Objective To explore the impact of sinomenine on bleomycin A5-induced pulmonary fibrosis (PF) in rats and the underlying mechanism. Methods MRC-5 cells were cultured and treated with sinomenine to determine its optimal concentration and time through the MTT assay. Subsequently, MRC-5 cells were incubated with 80 μmol/L sinomenine for 48 hours or transfected with miR-21 mimic/a disintegrin-like and metalloproteinase with thrombospondin type 1 motif (ADAMTS-1) siRNA prior to sinomenine treatment. The expression of miR-21, ADAMTS-1, collagen type 1 (Col1) and collagen type 3 (Col3) was detected by quantitative real-time PCR (qRT-PCR) and/or Western blot analysis. Thirty SD rats were randomly divided into control group, sinomenine group and sinomenine combined with miR-21 agomir group, with 10 animals in each group. Bleomycin A5 were intratracheally administered to establish the PF model. Then, rats in control group, sinomenine group and sinomenine +miR-21 agomir group were treated with 9 g/L sodium chloride solution, sinomenine and sinomenine+miR-21 agomir, respectively. On day 28, all rats were sacrificed. HE and Masson staining was performed in pulmonary tissue. The expression of ADAMTS-1, Col1 and Col3 in pulmonary tissue were detected by qRT-PCR and/or Western blot analysis. ELISA was used to measure serum procollagen type 1 carboxyterminal propeptide (P1CP) and procollagen type 3 aminoterminal propeptide (P3NP) levels. Results Administration of sinomenine decreased miR-21 levels, up-regulated ADAMTS-1 expression, and promoted Col1 and Col3 degradation in MRC-5 cells. Importantly, interfering with the miR-21/ADAMTS-1 signaling pathway partially reversed the promotive effect of sinomenine on Col1 and Col3 degradation. Treatment of SD rats with sinomenine reduced alveolitis and PF scores, decreased serum P1CP and P3NP levels, up-regulated pulmonary ADAMTS-1 expression, and down-regulated Col1 and Col3 expression. However, these effects were reversed by miR-21 agomir. Conclusion Sinomenine promotes Col1 and Col3 degradation and inhibits PF in rats by miR-21/ADAMTS-1 pathway.
Rats ; Animals ; Pulmonary Fibrosis/genetics* ; Procollagen/metabolism* ; Rats, Sprague-Dawley ; Signal Transduction ; Bleomycin/adverse effects* ; Collagen Type III/metabolism* ; MicroRNAs/metabolism*

BACKGROUND/AIMS: SKI306X, a mixed extract of three herbs, Clematis mandshurica (CM), Prunella vulgaris (PV), and Trichosanthes kirilowii (TK), is chondroprotective in animal models of osteoarthritis (OA). The objectives of this study were to investigate its effect on interleukin (IL)-1beta-induced degradation of glycosaminoglycan (GAG) and the basis of its action in human OA cartilage, as well as to screen for the presence of inhibitors of matrix metalloproteinase (MMP)-13 and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS)-4 in SKI306X and its component herbs, as well as in fractions from SKI306X. METHODS: Human OA chondrocytes and cartilage explants were obtained during total knee replacements and incubated with IL-1beta +/- oncostatin M with or without SKI306X or its component herb extracts. GAG degradation was assayed in cartilage explants using a commercial kit. Expression of genes involved in cartilage destruction was measured by real-time polymerase chain reaction using chondrocyte RNA. SKI306X was fractionated by preparative liquid chromatography to test for the presence of inhibitors of MMP-13 and ADAMTS-4. RESULTS: SKI306X and PV inhibited IL-1beta-induced GAG release from cartilage explants, and SKI306X, CM, PV, and TK inhibited IL-1beta-induced MMP gene expression. Unexpectedly, SKI306X greatly stimulated IL-1beta + oncostatin M-induced ADAMTS-4 gene expression, probably due to its TK component. Some fractions of SKI306X also inhibited ADAMTS-4 activity. CONCLUSIONS: SKI306X and its herbal components inhibit GAG degradation and catabolic gene expression in human OA chondrocytes and cartilage explants. SKI306X likely also contains one or more ADAMTS-4 inhibitor.
ADAM Proteins/antagonists & inhibitors ; Cartilage, Articular/*drug effects/*metabolism ; Cells, Cultured ; Chondrocytes/drug effects/metabolism ; Down-Regulation/drug effects ; Drugs, Chinese Herbal/*pharmacology ; Glycosaminoglycans/*metabolism ; Humans ; Interleukin-1beta/metabolism ; Matrix Metalloproteinase 13/metabolism ; Matrix Metalloproteinase Inhibitors/pharmacology ; Oncostatin M/metabolism ; Osteoarthritis, Knee/drug therapy/genetics/metabolism ; Procollagen N-Endopeptidase/antagonists & inhibitors

Biallelic inactivation of fumarate hydratase(FH) causes type 2 papillary renal cell carcinoma (PRCC2), uterine fibroids, and cutaneous leimyomas, a condition known as hereditary leiomyomatosis and renal cell cancer(HLRCC). The most direct effect of FH inactivation is intracellular fumarate accumulation. A majority of studies on FH inactivation over the past decade have focused on the theory that intracellular fumarate stabilizes hypoxia-inducible factor 1α(HIF1A) through competitive inhibition of HIF prolyl hydroxylases. Recently, a competing theory that intracellular fumarate activates nuclear factor (erythroid-derived 2)-like 2(NRF2) through post-translational modification of its negative regulator. Kelch-like ECH-associated protein 1(KEAP1) has emerged from a computational modeling study and mouse model studies. This review dissects the origin of these two governing theories and highlights the presence of chromatin-structure-regulated targets of transcription factors, which we refer to as "cryptic targets" of transcription factors. One such cryptic target is heme oxygenase I(HMOX1), the expression of which is known to be modulated by the gene product of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4, also known as BRG1).
Animals ; DNA Helicases ; metabolism ; Fumarate Hydratase ; genetics ; metabolism ; Fumarates ; metabolism ; Heme Oxygenase-1 ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Intracellular Signaling Peptides and Proteins ; metabolism ; Kelch-Like ECH-Associated Protein 1 ; Kidney Neoplasms ; genetics ; metabolism ; Leiomyomatosis ; genetics ; metabolism ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Neoplastic Syndromes, Hereditary ; genetics ; metabolism ; Nuclear Proteins ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Protein Processing, Post-Translational ; Skin Neoplasms ; Transcription Factors ; metabolism ; Uterine Neoplasms

OBJECTIVETo observe the expression of hypoxia-inducible factor-lalpha subunit (HIF-1alpha), HIF prolyl hydroxylase domain-containing protein(PHDs) and factor inhibiting HIF-1(FIH) in pulmonary arteries of patient with chronic obstructive pulmonary disease (COPD).

METHODSPulmonary specimens were obtained from patients undergoing lobectomy for lung cancer, 12 had concurrent COPD (COPD group) and 14 without COPD (control group). The ratio of vascular wall area to total vascular area (WA%) and pulmonary artery media thickness (PAMT) was observed, and HIF-1alpha and its hydroxylases(PHD1, PHD2, PHD3, FIH) mRNA and protein were detected by in situ hybridization and immunohistochemistry respectively.

RESULTSWA% and PAMT of COPD patients(50 microm +/- 9 microm, 40% +/- 5%, were statistically different from those of the control subjects (39 microm +/- 6 microm, 31% +/- 4%, P < 0.01). Relative quantification of mRNA and protein levels (absorbance, A) showed that HIF-lalpha mRNA and protein levels in COPD group (0.230 +/- 0.036,0.275 +/- 0.039) were statistically higher than those of the control subjects (0.174 +/- 0.029, 0.102 +/- 0.015, P < 0.01 ), and that the protein level increased more markedly. PHD1 mRNA in COPD subjects (0.180 +/- 0.030) was comparable to that in control group (0.191 +/- 0.029, P > 0.05); PHD2 and PHD3 mRNA levels in COPD (0.245 +/- 0.044, 0.252 +/- 0.023) were significantly higher than those in control group(0.182 +/- 0.028, 0.127 +/- 0.017, P < 0.01). On the other hand, in COPD subjects PHD1 protein (0.104 +/- 0.015) was significantly lower(P < 0.01), whereas PHD2 protein (0.274 +/- 0.044) was significantly higher(P < 0.01) than those in control group(0.209 +/- 0.023, 0.219+/- 0.043). As for PHD3 protein, no significant changes were observed between the two groups (0.161+/- 0.023 in COPD, 0.146 +/- 0.021 in control, P > 0.05). FIH mRNA and protein both showed no differences between the two groups. Linear correlation analysis showed that HIF1alpha protein was positively correlated with WA%, PAMT, PHD2 mRNA and protein, PHD3 mRNA, and that HIF1alpha protein was negatively correlated with PHD1 protein.

CONCLUSIONPHDs may be involved in the process of hypoxic pulmonary vascular remodeling in COPD via regulation of HIF-1alpha gene expression

Aged ; Case-Control Studies ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Lung ; blood supply ; metabolism ; Male ; Middle Aged ; Mixed Function Oxygenases ; metabolism ; Procollagen-Proline Dioxygenase ; metabolism ; Pulmonary Artery ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; RNA, Messenger ; genetics ; Repressor Proteins ; metabolism

AIMTo investigate the dynamic expression of hypoxia-inducible factor 1alpha, PHDs and OS-9 in pulmonary arteries of rats with hypoxia-induced pulmonary hypertension.

METHODSSD rats were randomly divided into 5 groups (n = 8) and exposed to hypoxia for 0, 3, 7, 14 or 21 d, respectively. RT-PCR and in situ hybridization were used to determine the expression of mRNA. Immunohistochemistry and Western blot were used to determine the expression of protein.

RESULTSHIF-1alpha protein was poorly positive in control, markedly up-regulated after 3 d and 7 d of hypoxia (P < 0.05, vs group C), and then declined slightly after 14 d and 21 d of hypoxia. HIF-1alpha mRNA increased dramatically after 14 d of hypoxia (P < 0.05, vs group C). PHD1, PHD2 mRNA and protein was positive in group C. PHD2 mRNA and protein were up-regulated after 3 d of hypoxia (P < 0.05, vs group C), reaching its peak after 14 d of hypoxia while PHD1 protein declined after 14 d of hypoxia (P < 0.05, vs group C) without statistic mRNA changing. PHD3 mRNA and protein were detected at low level in control, markedly up-regulated after 3 d of hypoxia (P < 0.05, vs group C), and then PHD3 mRNA kept at high level while PHD3 protein declined after 14 d of hypoxia (P < 0.05, vs 7 d). OS-9 mRNA was positively in control, markedly decreased after 3 d of hypoxia (P < 0.05, vs group C), reaching its lowest lever after 14 d of hypoxia. Linear correlation analysis showed that OS-9 protein was positively correlated with OS-9 mRNA (r = 0.82, P < 0.01) and HIF-1alpha protein (r = 0.57, P < 0.01).

CONCLUSIONHIF-1alpha, PHDs and OS-9 are all involved in the pathogenesis of hypoxic pulmonary hypertension in rats. OS-9 may interact with both HIF-1alpha and PHDs to promote PHD-mediated hydroxylation of HIF-1alpha.

Animals ; Female ; Hypertension, Pulmonary ; metabolism ; physiopathology ; Hypoxia ; complications ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Lectins ; genetics ; metabolism ; Male ; Procollagen-Proline Dioxygenase ; genetics ; metabolism ; Pulmonary Artery ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo study the effect of RNA interference (RNAi)-mediated aggrecanase-1 gene silencing on extracellular matrix metabolism of cultured rat costochondral chondrocytes.

METHODSRat costochondral chondrocyte monolayers were obtained by microdissection and digestion. The growth and morphological changes of the chondrocytes were observed after RNAi of aggrecanase-1 gene. The mRNA expression of aggrecanase-1 was detected by RT-PCR method, and aggrecan content was determined by Western blotting.

RESULTSThe specific inhibition of aggrecanase-1 by RNAi produced no adverse effect on the morphology and growth of the chondrocytes. The mRNA of aggrecanase-1 decreased and aggrecan content increased significantly after transfection of the chondrocytes.

CONCLUSIONInhibition of aggrecanase-1 decreases aggrecan degradation in cultured rat chondrocytes. RNAi technique can be a useful means for studying extracellular matrix metabolism in the cartilage.

ADAM Proteins ; genetics ; metabolism ; ADAMTS4 Protein ; Aggrecans ; metabolism ; Animals ; Cells, Cultured ; Chondrocytes ; cytology ; metabolism ; Extracellular Matrix ; metabolism ; Female ; Procollagen N-Endopeptidase ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection

BACKGROUNDS/AIMS: The serum level of hyaluronic acid (HA) has been suggested as a useful serologic marker for hepatic fibrosis. However, the relationship between serum HA levels and quantitative markers of fibrosis from liver tissue has not been reported. The aim of this study was to determine the correlation between serum HA level and quantitative measurement of hepatic fibrosis in a cirrhotic rat model. METHODS: Cirrhosis was produced by common bile duct ligation (BDL) in adult Sprague-Dawley rats. The animals were classified into four groups: (1) G1, sham operated (n=5); (2) G2, BDL for 2 weeks (n=6); (3) G3, BDL for 3 weeks (n=6); and (4) G4, BDL for 4 weeks (n=6). Hepatic fibrosis was analyzed histomorphologically using the Batts and Ludwig scoring system. Serum HA level and hepatic hydroxyproline content were quantified. The gene expressions in the liver of procollagen, collagen, and transforming growth factor-beta (TGF-beta) were measured by reverse transcriptase-polymerase chain reaction. RESULTS: In groups G1, G2, G3, and G4, the Batts and Ludwig scores (mean+/-SD) were 0, 1.3+/-0.5, 2.6+/-0.5, and 3.4+/-0.5, respectively (P<0.05), serum HA levels were 12.5+/-3.2, 30.0+/-4.3, 228.6+/-157.7, and 391.3+/-207.7 ng/mL (P<0.05), and the concentration of hydroxyproline was 12.4+/-2.8, 17.6+/-3.8, 17.9+/-2.4, and 33.4+/-3.4 microgram/g liver tissue, and it was significantly higher in group G4 than in the other groups (P<0.05). The gene expressions of collagen, procollagen, and TGF-beta1 in the liver were also significantly higher in group G4 compared with the other groups (P<0.05). Direct linear correlations were observed between serum HA level and hepatic hydroxyproline content, hepatic gene expressions of collagen, procollagen, TGF-beta1, and histomorphological grade of hepatic fibrosis (P<0.001). CONCLUSIONS: These results indicate that serum HA is a useful and noninvasive serologic marker for the evaluation of advanced hepatic fibrosis.
Animals ; Bile Ducts/pathology/surgery ; Biological Markers/blood ; Collagen/analysis/genetics ; Hyaluronic Acid/*blood ; Hydroxyproline/blood ; Ligation ; Liver/metabolism/*pathology ; Liver Cirrhosis, Experimental/*diagnosis/metabolism/pathology ; Male ; Models, Animal ; Procollagen/analysis/genetics ; RNA, Messenger/analysis ; Rats ; Rats, Sprague-Dawley ; Sickness Impact Profile ; Transforming Growth Factor beta/analysis/genetics

OBJECTIVETo investigate type I collagen metabolism in patients with arsenic poisoning in Dazhuyuan town, Ankang area in southern Shaanxi province known for high-level exposure to arsenic (As), fluoride (F) and selenium (Se).

METHODSThis case-control study involved 29 patients with arsenic poisoning from Dazhuyuan, a town in Ankang area of Shaanxi province affected by coal-borne endemic arsenicosis. Thirty individuals without arsenic poisoning from Chang'an county of Xi'an city were included as the control group. The serum concentrations of As and Se were determined using a graphite furnace atomic absorption spectrophotometer, and the levels of F detected with a fluoride ion-selective electrode. Enzyme-linked immunosorbent assay (ELISA) was used for assessing serum contents of PICP and PINP, and the sequence of COL1alpha1 gene was analyzed with ABI sequence analyzer.

RESULTSThe contents of PICP and PINP were similar between the two groups, which had also identical COL1alpha1 DNA sequences. After controlling for all the other factors, an inverse correlation was noted between PICP and serum F contents (r=-0.4, P<0.05) and between PINP and hair F content (r=-0.5, P<0.05), but a positive correlation was found between PINP and hair Se content (r=0.5, P<0.05).

CONCLUSIONHair F and Se levels are the major factors affecting PINP synthesis, and increased hair F level is associated with elevated PINP content, whereas hair Se level is inversely correlated to PINP content. But in the patients with arsenic poisoning from Dazhuyuan town, the PICP, PINP contents and COL1alpha1 DNA sequence show no significant alterations, suggesting that hair Se increment abolishes the effect of hair F increment in inducing the decrease in PINP content.

Adult ; Aged ; Aged, 80 and over ; Arsenic Poisoning ; blood ; genetics ; Case-Control Studies ; China ; Collagen Type I ; genetics ; Female ; Humans ; Male ; Middle Aged ; Peptide Fragments ; blood ; Procollagen ; blood ; Sequence Analysis, DNA

Forkhead Transcription Factors ; genetics ; Gene Expression Regulation ; Humans ; Procollagen ; genetics ; Transcription, Genetic

BACKGROUNDLower fluence of 585-nm flashlamp-pumped pulsed dye laser has been successfully used as a nonablative technique in the treatment of wrinkles. The objective of this study was to evaluate the effect of the pulsed dye laser (585 nm) on the production of collagen and the mRNA expression of collagen related gene in fibroblasts in vitro.

METHODSCultured fibroblasts were treated with a 585-nm flashlamp-pumped pulsed dye laser (fluence 3 J/cm(2), 4 J/cm(2), spot size 7 mm, pulse duration 450 micros). The production of collagen and the mRNA expression of transforming growth factor (TGF)-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen alpha1, alpha2 in fibroblasts were investigated by colorimetry or real time polymerase chain reaction.

RESULTSThe production of collagen was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm(2) (P < 0.001). The mRNA expression of TGF-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and procollagen I was significantly up-regulated after treatment with a 585-nm flashlamp-pumped pulsed dye laser with a fluence of 3 J/cm(2) (P < 0.001). No significant difference of mRNA expression of SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen was found between controls and fibroblasts treated with pulsed dye laser with a fluence of 4 J/cm(2) (P > 0.05).

CONCLUSIONSLower fluence (3 J/cm(2)) pulsed dye laser increased the collagen production in fibroblasts by up-regulating TGF-beta1, SMAD2, SMAD3, SMAD4, SMAD7 and type I procollagen mRNA expression. These may be the reason it can be effectively used in the treatment of wrinkles.

Analysis of Variance ; Cells, Cultured ; Collagen ; biosynthesis ; Fibroblasts ; cytology ; metabolism ; radiation effects ; Gene Expression ; radiation effects ; Humans ; Lasers ; Procollagen ; genetics ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Smad2 Protein ; genetics ; Smad3 Protein ; genetics ; Smad4 Protein ; genetics ; Smad7 Protein ; genetics ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1