OBJECTIVE: To explore the effect of miR-143 regulating matrix metalloproteinase(MMP)-13 expression on migration and invasion of osteosarcoma cells. METHODS: The mouse osteosarcoma cell line 143B cells were cultured in 96-well plates, and blank group, negative group, positive group, and intervention group were set up. Then, the blank group did no treatment 50 μg miR-143 mimic was added to positive group, negative group added equal mimic NC (control sequence of miR-143 mimic), the intervention group was added 50 μg miR-143 mimic and 10 μg MMP-13 protein, all groups continued to culture for 3 to 6 hours, and finally the serum was aspirated to treat for half an hour. The protein expressions of miR-143 and MMP-13 in each group were measured by fluorescence quantitative PCR experiment and Western blot experiment, respectively, and the invasion and migration abilities of cells were measured by Transwell and scratch experiments. RESULTS: The expression of MMP-13 protein in the positive group and the intervention group was significantly lower than that in the blank group, and the positive group was lower than the intervention group (P<0.05);The mean numbers of invasive cells in blank group, negative group, positive group and intervention group were (1 000.01±44.77), (959.25±46.32), (245.04±4.33), (634.06±33.78) cells/field, respectively;the scratch healing rate of the positive group and the intervention group was significantly lower than that of the blank group, and the positive group was lower than the intervention group (P<0.05). CONCLUSION MMP-13 is a target of miR-143, which can reduce the migration and invasion ability of osteosarcoma cells by inhibiting the expression of MMP-13.
Osteosarcoma/pathology* ; MicroRNAs/genetics* ; Matrix Metalloproteinase 13/genetics* ; Neoplasm Invasiveness ; Animals ; Mice ; Cell Line, Tumor ; Cell Movement

BACKGROUND: Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway. METHODS: Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting. RESULTS: IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells. CONCLUSION HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.
Cell Line, Tumor ; Cells, Cultured ; Chondrocytes/metabolism* ; Glycogen Synthase Kinase 3 beta/genetics* ; Histone Deacetylases/genetics* ; Humans ; Interleukin-1beta/pharmacology* ; Matrix Metalloproteinase 13/metabolism* ; Matrix Metalloproteinase 3 ; Repressor Proteins ; Wnt Signaling Pathway ; Wnt3A Protein/genetics* ; beta Catenin/metabolism*

Fibroblast-like synoviocytes (FLS) play a pivotal role in Rheumatoid arthritis (RA) pathogenesis through aggressive migration and invasion. Madecassoside (Madec), a triterpenoid saponin present in Centella asiatica herbs, has a potent anti-inflammatory effect. In the present study, Madec exerted an obvious therapeutic effect in reversing the histological lesions in adjuvant-induced arthritis (AIA) rats. To recognize the anti-rheumatoid potentials of Madec, we further investigated whether Madec interfered with FLS invasion and metalloproteinase (MMP) expression. In cultures of primary FLS isolated from the AIA rats, Madec (10 and 30 μmol·L) was proven to considerably inhibit migration and invasion of FLS induced by interleukin 1β (IL-1β), but exhibiting no obvious effect on cell proliferation. Madec repressed IL-1β-triggered FLS invasion by prohibiting the expression of MMP-13. Additionally, Madec suppressed MMP-13 transcription via inhibiting the MMP-13 promoter-binding activity of NF-κB. Our results further showed that Madec down-regulated the translocation and phosphorylation of NF-κB as demonstrated by Western blotting and immunofluorescence assays. In conclusion, our results suggest that Madec exerts anti-RA activity via inhibiting the NF-κB/MMP-13 pathway.
Animals ; Antirheumatic Agents ; chemistry ; pharmacology ; therapeutic use ; Arthritis, Experimental ; chemically induced ; drug therapy ; pathology ; Cell Movement ; drug effects ; Cell Nucleus ; metabolism ; Cells, Cultured ; Gene Expression Regulation, Enzymologic ; drug effects ; Interleukin-1beta ; pharmacology ; Matrix Metalloproteinase 13 ; genetics ; NF-kappa B ; genetics ; metabolism ; Phosphorylation ; drug effects ; Protein Transport ; drug effects ; Rats ; Signal Transduction ; drug effects ; Synoviocytes ; drug effects ; metabolism ; Transcriptional Activation ; drug effects ; Triterpenes ; chemistry ; pharmacology ; therapeutic use

OBJECTIVETo establish a reliable model for drug screening and therapy by culturing rat femoral head and inducing cartilage degeneration quickly in vitro.

METHODSThe femoral heads from the same SD rats of two-month old were divided into control group and experimental group respectively. They were cultured with DMEM medium plus 10% fetal bovine serum or DMEM medium plus 10% fetal bovine serum plus 50 ng/ml IL-1β for three days. Femoral heads were fixed in 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin and cut into slices. Specimens were stained with Toluidine blue and Safranine O-Fast Green FCF. The protein expression levels of type II collagen, MMP13, Sox9 and ADAMTS5 were analyzed by immunofluorescence.

RESULTSBoth the Toluidine blue and Safranine O staining were pale in the margin of femoral heads which were stimulated with IL-1β for three days compared to that in control group. The Fast Green FCF staining was positive at the edge of the femoral head in experimental group, which indicated that cartilage became degenerated. The expression levels of both type H collagen and Sox9 were decreased significantly while the expression levels of MMP13 and ADAMTS5 were increased in experimental group.

CONCLUSIONThe model of cartilage degeneration is established by culturing and inducing the degeneration of the femoral heads quickly in vitro.

Animals ; Cartilage Diseases ; genetics ; metabolism ; Collagen Type II ; genetics ; metabolism ; Disease Models, Animal ; Femur Head ; metabolism ; Humans ; In Vitro Techniques ; Interleukin-1beta ; genetics ; metabolism ; Male ; Matrix Metalloproteinase 13 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; SOX9 Transcription Factor ; genetics ; 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

OBJECTIVETo investigate the effects of a proliferation-inducing ligand (APRlL) on colorectal cancer (CRC) cell growth and migration, and to observe the role of APRIL in CRC biological behavior.

METHODSThe siRNA plasmid vector targeting APRIL gene (APRIL-siRNA) was transfected into human colorectal cancer SW480 cells and recombinant human APRIL (rhAPRIL) was used to stimulate human colorectal cancer HCT-116 cells. Cell proliferation activity was analyzed using cell counting kit-8 (CCK-8), cell cycle was detected by flow cytometry, and the protein expression of cyclin D1, p21 and Bcl-2 was detected by Western blot analysis. Tumor cell migration and invasion were measured by Transwell chambers. RT-PCR was applied to examine the mRNA expression level of MMP-2 and MMP-9. APRIL-siRNA was used to transfect directly SW480 cells, which were injected subcutaneously into nude mice, then the tumor growth and metastasis were observed.

RESULTSCell proliferation ability of APRIL-siRNA-transfected SW480 cells was drastically repressed, and the percentage of G0/G1 phase cells was significantly increased (t = 4.12, P < 0.05), accompanied with depressed cyclin D1, Bcl-2 expression and elevated p21 expression. Cell proliferation ability of rhAPRIL-stimulated HCT-116 cells was promoted with a decreased G0/G1 phase ratio (t = 3.31, P < 0.05). cyclin D1 and Bcl-2 protein expression was up-regulated while p21 was down-regulated by rhAPRIL stimulation. Metastatic and invasive capacities of APRIL-siRNA-transfected SW480 cells were significantly inhibited compared with their respective controls (both P < 0.05), accompanied with the deregulated MMP-2 and MMP-9 mRNA expression. Metastatic and invasive capacities of rhAPRIL-stimulated HCT-116 cells were promoted with up-regulated MMP-2 and MMP-9 mRNA expression(both P < 0.05). Tumor growth in the group transfected with APRIL-siRNA appeared to be slower than that in the control groups and the expression of MMP-2, MMP-9 in tumor tissues was depressed in the APRIL-siRNA group.

CONCLUSIONSAPRIL facilitates tumor growth and metastasis, and is associated with carcinogenesis and prognosis. Our findings suggest that APRIL might be used as a novel target for the intervention and therapy of colorectal cancer.

Animals ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Cyclin D1 ; metabolism ; Female ; Genetic Vectors ; HCT116 Cells ; HT29 Cells ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Neoplasm Transplantation ; Plasmids ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Burden ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; metabolism

OBJECTIVETo study the effect of laminarin polysaccharide (LP) on the activity of matrix metalloproteinase of photoaging skins.

METHODKunming SPF mice were prepared with back hair shaved, and randomly divided into the control group, the model group, the LP low does group (LP-L, 1 mg x kg(-1)), the LP high dose group (LP-H, 5 mg x kg(-1)) and the Vit E (100 mg x kg(-1)) group. They were abdominally injected with drugs twice on a daily basis. Except for the control group, all groups were exposed to ultraviolet rays for 1 hour every day, five times on a weekly basis, with accumulated exposure dose of UVB being 21.60 J x cm(-2) and accumulated exposure dose of UVA being 84.02 J x cm(-2). Eight weeks later, exposed back skins were collected to detect thickness of dermis by HE stain, content of hydroxyproline (Hyp) by chemical colorimetry, and serum MMP-1 and TIMP-1 content by ELISA. In addition, matrix metalloproteinase-1 (MMP-1) mRNA and relative content of tissue inhibitor of metalloproteinase-1 (TIMP1) mRNA was analyzed with Real-time PCR.

RESULTCompared with the model group, the LP-H group could significantly increase the thickness of dermis, skin Hyp content and serum TIMP-1 level, and decrease relative content of MMP-1 mRNA in skin and MMP-1 content in serum.

CONCLUSIONLP can regulate the metabolism of collagen photoaging skins by adjusting the activity of matrix metalloproteinase.

Animals ; Female ; Glucans ; Matrix Metalloproteinase 13 ; biosynthesis ; genetics ; metabolism ; Mice ; Plant Extracts ; chemistry ; pharmacology ; Plants, Medicinal ; chemistry ; Polysaccharides ; chemistry ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Skin Aging ; drug effects ; physiology ; radiation effects ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; metabolism ; Ultraviolet Rays

This study was done to evaluate whether injections of resveratrol, a natural compound found in the skin of grapes, had anabolic effects on degenerated intervertebral discs in a rabbit model. Two non-continuous lumbar discs were punctured in rabbits to induce disc degeneration. Four weeks and 6 weeks after puncture, the rabbits were treated by injections with dimethylsulfoxide (DMSO) or resveratrol. At 4, 8, and 16 weeks after initial injection, rabbits were sacrificed and the spine was extracted for magnetic resonance image (MRI), mRNA expression, and histological staining. Resveratrol treatment resulted in stronger signal intensity in T2-weighted images. MRI grade showed significantly lower in the resveratrol group than the DMSO group (P = 0.039). In the resveratrol group, aggrecan gene expression was significantly increased than that in the DMSO group at 16 weeks after injection (P = 0.027). MMP-13 mRNA levels in the resveratrol group were significantly decreased than those in the DMSO group at 8 and 16 weeks (P = 0.006 and P = 0.048, respectively). In hematoxylin and eosin stain, resveratrol-treated discs showed the features of regeneration. Histologic grade revealed improvement in resveratrol-treated discs, compared with DMSO-treated discs (P = 0.024). These anabolic effects on degenerated discs indicate that resveratrol is a promising candidate for treatment of degenerative disc disease.
Aggrecans/genetics/metabolism ; Anabolic Agents/*administration & dosage ; Animals ; Disease Models, Animal ; Drug Administration Schedule ; Intervertebral Disc Degeneration/*drug therapy/pathology ; Magnetic Resonance Imaging ; Matrix Metalloproteinase 13/genetics/metabolism ; RNA, Messenger/metabolism ; Rabbits ; Spine/radiography ; Stilbenes/*administration & dosage

OBJECTIVETo investigate the effect of Panax notoginseng saponins (PNS) on the expressions of matrix metalloproteinase (MMP)-13 and its tissue inhibitor of metalloproteinase (TIMP)-1 in carbon tetrachloride (CCl4)-induced hepatic fibrosis rats, and explore the possible mechanism of PNS's effect against hepatic fibrosis.

METHODThe rats were randomly divided into 6 groups: the normal group, the model group, PNS (50, 100, 200 mg x kg(-1)) treatment groups and the Col (0.1 mg x kg(-1)) group. Apart from the normal group, all of the remaining groups were subcutaneously injected with CCl4 twice a week for 18 weeks, in order to establish the hepatic fibrosis rat model. Since the 9th weeks, each treatment group was orally administered with corresponding drugs, and the normal group and the model group were orally administered with equal volume of normal saline for 10 weeks. After the end of the experiment, liver and spleen indexes were calculated; the levels of serum ALT and AST were measured by chromatometry. Liver tissues were collected to detect the pathological alteration HE staining; protein expressions of MMP-13 and TIMP-1 were determined with immuninochemistry. Moreover, MMP-13 and TIMP-1 mRNA expressions was detected by RT-PCR technology.

RESULTCompared with the model group, PNS (100, 200 mg x kg(-1)) significantly mitigated hepatic fibrosis in rats, reduced liver and spleen indexes, ALT and AST contents in serum, and TIMP-1 expression, and notably increased MMP-13 expression in rats with hepatic fibrosis.

CONCLUSIONP. notoginseng saponins have certain protective effect in rats with hepatic fibrosis. Its mechanism is related to up-regulating MMP-3, inhibiting TIMP-1 expression and improving collagen degradation.

Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Gene Expression Regulation ; drug effects ; Liver Cirrhosis, Experimental ; drug therapy ; metabolism ; Male ; Matrix Metalloproteinase 13 ; genetics ; metabolism ; Panax notoginseng ; chemistry ; RNA, Messenger ; genetics ; Rats ; Saponins ; administration & dosage ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism

BACKGROUNDThere is a difficulty in evaluating the in vivo functionality of individual chondrocytes, and there is much heterogeneity among cartilage affected by osteoarthritis (OA). In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.

METHODSCartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan (GAG) content were measured. Cell morphology, proliferation, and gene expression of collagen type I, II, and X, aggrecan, matrix metalloproteinase 13 (MMP-13), and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.

RESULTSBoth the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable.

CONCLUSIONSExpression of functional genes such as collagen type II and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.

ADAM Proteins ; ADAMTS5 Protein ; Cartilage ; pathology ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Chondrocytes ; metabolism ; Collagen Type II ; genetics ; Collagen Type X ; genetics ; Glycosaminoglycans ; metabolism ; Humans ; Matrix Metalloproteinase 13 ; genetics ; Osteoarthritis ; genetics ; pathology