This study investigated the effects of SIRT1 gene knock-out on osteoarthritis in mice, and the possible roles of SREBP2 protein and the PI3K/AKT signaling pathway in the effects. Mice were randomly divided into a normal group and a SIRT1 gene knock-out group (6 mice in each group). In these groups, one side of the knee anterior cruciate ligament was traversed, and the ipsilateral medial meniscus was cut to establish an osteoarthritis model of knee joint. The countralateral synovial bursa was cut out, serving as controls. The knee joint specimens were then divided into four groups: SIRT1control group (group A, n=6); SIRT1osteoarthritis group (group B, n=6); SIRT1control group (group C, n=6); SIRT1osteoarthritis group (group D, n=6). HE staining, Masson staining, Safranin O-Fast Green staining and Van Gieson staining were used to observe the morphological changes in the articular cartilage of the knee. Immunohistochemical staining was employed to detect the expression of SIRT1, SREBP2, VEGF, AKT, HMGCR and type II collagen proteins. SA-β-gal staining was utilized to evaluate chondrocyte aging. The results showed clear knee joint cartilage destruction and degeneration in the SIRT1osteoarthritis group. The tidal line was twisted and displaced anteriorly. Type II collagen was destroyed and distributed unevenly. Compared with the SIRT1osteoarthritis group and SIRT1control group, SIRT1 protein expression was not obviously changed in the SIRT1osteoarthritis group (P>0.05), while the expression levels of the SREBP2, VEGF and HMGCR proteins were significantly increased (P<0.05) and the levels of AKT and type II collagen proteins were significantly decreased (P<0.05). SIRT1 gene knock-out may aggravate cartilage degeneration in osteoarthritis by activating the SREBP2 protein-mediated PI3K/AKT signalling pathway, suggesting that SIRT1 gene may play a protective role against osteoarthritis.
Animals ; Cartilage ; pathology ; Chondrocytes ; metabolism ; Collagen Type II ; metabolism ; Disease Models, Animal ; Humans ; Knee Joint ; metabolism ; pathology ; Mice ; Mice, Knockout ; Oncogene Protein v-akt ; genetics ; Osteoarthritis ; genetics ; pathology ; Phosphatidylinositol 3-Kinases ; genetics ; Signal Transduction ; genetics ; Sirtuin 1 ; genetics ; Sterol Regulatory Element Binding Protein 2 ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis

Eupatilin is the main active component of DA-9601, an extract from Artemisia. Recently, eupatilin was reported to have anti-inflammatory properties. We investigated the anti-arthritic effect of eupatilin in a murine arthritis model and human rheumatoid synoviocytes. DA-9601 was injected into collagen-induced arthritis (CIA) mice. Arthritis score was regularly evaluated. Mouse monocytes were differentiated into osteoclasts when eupatilin was added simultaneously. Osteoclasts were stained with tartrate-resistant acid phosphatase and then manually counted. Rheumatoid synoviocytes were stimulated with TNF-alpha and then treated with eupatilin, and the levels of IL-6 and IL-1beta mRNA expression in synoviocytes were measured by RT-PCR. Intraperitoneal injection of DA-9601 reduced arthritis scores in CIA mice. TNF-alpha treatment of synoviocytes increased the expression of IL-6 and IL-1beta mRNAs, which was inhibited by eupatilin. Eupatilin decreased the number of osteoclasts in a concentration dependent manner. These findings, showing that eupatilin and DA-9601 inhibited the expression of inflammatory cytokines and the differentiation of osteoclasts, suggest that eupatilin and DA-9601 is a candidate anti-inflammatory agent.
Animals ; Anti-Inflammatory Agents/pharmacology/*therapeutic use ; Arthritis, Experimental/chemically induced/*drug therapy ; Arthritis, Rheumatoid/drug therapy/pathology ; Cell Differentiation/*drug effects ; Cells, Cultured ; Collagen Type II ; Cytokines/biosynthesis ; Disease Models, Animal ; Drugs, Chinese Herbal/therapeutic use ; Female ; Flavonoids/pharmacology/*therapeutic use ; Humans ; Inflammation/drug therapy/immunology ; Interleukin-1beta/genetics/metabolism ; Interleukin-6/genetics/metabolism ; Lymph Nodes/cytology ; Mice ; Mice, Inbred DBA ; Monocytes/cytology ; Osteoclasts/*cytology ; Plant Extracts/pharmacology ; RNA, Messenger/biosynthesis ; Synovial Membrane/cytology ; T-Lymphocytes, Regulatory/cytology/immunology ; Tumor Necrosis Factor-alpha/pharmacology

OBJECTIVETo observe the effects of low-intensity pulsed ultrasound (LIPUS) on repairing extracellular matrix in rabbit knee osteoarthritis and analyze its mechanism.

METHODSSixty adult female rabbits with an average weight of (2.0 ± 0.2) kg, were divided randomly into two groups (experimental group and control group, 30 rabbits in each group). All rabbits were replicated in right knees by Hulth method for knee osteoarthritis model. Two weeks after operation, the rabbits in experimental group were treated with LIPUS, and the ultrasonic frequency was (800 ± 5%)KHz and the maximum intensities of spatially averaged and time averaged (SATA) was (50 ± 10%) mw/cm2, for 1 time a day and every time 20 min, while the rabbits in control group were treated with sham LIPUS,the same operation with experimental group but without energy output. At the 2, 4, 8 weeks after treatment, 10 rabbits in each group were randomly killed for each time. The general changes of cartilage and its histopathological changes by HE staining were observed; the expression of collagen type II, proteoglycan, MMP-3, 7, 13 in cartilage were analyzed by immunohistochemical and RT-PCR technique; and the expression of NO in cartilage was analyzed by nitrate reduction method.

RESULTSOn the same observed time point, the damage degree of cartilage in experimental group was slighter than that of control group (P < 0.01), the expression of MMP-3, 7, 13 and NO in cartilage in experimental group was lower than that of control group (P < 0.01) while collagen type II and proteoglycan was higher than that of control group (P < 0.01).

CONCLUSIONLow-intensity pulsed ultrasound can repair the damaged cartilage by reducing the expression of MMP-3, 7, 13, inhibiting the secretion of NO and promoting the synthesis of collagen type II and proteoglycan in cartilage.

Animals ; Cartilage, Articular ; pathology ; Collagen Type II ; biosynthesis ; Extracellular Matrix ; metabolism ; Female ; Matrix Metalloproteinases ; analysis ; Nitric Oxide ; biosynthesis ; Osteoarthritis, Knee ; metabolism ; therapy ; Rabbits ; Ultrasonic Therapy ; methods

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 study the effects of peroxisome proliferator-activated receptor gamma agonists on angiotensin II-induced cellular response in cultured fibroblasts derived from patients with hypertrophic scars, so as to investigate its effects on preventing the formation of hypertrophic scars.

METHODSFibroblasts were freshly isolated from hypertrophic scars and cultured with angiotensin II, rosiglitazone and GW9662 at a certain concentration. Fibroblasts proliferation were assessed via Cell Counting Kit-8; the mRNA and protein expressions of Collagen I and Fibronectin (FN) were determined by quantitative real-time RT-PCR and Western blotting.

RESULTSThe absorbance of CCK-8 and relative expression of Collagen I, FN mRNA and protein were 1.082 5 +/- 0.007, 6.45 +/- 0.97, 4.92 +/- 0.86, 2.92 +/- 0.41, 2.78 +/- 1.04 in Ang II group; 0.722 4 +/- 0.012, 1.82 +/- 0.34, 1.78 +/- 0.27, 1.57 +/- 0.46, 1.68 +/- 0.39 in Ros + Ang II group; 0.554 7 +/- 0.012, 0.97 +/- 0.12, 1.07 +/- 1.08, 1.05 +/- 0.43, 1.14 +/- 0.36 in Ros group; 1.056 0 +/- 0.005, 5.83 +/- 0.24, 4.47 +/- 0.32, 2.69 +/- 0.35, 2.62 +/- 0.27 in GW9662 + ros + Ang II group. The results showed a significant difference between the Ang II group and the control group (P < 0.05). The effect of Ang II could be markedly inhibited by Ros (P < 0.05). In addition, Ros did not influence cell proliferation and production of extracellular matrix (P > 0.05). There was a significant difference between the GW9662 + Ros + Ang II group and the Ros + Ang II (P < 0.05).

CONCLUSIONSPPAR-gamma agonists inhibit Ang II-induced proliferation and extracellular matrix synthesis effectively in the hypertrophic scar fibroblasts. Thus PPAR-gamma agonists may have potential therapeutic effect for hypertrophic scar.

Angiotensin II ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; Collagen Type I ; biosynthesis ; Extracellular Matrix ; drug effects ; Fibroblasts ; drug effects ; metabolism ; pathology ; Fibronectins ; biosynthesis ; Humans ; PPAR gamma ; agonists

BACKGROUNDLow back pain has emerged as a widespread disease often caused by intervertebral disc degeneration. This study aimed to establish an in vitro cell culture model of rhesus monkey lumbar intervertebral discs and to investigate the effect of combined connective tissue growth factor (CTGF) and tissue inhibitor of metalloprotease-1 (TIMP-1) expression mediated by adeno-associated virus (AAV) on collagen type II and proteoglycan levels. The purpose of these investigations was to explore potential methods for relieving the degeneration of lumbar intervertebral disc cells.

METHODSRhesus monkey lumbar intervertebral disc nucleus pulposus cells (NPCs) were isolated by enzyme digestion, cultured, and transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, or rAAV2-TIMP-1 at a multiplicity of infection (MOI) of 10(6). The expression of collagen type II and proteoglycan was measured using RT-PCR and Western blotting. The synthetic rate of proteoglycan was measured using (35)S incorporation.

RESULTSRhesus monkey lumbar intervertebral disc NPCs were transduced with rAAV2-CTGF-IRES-TIMP-1, rAAV2-CTGF, and rAAV2-TIMP-1 and the transduced genes were expressed and detected. Compared to the control, CTGF promoted the synthesis of collagen type II and proteoglycan. TIMP-1 showed an enhancing effect on the expression of proteoglycan but no effect on collagen type II. Expression of both genes in rhesus monkey lumbar intervertebral disc NPCs significantly enhances the synthesis of proteoglycan and collagen type II.

CONCLUSIONSSingle gene transduction of CTGF or TIMP-1 can enhanced synthesis of proteoglycan. CTGF expression can also enhance collagen type II protein synthesis. Combined transduction of both CTGF and TIMP1 can significantly promote the expression of proteoglycan and collagen type II to levels greater than transduction of a single gene alone. Our study provides a good basis for multi-gene therapy to treat lumbar intervertebral disc degeneration.

Animals ; Blotting, Western ; Cells, Cultured ; Collagen Type II ; biosynthesis ; Connective Tissue Growth Factor ; genetics ; metabolism ; Intervertebral Disc ; cytology ; Macaca mulatta ; Proteoglycans ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Transduction, Genetic

BACKGROUND: The mechanism by which mutant cartilage oligomeric matrix protein (COMP) induces a pseudoachondroplasia phenotype remains unknown, and the reason why a mutation of a minor protein of the growth plate cartilage causes total disruption of endochondral bone formation has not yet been determined. The current study was performed to investigate the effects of mutated COMP on the synthesis of the cartilage-specific major matrix proteins of Swarm rat chondrosarcoma chondrocytes. METHODS: The Swarm rat chondrosarcoma chondrocytes transfected with a chimeric construct, which consisted of a mutant gene of human COMP and an amino acid FLAG tag sequence, were cultured in agarose gel. Formation of extracellular proteoglycan and type-II collagen by the cells was evaluated by immunohistochemical staining and measuring the (35)S-sulfate incorporation. RESULTS: No difference was observed for the detection of type-II collagen among the cell lines expressing mutant COMP and the control cell lines. Histochemical staining of sulfated proteoglycans with safranin-O showed that lesser amounts of proteoglycans were incorporated into the extracellular matrix of the chondrocytes transfected with the mutant gene. (35)S-sulfate incorporation into the cell/matrix fractions demonstrated markedly lower radiolabel incorporation, as compared to that of the control cells. CONCLUSIONS: Mutation of COMP has an important impact on the processing of proteoglycans, rather than type-II collagen, in the three-dimensional culture of Swarm rat chondrosarcoma chondrocytes.
Aggrecans/analysis/*biosynthesis ; Animals ; Cells, Cultured ; Chondrocytes/*metabolism ; Chondrosarcoma/metabolism ; Collagen Type II/*biosynthesis ; Extracellular Matrix/*metabolism ; Extracellular Matrix Proteins/*genetics ; Glycoproteins/*genetics ; Humans ; Mutation ; Rats ; Transfection

BACKGROUNDAngiotensin II (Ang II) is a very important vasoactive peptide that acts upon hepatic stellate cells (HSCs), which are major effector cells in hepatic cirrhosis and portal hypertension. The present study was aimed to investigate the effects of Ang II and angiotensin II type 1 receptor antagonist (AT(1)RA) on the proliferation, contraction and collagen synthesis in HSCs.

METHODSHSC-T6 rat hepatic stellate cell line was studied. The proliferation of the HSC cells was evaluated by MTT colorimetric assay while HSC DNA synthesis was measured by (3)H-thymidine incorporation. The effects of angiotensin II and AT(1)RA on HSCs contraction were studied by analysis of the contraction of the collagen lattice. Cell culture media were analyzed by RT-PCR to detect secretion of collagen I (Col I), collagen III (Col III) and transforming growth factor beta1 (TGF-beta1) by enzyme linked immunosorbent assay. HSC was harvested to measure collagen I, collagen III and tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression.

RESULTSAng II ((1 x 10(-10) - 1 x 10(-4)) mol/L) stimulated DNA synthesis and proliferation in HSCs compared with untreated control cells. AT(1)RA inhibited angiotensin II induced proliferation of HSCs. A linear increase in the contractive area of collagen lattice correlated with the concentration of angiotensin II (1 x 10(-9) - 1 x 10(-5) mol/L) and with time over 48 hours. AT(1)RA blocks angiotensin II induced contraction of collagen lattice. Col I, Col III and TGF-beta1 levels of the Ang II group were higher than those of control group and this increase was downregulated by AT(1)RA. The mRNA expressions of Col I, Col III and TIMP-1 were higher in HSCs from the Ang II group than the control group and downregulated by AT(1)RA.

CONCLUSIONSAngiotensin II increased DNA synthesis and proliferation of HSCs in a dose-dependent manner, stimulated the contraction of HSCs dose- and time-dependently. Angiotensin also promoted excretion of Col I, Col III and TGF-beta1 levels and stimulated Col I, Col III and TIMP-1 expression in HSCs. Angiotensin acts via the angiotensin II receptor because all of these effects are blocked by angiotensin II type 1 receptor antagonist.

Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Calcium ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Liver ; cytology ; drug effects ; metabolism ; Rats ; Transforming Growth Factor beta1 ; biosynthesis

Cyclooxygenase-2 (COX-2) is known to modulate bone metabolism, including bone formation and resorption. Because cartilage serves as a template for endochondral bone formation and because cartilage development is initiated by the differentiation of mesenchymal cells into chondrocytes (Ahrens et al., 1977; Sandell and Adler, 1999; Solursh, 1989), it is of interest to know whether COX-2 expression affect chondrocyte differentiation. Therefore, we investigated the effects of COX-2 protein on differentiation in rabbit articular chondrocyte and chick limb bud mesenchymal cells. Overexpression of COX-2 protein was induced by the COX-2 cDNA transfection. Ectopic expression of COX-2 was sufficient to causes dedifferentiation in articular chondrocytes as determined by the expression of type II collagen via Alcian blue staining and Western blot. Also, COX-2 overexpression caused suppression of SOX-9 expression, a major transcription factor that regulates type II collagen expression, as indicated by the Western blot and RT-PCR. We further examined ectopic expression of COX-2 in chondrifying mesenchymal cells. As expected, COX-2 cDNA transfection blocked cartilage nodule formation as determined by Alcian blue staining. Our results collectively suggest that COX-2 overexpression causes dedifferentiation in articular chondrocytes and inhibits chondrogenic differentiation of mesenchymal cells.
Animals ; Cartilage, Articular/cytology ; Cell Differentiation ; Cells, Cultured ; Chick Embryo ; Chondrocytes/*cytology/enzymology ; Chondrogenesis ; Collagen Type II/metabolism ; Cyclooxygenase 2/*biosynthesis/genetics ; Interleukin-1beta/pharmacology ; Mesenchymal Stem Cells/*cytology/enzymology ; Rabbits ; SOX9 Transcription Factor/metabolism

OBJECTIVETo explore the effects of adrenomedullin (ADM) on Angiotensin II (AngII)-induced collagen synthesis in cultured rat vascular adventitial fibroblasts.

METHODSRat vascular adventitial fibroblasts were cultured in vitro. ADM produced and secreted from adventitia in the presence of AngII was detected by radioimmunoassay, type I, III collagen contents in adventitia fibroblasts were measured by ELISA and the expressions of TGFbeta1 and MMP-2 were determined by RT-PCR and Western blotting.

RESULTSAngII significantly induced ADM secretion in adventitial fibroblasts in a dose-dependent manner. These effects could be reduced by 45%, 3% and 46%, through pre-treatment with Losartan, PD123319 or both, respectively for 30 min in culture medium. The AngII-induced type I, III collagen secretion in adventitial fibroblasts was significantly reduced by AMD in a dose-dependent manner, (P < 0.01) while ADM agonist ADM(22 - 52) significantly potentiated the effect of AngII; ADM also reduced AngII-induced expression of TGFbeta1 at mRNA and protein levels in a dose-dependent manner. AngII reduced the expression of MMP-2 at mRNA and protein levels in adventitial fibroblasts and these effects could be reversed by AMD (10(-8) mol/L).

CONCLUSIONAngII stimulated ADM secretion in adventitia fibroblasts, ADM in turn can inhibit AngII-induced type I, III collagen synthesis in adventitial fibroblasts probably by downregulating the TGFbeta1 expression and upregulating MMP-2 expression. ADM therefore served as an antifibrotic factor in vascular remodeling process.

Adrenomedullin ; metabolism ; Angiotensin II ; metabolism ; Animals ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Extracellular Matrix ; metabolism ; Fibroblasts ; metabolism ; Gene Expression Regulation ; Male ; Matrix Metalloproteinase 2 ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Transforming Growth Factor beta ; metabolism