and transforming growth factor-β1 (TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. RESULTS: Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts (Plt;0.05 or Plt;0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group (Plt;0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group (Plt;0.05 or Plt;0.01). CONCLUSION A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.
Animals ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Fibroblasts ; drug effects ; Humans ; Male ; Mice ; Polysaccharides ; pharmacology ; Reishi ; chemistry ; Skin ; drug effects ; injuries ; Transforming Growth Factor beta1 ; physiology ; Wound Healing ; drug effects ; beta Catenin ; physiology

The present study was designed to elucidate whether the mechanism by which osthole decreases collagenI/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts (CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pcDNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·mL of osthole for 24 h, the mRNA and protein expression levels of collagensIand III were reduced. The collagen I/III ratio was also reduced. The mRNA and protein expression levels of TGF-β1, TβRI, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen I and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.
Actins ; genetics ; Animals ; Cells, Cultured ; Collagen ; biosynthesis ; genetics ; Coumarins ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Mice ; Myocardium ; cytology ; Protein-Serine-Threonine Kinases ; genetics ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Receptor, Transforming Growth Factor-beta Type I ; Receptors, Transforming Growth Factor beta ; genetics ; Signal Transduction ; drug effects ; Smad Proteins ; genetics ; Transforming Growth Factor beta1 ; genetics

PURPOSE: The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and alpha-smooth muscle actin (alpha-SMA) protein in the liver, transforming growth factor beta1 (TGF-beta1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-beta1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction. RESULTS: Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-beta1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group. CONCLUSION: Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-beta1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats.
Actins ; Animals ; Carbon Tetrachloride/toxicity ; Collagen Type III/metabolism ; Down-Regulation ; Extracellular Matrix/metabolism ; Immunohistochemistry ; Immunosuppressive Agents/*pharmacology ; Liver Cirrhosis, Experimental/chemically induced/*metabolism/pathology/*prevention & control ; Male ; RNA, Messenger/analysis/metabolism ; Rats ; Rats, Wistar ; Thalidomide/*pharmacology ; Tissue Inhibitor of Metalloproteinase-1/biosynthesis/*drug effects ; Transcription Factor RelA/biosynthesis/drug effects ; Transforming Growth Factor beta1/biosynthesis/*drug effects ; Transforming Growth Factors/metabolism

OBJECTIVETo investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose.

METHODSThe third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril (10(-7)-10(-3) mmol/L) was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor beta 1 (TGF-β1), and hepatocyte growth factor (HGF) were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-β1 and HGF mRNA expression by reverse transcription polymerase chain reaction.

RESULTSCpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 (P<0.01). Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-β1 were reduced markedly (P<0.05). The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 (2.00 g/L) and benazepril (10(-5) mmol/L).

CONCLUSIONThe anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type IV ; biosynthesis ; secretion ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Glucose ; toxicity ; Hepatocyte Growth Factor ; secretion ; Matrix Metalloproteinase 9 ; metabolism ; Mesangial Cells ; cytology ; drug effects ; enzymology ; metabolism ; Polymerase Chain Reaction ; Proteolysis ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transforming Growth Factor beta1 ; secretion

OBJECTIVETo observe the effect of sodium tanshinone II (A) sulfonate (STS) on Ang II -induced atrial fibroblast collagen synthesis and TGF-beta1 activation.

METHODAtrial fibroblasts of neonatal rats were cultured to determine the content of collagen protein. The original synthesis rate determined by the [3H]-proline incorporation method was taken as the index for myocardial fibrosis. The content of active TGF-beta1 and total TGF-beta1 in cell culture supernatants were tested and cultured by ELISA. The expression of thrombospondin-1 (TSP-1) was assessed by using Western blot.

RESULTAng II could significantly increase the content of atrial fibroblast collagen and the collagen synthesis rate, the TSP-1 expression and the concentration of active TGF-beta1, without any obvious change in total TGF-beta1. After the STS treatment, all of the indexes, apart from total TGF-beta1, were obviously down-regulated.

CONCLUSIONSTS could decrease the secretion of Ang II -induced atrial fibroblast collagen and the synthesis rate. Its mechanism is related to the inhibition of TSP-1/TGF-beta1 pathway.

Angiotensin II ; pharmacology ; Animals ; Collagen ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Heart Atria ; cytology ; Phenanthrenes ; pharmacology ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Thrombospondin 1 ; metabolism ; Transforming Growth Factor beta1 ; metabolism

T-helper (Th)17 cell responses are important for the development of neutrophilic inflammatory disease. Recently, we found that acetyl salicylic acid (ASA) inhibited Th17 airway inflammation in an asthma mouse model induced by sensitization with lipopolysaccharide (LPS)-containing allergens. To investigate the mechanism(s) of the inhibitory effect of ASA on the development of Th17 airway inflammation, a neutrophilic asthma mouse model was generated by intranasal sensitization with LPS plus ovalbumin (OVA) and then challenged with OVA alone. Immunologic parameters and airway inflammation were evaluated 6 and 48 h after the last OVA challenge. ASA inhibited the production of interleukin (IL)-17 from lung T cells as well as in vitro Th17 polarization induced by IL-6. Additionally, ASA, but not salicylic acid, suppressed Th17 airway inflammation, which was associated with decreased expression of acetyl-STAT3 (downstream signaling of IL-6) in the lung. Moreover, the production of IL-6 from inflammatory cells, induced by IL-17, was abolished by treatment with ASA, whereas that induced by LPS was not. Altogether, ASA, likely via its acetyl moiety, inhibits Th17 airway inflammation by blockade of IL-6 and IL-17 positive feedback.
Animals ; Aspirin/pharmacology/*therapeutic use ; Cell Polarity/drug effects/immunology ; Feedback, Physiological/*drug effects ; Interferon-gamma/deficiency/metabolism ; Interleukin-17/*metabolism/pharmacology ; Interleukin-6/biosynthesis/*metabolism ; Lipopolysaccharides/pharmacology ; Lung/drug effects/metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Pneumonia/*drug therapy/*immunology/pathology ; Th17 Cells/drug effects/*immunology/pathology ; Transforming Growth Factor beta1/pharmacology

The aim of this study was to investigate the mechanism of deposition of extracellular matrix induced by TGF-β1 in skeletal muscle-derived stem cells (MDSCs). Rat skeletal MDSCs were obtained by using preplate technique, and divided into four groups: group A (control group), group B (treated with TGF-β1, 10 ng/mL), group C (treated with TGF-β1 and anti-connective tissue growth factor (CTGF), both in 10 ng/mL), and group D (treated with anti-CTGF, 10 ng/mL). The expression of CTGF, collagen type-I (COL-I) and collagen type-III (COL-III) in MDSCs was examined by using RT-PCR, Western blot and immunofluorescent stain. It was found that one day after TGF-β1 treatment, the expression of CTGF, COL-I and COL-III was increased dramatically. CTGF expression reached the peak on the day 2, and then decreased rapidly to a level of control group on the day 5. COL-I and COL-III mRNA levels were overexpresed on the day 2 and 3 respectively, while their protein expression levels were up-regulated on the day 2 and reached the peak on the day 7. In group C, anti-CTGF could partly suppress the overexpression of COL-I and COL-II induced by TGF-β1 one day after adding CTGF antibody. It was concluded that TGF-β1 could induce MDSCs to express CTGF, and promote the production of COL-I and COL-III. In contrast, CTGF antibody could partially inhibit the effect of TGF-β1 on the MDSCs by reducing the expression of COL-I and COL-III. Taken together, we demonstrated that TGF-β1-CTGF signaling played a crucial role in MDSCs synthesizing collagen proteins in vitro, which provided theoretical basis for exploring the methods postponing skeletal muscle fibrosis after nerve injury.
Animals ; Cell Differentiation ; drug effects ; physiology ; Cells, Cultured ; Fibrillar Collagens ; biosynthesis ; Male ; Myoblasts, Skeletal ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; drug effects ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

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

OBJECTIVETo investigate the effect of an anti-fibrotic tetra peptide Ac-SDKP on vascular fibrosis by regulating extracellular regulated protein kinase (ERK1/2) activity through Ang II.

METHODSRat vascular adventitial fibroblasts were cultured in vitro. They were randomly divided into control group, Ang II (10(-6) mmol/L) group, Ang II and Ac-SDKP joint action group, PD98059 group. Type I, III collagen contents in adventitia fibroblasts were measured by RT-PCR and the expressions of matrix metalloproteinases (MMP-2) and transforming growth factor-beta1 (TGF-beta1) were determined by Western blot.

RESULTSAc-SDKP could reduced Ang II-induced expression of type I, III collagen secretion and TGF-beta1 at mRNA,and increase MMP-2 expression, PD98059 could inhibit the above effect.

CONCLUSIONThe results suggested that Ac-SDKP could inhibit the formation and development of vascular fibrosis through blocking ERK1/2 pathway mediated by Ang II. Ac-SDKP therefore served as an antifibrotic factor in vascular fibrosis.

Angiotensin II ; adverse effects ; Animals ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Collagen Type III ; biosynthesis ; Fibroblasts ; cytology ; drug effects ; metabolism ; Flavonoids ; pharmacology ; MAP Kinase Signaling System ; Male ; Matrix Metalloproteinase 2 ; metabolism ; Oligopeptides ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate the effect of Qindan capsule (QC) on collagen synthesis and the mechanism underlying the process in spontaneously hypertensive rats (SHRs).

METHODSTwentyfour SHRs were divided into three groups: the hypertension model group, the QC treatment group, and the losartan treatment group. Eight Wistar Kyoto (WKY) rats were used as the normal control group. The systolic blood pressure (SBP) of the rats was monitored, and the thoracic aorta adventitia of the rats was segregated. The expressions of transforming growth factor 1 (TGF-β1), Smad3, and collagens I and were measured by histological staining and reverse transcription polymerase chain reaction.

RESULTSThe SBP was significantly higher in the model group than in the normal control group (P<0.01). However, a significant SBP-lowering effect was observed in QC or losartan treatment groups (P<0.05 or P<0.01) after 3 weeks of treatment. QC-treated rats showed a decrease of approximately 40 mm Hg, and the losartan-treated rats showed a decrease of approximately 50 mm Hg at the end of treatment compared with the beginning of treatment. The protein and gene levels of TGF-β1, Smad3, and collagens I and in the model group were significantly increased compared with those in the normal control group (P<0.01). However, the levels were significantly decreased in the QC or losartan treatment group compared with the model group (P<0.05 or P<0.01). However, there was no significant difference between the QC and losartan treatment groups (P<0.05).

CONCLUSIONSQC could exert its antihypertensive effect through down-regulating TGF-β1-stimulated collagen expressions. The TGF-β1/Smad3 signaling pathway may be involved in this process.

Adventitia ; drug effects ; metabolism ; pathology ; Animals ; Blood Pressure ; drug effects ; Blood Vessels ; drug effects ; metabolism ; pathology ; Capsules ; Collagen ; biosynthesis ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Losartan ; pharmacology ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Smad3 Protein ; genetics ; metabolism ; Staining and Labeling ; Systole ; drug effects ; Transforming Growth Factor beta1 ; genetics ; metabolism