Experimental Study on the Mechanism of Mangiferin Inhibiting Malignant Biological Characteristics of Multiple Myeloma and Exerting Anticancer Effect / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of pure Chinese herbal extract Mangiferin on the malignant biological behaviors of multiple myeloma (MM) cells, and to analyze the molecular mechanism of the anti-myeloma effect of Mangiferin, so as to provide experimental basis for MM replacement therapy.@*METHODS@#U266 and RPMI8226 of human MM cell lines were intervened with different concentrations of Mangiferin. Cell proliferation was detected by CCK-8 method. Annexin V/PI double staining flow cytometry was used to detect cell apoptosis. Western blot was used to detect the expression of apoptosis and related signaling pathway proteins, and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of matrix metalloproteinase (MMP) and CXC chemokine receptor (CXCR) family.@*RESULTS@#Mangiferin could inhibit the proliferation activity of U266 and RPMI8226 cells and induce cells apoptosis. After Mangiferin intervened in U266, RPMI8226 cells for 48 h, the expression of Bcl-2 family pro-apoptotic protein Bax was up-regulated, while the expression of survivin and Bcl-xL proteins was down-regulated and caspase-3 was hydrolyzed and activated to promote cell apoptosis, besides, the expression of Bcl-2 protein in U266 cells was also significantly down-regulated to induce apoptosis (P<0.05). After Mangiferin intervenes in MM cells, it can not only increase the expression level of tumor suppressor p53, but also induce programmed cell death of MM cells by inhibiting the expression of anti-apoptotic molecules and down-regulating the phosphorylation levels of AKT and NF-κB. In addition, after the intervention of Mangiferin, the expressions of CXCR4, MMP2 and MMP9 in U266 cells were down-regulated (P<0.05), while there is no effect on the expressions of CXCR2, CXCR7 and MMP13 (P>0.05). However, the expressions of CXCR4, MMP9, and MMP13 in RPMI8226 cells were down-regulated (P<0.01), the expression of MMP2 was weakly affected, and the expression of CXCR2 and CXCR7 was basically not affected (P>0.05).@*CONCLUSION@#Mangiferin can inhibit the proliferation and induce apoptosis of MM cells, and its mechanism may be related to inhibiting the activation of NF-κB signaling pathway, affecting the expression of Bcl-2 family proteins, and inhibiting the expression of core members of MMP and CXCR family.

The expression of matrix metalloproteinase-13 (MMP-13) on xenograft and PRF in bone regeneration / Efeito da combinação de xenoenxerto e PRP na expressão de metaloproteinase de matriz -13 (MMP-13) durante regeneração óssea

Objective: inflammation may play a role in bone loss by altering the boné remodelling process, favouring bone resorption by osteoclasts over bone synthesis by osteoblasts. Matrix metalloproteinase 13 (MMP-13) has the ability to activate osteoclasts, leading to bone resorption. Regenerative treatments have been widely used in periodontology. When combined with Platelet-rich fibrin (PRF), xenografts will give better results in bone regeneration. The aim of this study was to evaluate the effect of xenograft combined with PRF on MMP-13 expression in a bone defect using an experimentally created bone defect. Material and Methods: eighteen New Zealand rabbits were assigned to three groups. Each group consisted of six New Zealand rabbits. A critical bone defect with a diameter size of 5 mm was created in the right tibia of each rabbit in group 1 (application: xenograft), group 2 (application: PRF), and group 3 (application: xenograft and PRF). The PRF was produced from 5 ml of blood taken from each rabbit's ears. After 30 days, the rabbits were euthanized. The tissue samples were evaluated by immunohistochemical staining. Results: group 3 showed the lowest mean expression of MMP-13 (4.50) compared to group 1 (20.50) and group 2 (11.70). Group 3 showed a significant difference in the MMP-13 expression compared to group 1 and group 2 (P = 0.000) (P < 0.05). Conclusion: this research showed that the combination of xenograft and PRF had the lowest expression of MMP-13. The application of a xenograft and PRF has better osteogenesis ability in bone regeneration.(AU)

Objetivo: inflamação pode interferir na perda óssea através de alterações no processo de remodelação, favorecendo a reabsorção óssea pelos osteoclastos ao invés da síntese pelos osteoblastos. A metaloproteinases de matriz 13 (MMP-13) ativa osteoclastos causando reabsorção óssea. Tratamentos regenerativos têm sido amplamente usados na periodontia. Quando combinamos Plasma rico em plaquetas (PRP) e xenoenxerto levam a melhores resultados de regeneração óssea. O objetivo deste estudo foi avaliar os efeitos de xenoenxerto combinado com PRP na expressão de MMP-13 em defeitos ósseos experimentais. Material e Métodos: dezoitos coelhos Nova Zelândia foram distribuídos em 3 grupos de 6 coelhos cada. Um defeito ósseo de 5 mm de diâmetro foi feito na tíbia direita dos animais do grupo 1 (xenoenxerto), grupo 2 (PRP) e grupo 3 (Xenoenxerto+PRP). O PRP foi obtido pela coleta de 5mL de sangue das orelhas dos coelhos. Após 30 dias, os coelhos foram eutanasiados. As amostras foram submetidas a coloração imuno-histoquímica. Resultados: o grupo 3 apresentou a menor expressão de MMP-13 (4.50) quando comparado ao grupo 1 (20.50) e ao grupo 2 (11.70). O grupo 3 mostrou diferença estatística significante em relação a expressão de MMP-13 quando comparado aos grupos 1 e 2 (p=0.000) (p< 0.05). Conclusão: esta pesquisa mostra que a combinação de xenoenxerto e PRP teve a menor expressão de MMP-13. A combinação de xenoenxerto e PRP têm maior habilidade de osteogênese na regeneração óssea (AU)

Effects of HDAC4 on IL-1β-induced matrix metalloproteinase expression regulated partially through the WNT3A/β-catenin pathway / 中华医学杂志(英文版)

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.

Madecassoside impedes invasion of rheumatoid fibroblast-like synoviocyte from adjuvant arthritis rats via inhibition of NF-κB-mediated matrix metalloproteinase-13 expression / 中国天然药物

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.

Madecassoside impedes invasion of rheumatoid fibroblast-like synoviocyte from adjuvant arthritis rats via inhibition of NF-κB-mediated matrix metalloproteinase-13 expression / 中国天然药物

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.

Topical or oral treatment of peach flower extract attenuates UV-induced epidermal thickening, matrix metalloproteinase-13 expression and pro-inflammatory cytokine production in hairless mice skin

BACKGROUND/OBJECTIVES: Ultraviolet radiation (UV) is a major cause of skin photoaging. Previous studies reported that ethanol extract (PET) of Prunus persica (L.) Batsch flowers (PPF, peach flowers) and its subfractions, particularly the ethylacetate (PEA) and n-butanol extracts (PBT), have potent antioxidant activity and attenuate the UV-induced matrix metalloproteinase (MMP) expression in human skin cells. In this study, we investigated the protective activity of PPF extract against UV-induced photoaging in a mouse model. MATERIALS/METHODS: Hairless mice were treated with PET or a mixture of PEA and PBT either topically or orally along with UV irradiation. Histological changes and biochemical alterations of mouse skin were examined. Major phenolic compounds in PPF extract were analyzed using an ACQUITY UPLC system. RESULTS: The overall effects of topical and oral treatments with PPF extract on the UV-induced skin responses exhibited similar patterns. In both experiments, the mixture of PEA and PBT significantly inhibited the UV-induced skin and epidermal thickening, while PET inhibited only the UV-induced epidermal thickening. Treatment of PET or the mixture of PEA and PBT significantly inhibited the UV-induced MMP-13 expression, but not typeⅠ collagen expression. Topical treatment of the mixture of PEA and PBT with UV irradiation significantly elevated catalase, superoxide dismutase (SOD) and glutathione-peroxidase (GPx) activities in the skin compared to those in the UV irradiated control group, while oral treatment of the mixture of PEA and PBT or PET elevated only catalase and SOD activities, but not GPx. Thirteen phytochemical compounds including 4-O-caffeoylquinic acid, cimicifugic acid E and B, quercetin-3-O-rhamnoside and kaempferol glycoside derivatives were identified in the PPF extract. CONCLUSIONS: These results demonstrate that treatment with PET or the mixture of PEA and PBT, both topically or orally, attenuates UV-induced photoaging via the cooperative interactions of phenolic components having anti-oxidative and collagen-protective activities.

Primary cultures of mouse small intestinal epithelial cells using the dissociating enzyme type I collagenase and hyaluronidase

The epithelium is a highly dynamic system, which plays a crucial role in the homeostasis of the intestinal tract. However, studies on the physiological and pathophysiological functions of intestinal epithelial cells (IECs) have been hampered due to lack of normal epithelial cell models. In the present study, we established a reproducible method for primary culture of mouse IECs, which were isolated from the viable small intestinal crypts of murine fetuses (on embryonic day 19), using type I collagenase and hyaluronidase in a short span of time (≤20 min). With this method, continuously growing mouse IECs, which can be subcultured over a number of passages, were obtained. The obtained cell lines formed a tight cobblestone-like arrangement, displayed long and slender microvilli, expressed characteristic markers (cytokeratin 18 and Notch-1), and generated increasing transepithelial electrical resistance and low paracellular permeability during in vitro culture. The cells also had enzymatic activities of alkaline phosphatase and sucrase-isomaltase, and secreted various cytokines (IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1), responding to the stimulation of Escherichia coli. These results show that the primary-cultured mouse IECs obtained by the method established here had the morphological and immunological characteristics of IECs. This culture system can be a beneficial in vitro model for studies on mucosal immunology and toxicology.

Etanercept protects rat cardiomyocytes against hypertrophy by regulating inflammatory cytokines secretion and cell apoptosis

We aimed to investigate the effect of etanercept, a tumor necrosis factor-α (TNF-α) inhibitor, on rat cardiomyocyte hypertrophy and its underlying mechanism. Primary neonatal rat cardiomyocytes were isolated from Sprague-Dawley rats. The model of rat cardiomyocyte hypertrophy was induced by endothelin, and then treated with different concentrations of etanercept (1, 10, and 50 μM). After treatment, cell counts, viability and cell apoptosis were evaluated. The mRNA levels of myocardial hypertrophy marker genes, including atrial natriuretic factor (ANF), matrix metalloproteinase (MMP)-9 and MMP-13, were detected by qRT-PCR, and the expressions of apoptosis-related proteins (Bcl-2 and Bax) were measured by western blotting. The protein levels of transforming growth factor-β1 (TGF-β1), interleukin (IL)-1β, IL-6, leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1) were determined using enzyme linked immunosorbent assay (ELISA) kits. In the present study, TNF-α level in cardiomyocytes with hypertrophy was significantly enhanced (P<0.05). Compared to the model group, cell number and viability were significantly increased and ratio of apoptotic cells was reduced by etanercept (P<0.05, P<0.01, or P<0.001). In addition, etanercept remarkably reduced the mRNA levels of ANF, MMP-9 and MMP-13, inhibited the expression of Bax, and increased the expression of Bcl-2 compared to the model group (P<0.05). ELISA results further showed that etanercept lowered the levels of IL-1β, IL-6, LIF and CT-1 but not TGF-β1 compared to the model group (P<0.05). Etanercept may protect rat cardiomyocytes from hypertrophy by inhibiting inflammatory cytokines secretion and cell apoptosis.

Effect of Matrix Metallopeptidase 13 on the Function of Mouse Bone Marrow-derived Dendritic Cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Dendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 (MMP-13), a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells (DCs). The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis.</p><p><b>METHODS</b>Bone marrow-derived dendritic cells were obtained from C57BL/6 mice. One short-interfering RNA specific for MMP-13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry.</p><p><b>RESULTS</b>Compared to the control DCs, MMP-13-silenced DCs increased expression of anti-apoptosis-related genes, BAG1 (control group vs. MMP-13-silenced group: 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008), BCL-2 (control group vs. MMP-13-silenced group: 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036), and TP73 (control group vs. MMP-13-silenced group: 4.33 ± 0.29 vs. 5.60 ± 0.32, P = 0.001) and decreased apoptosis-related genes, CASP1 (control group vs. MMP-13-silenced group: 3.79 ± 0.67 vs. 2.54 ± 0.39, P = 0.019), LTBR (control group vs. MMP-13-silenced group: 9.23 ± 1.25 vs. 6.24 ± 1.15, P = 0.012), and CASP4 (control group vs. MMP-13-silenced group: 2.07 ± 0.56 vs. 0.35 ± 0.35, P = 0.002). Protein levels confirmed the same expression pattern. MMP-13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability.</p><p><b>CONCLUSION</b>These results indicate that MMP-13 inhibition dampens DC maturation, apoptosis, and phagocytosis.</p>

MMP13, TIMP2 and TGFB3 Gene Polymorphisms in Brazilian Chronic Periodontitis and Periimplantitis Subjects

Abstract Subjects susceptible to chronic periodontitis (CP) show a high risk for the development of peiimplantitis (PI). Both diseases are multifactorial, presenting similarities in their pathophysiology and polygenic profile. MMP-13 (matrix metalloproteinases 13/ collagenase 3) is a collagenolytic enzyme, which expression is induced by TGF beta 3 (transforming growth factor type 3) in human gingival fibroblasts and inhibited by TIMP-2 (tissue inhibitor of metalloproteinase type 2). The aim of this study was to investigate the occurrence of peiimplantitis (PI) in subjects with history of chronic periodontitis (CP) and polymorphisms frequency in MMP13, TIMP2 and TGFB3 genes. One hundred and sixty-three volunteers received dental implant placement were submitted to oral and radiographic examination in order to identify past history of CP or presence of PI. Volunteers were divided into 4 groups: Control (without PI and CP, n=72), CP (with CP and without PI, n=28), PI (with PI and without CP, n=28) and diseased (with CP and PI, n=35). The chi-square test correlated genotypes in specific regions of MMP13 (rs2252070), TIMP2 (rs7501477) and TGFB3 (rs2268626) genes, considering the interaction between CP and PI. The results showed that volunteers with CP had 3.2 times more susceptibility to develop PI (p=0.0004) compared to those without CP. No significant association was observed in MMP13, TIMP2 and TGFB3 genes with CP or PI. CP is a risk factor to develop PI, however, there is no association of both diseases with polymorphisms in the MMP13, TIMP2 and TGFB3 genes.

Resumo Indivíduos susceptíveis à periodontite crônica (CP) apresentam alto risco para o desenvolvimento de periimplantite (PI). Ambas doenças são multifatoriais e apresentam similaridades na patofisiologia e perfil poligênico. A MMP-13 (metaloproteinase da matriz tipo 13) é uma enzima colagenolítica cuja expressão é induzida por TGF beta 3 (fator transformador do crescimento tipo 3) nos fibroblastos gengivais humanos e inibida por TIMP-2 (inibidor tecidual de metaloproteinase tipo 2). O objetivo deste estudo foi investigar a ocorrência de periimplantite em sujeitos com periodontite crônica e a frequência dos polimorfismos nos genes MMP13, TIMP2 e TGFB3. Cento e sessenta e três voluntários submetidos à instalação de implantes endósseos foram analisados clínica e radiograficamente quanto à presença de histórico de CP e PI, sendo divididos em 4 grupos: Controle (sem história de CP e PI, n=72), CP (com CP e sem PI, n=28), PI (com PI e sem CP, n=28) e Doentes (com CP e PI, n=35). O teste do qui-quadrado correlacionou os genótipos nas regiões dos genes MMP13 (rs2252070), TIMP2 (rs7501477) e TGFB3 (rs2268626), considerando a interação entre CP e PI. Os resultados mostraram que voluntários com CP possuem 3.2 vezes mais chances de desenvolver PI (p=0.0004) comparados aos sem CP. Nenhuma associação significativa foi observada entre os genes MMP13, TIMP2 e TGFB3 e CP ou PI. A CP é um fator de risco ao desenvolvimento de PI, no entanto, não há associação entre ambas as doenças com polimorfismos nos genes MMP13, TIMP2 e TGFB3.

Protective effect of LR-90 on articular cartilage in rabbit model of osteoarthritis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the protective effect of LR-90 on articular cartilage in rabbit model of osteoarthritis.</p><p><b>METHODS</b>The cultured rabbits chondrocytes were assigned to be treated with IL-1β (10ng/ml) or IL-1β (10ng/ml)+LR-90 (50 mg/L). The mRNA expression of MMP-13, ADAMTS-5, aggrecan and collagen II in chondrocytes were assessed by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Twenty male New Zealand white rabbits underwent bilateral anterior cruciate ligament transection (ACLT) to establish a animal model of osteoarthritis. Four weeks after model established, on the basis of randomization one knee of each rabbit was treated with 50 mg/L LR-90 in normal saline (NS) (experimental group) and the other knee was treated with same volume of NS (control group), 1/week × 5. Nine weeks after ACLT all rabbits were sacrificed and the knee joints were evaluated by gross morphology and histology. The mRNA expression of IL-1β, MMP-13, ADAMTS-5, aggrecan and collagen Ⅱ in articular cartilage was analyzed by RT-PCR.</p><p><b>RESULTS</b>Gross morphology and Mankin histological evaluation showed that the extent and grade of cartilage damage in the experimental group were less severe than those in the control group.Compared to IL-1β group, LR-90 treatment suppressed the mRNA expression of MMP-13 and ADAMTS-5, and enhanced aggrecan and collagen Ⅱ mRNA expression. Consistent with the in vitro results, the intraarticular LR-90 administration suppressed the mRNA expression of IL-1β,MMP-13 and ADAMTS-5 (all P<0.01), while enhanced mRNA expression of aggrecan and collagen Ⅱ in cartilage (all P<0.01).</p><p><b>CONCLUSION</b>LR-90 protects against cartilage degradation and inhibits the progression of osteoarthritis in rabbit mode1 of osteoarthritis, which is associated with the suppressing IL-1β, MMP-13, ADAMTS-5 and promoting aggrecan and collagen Ⅱ mRNA expression in cartilage.</p>

The role of MCP-1-CCR2 ligand-receptor axis in chondrocyte degradation and disease progress in knee osteoarthritis

BACKGROUND: Osteoarthritis (OA) is a common arthritic disease and multifactorial whole-joint disease. Interactions of chemokines and OA is inadequately documented. RESULTS: In vivo and in vitro studies were conducted to investigate monocyte chemoattractant protein 1 (MCP-1) and receptor chemokine (C-C motif) receptor 2 (CCR2) in chondrocyte degradation and cartilage degeneration. Chondrocytes from 16 OA patients and 6 normal controls were involved in this study. After stimulation of MCP-1, the expression of MCP-1 and CCR2 increased significantly (P < 0.001) and the expression of MMP-13 also increased (P < 0.05). MCP-1 stimulation also induced (or enhanced) the apoptosis of OA chondrocytes (P < 0.05). Additionally, the degradation of cartilage matrix markers (metalloproteinase 3 and 13, MMP3 and MMP13) in the culture medium of normal chondrocytes was also assessed. Furthermore, intra-articular injection of MCP-1 in mouse knees induced cartilage degradation and the CCR2 antagonist did not impede cartilage destroy in rats knees of monosodium iodoacetate (MIA) model. CONCLUSIONS: The results of this study demonstrate that the MCP-1-CCR2 ligand-receptor axis plays a special role in the initiation and progression of OA pathology. Patients with ambiguous etiology can gain some insight from the MCP-1-CCR2 ligand-receptor axis.

Establishment of chondrocyte degeneration model in vitro by rat serum / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a model of chondrocyte degeneration in vitro.</p><p><b>METHODS</b>Chondrocytes were isolated from articular cartilages of newly born SD rats by digestion with typeⅡ collagenase. The chondrocytes were cultured with H-DMEM medium containing 10%FBS, 50 ng/mL IL-1β+10%FBS, 2.5% rat serum and 5% rat serum, respectively; and the chondrocytes at passage one were used in the experiments. The morphology changes were investigated under phase contrast microscope after chondrocytes were treated with rat serum and IL-1β. Proliferation of chondrocytes was detected by MTT method. The protein expression levels of PCNA, typeⅡ collagen and MMP-13 were examined by Western blotting. The levels of ADAMTS5, MMP-9, Aggrecan and SOX-9 mRNA were detected by real-time PCR.</p><p><b>RESULTS</b>The cell morphology was changed from polygon to spindle in both rat serum groups and IL-1β group, and the proliferation of chondrocytes in these groups was much higher than that in control group. The results showed that the expression levels of typeⅡ collagen, Aggrecan and SOX-9 decreased while the expression levels of MMP-13, MMP-9 and ADMATS5 were up-regulated in rat serum and IL-1β-treated groups compared with control group.</p><p><b>CONCLUSION</b>The results indicate that rat serum can induce chondrocyte degeneration and may be used for osteoarthritis model in vitro.</p>

Etablishment of cartilage degeneration model by IL-1 beta in vitro / 中国骨伤

<p><b>OBJECTIVE</b>To establish a reliable model for drug screening and therapy by culturing rat femoral head and inducing cartilage degeneration quickly in vitro.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Both 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.</p><p><b>CONCLUSION</b>The model of cartilage degeneration is established by culturing and inducing the degeneration of the femoral heads quickly in vitro.</p>

Suppression of E3 ubiquitin ligase Cbl-b in interleukin-1 signaling / 生理学报

The present study aims to investigate the effect of Cbl-b, a member of E3 ubiquitin ligase family, on interleukin-1 (IL-1) pathway in synoviocytes. The protein expression levels of Cbl-b and IL-1-induced matrix metalloproteinase 13 (MMP-13) in synoviocytes were analyzed by Western blot. Collagen substrates were incubated with the conditioned medium collected from synoviocytes cultures and then subjected to SDS-PAGE for analysis of collagen degradation. The results showed that compared with wild-type cells, Cbl-b-deficient cells expressed more MMP-13 protein and had enhanced ability to degrade collagens under IL-1 stimulation. These data suggest that Cbl-b may negatively regulate IL-1-triggered degradation of collagen matrix in synoviocytes.

Research on application of determination of MMP-13 in osteoarthritis / 中国骨伤

Osteoarthritis (OA) is a complex chronic progressive disease attacked by biological and mechanical factors and a result from the anabolic and catabolic imbalance in chondrocyte, subchondral bone and extracellular matrix(ECM). Etiology and pathological of OA are not yet entirely clear. The degradation and destruction of collagen II caused by matrix metalloproteinase -13 (MMP-13) is considered the core factor in the occurrence and development of OA. The research of MMP-13 inhibitor provide ideas and methods for the treatment of OA. In this article,the role and determination of MMP-13 in OA and the development prospect of MMP-13 inhibitor in the treatment of OA research progress were reviewed.

Expression of methionine sulfoxide reductase in colorectal cancer stem cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of methionine sulfoxide reductase (MsrA) in colorectal cancer stem cells and its association with the tumorigenesis and progression of colorectal cancer.</p><p><b>METHODS</b>The CD133⁺/CD44⁺/ESA⁺ subpopulation of colorectal cancer cell line SW480 was obtained by magnetic activated cell sorting (MACS). The expression of MsrA, VEGF, MMP-13 and CXCR4 in the cancer cells, cancer stem cells and normal colon mucosa cells were detected using RT-PCR. The proliferation of colorectal cancer stem cells was evaluated with MTT assay.</p><p><b>RESULTS</b>The expression of MsrA was significantly higher in cancer stem cells than in the cancer cells and normal mucosa cells. Overexpression of MsrA inhibited the proliferation of colorectal cancer stem cells and down-regulated the expression of VEGF, MMP-13 and CXCR4.</p><p><b>CONCLUSIONS</b>MsrA suppresses the tumorigenesis and progression of colorectal cancer cells possibly by inhibiting cell proliferation and down-regulating VEGF, MMP-13 and CXCR4.</p>

Effect of Rougan Huaqian granules combined with human mesenchymal stem cell transplantation on liver fibrosis in cirrhosis rats

OBJECTIVE@#To observe the effect of Rougan Huaqian granules combined with human mesenchymal stem cell (hMSC) transplantation on the liver fibrosis in carbon tetrachloride-induced cirrhosis rats.@*METHODS@#Sixty SD rats were randomly divided into five groups. The rats in control group received intraperitoneal injection of saline, while those in model control group, treatment group A, group B and group C received intraperitoneal injection of carbon tetrachloride oily solution to induce liver cirrhosis within 8 weeks. Then, the rats in the model control group, treatment group A, treatment group B, treatment group C received vein tail injection of saline, Rougan Huaqian granules, hMSC suspension and Rougan Huaqian granules combined with hMSC suspension.@*RESULTS@#The treatment groups had significantly different liver function (AST levels), liver fibrosis index (laminin and HA), hepatic sinusoidal wallsα-smooth muscle actin, IV collagen and laminin protein expression and I, III collagen from the model group (P<0.05). The transplanted cells showed human hepatocyte-like cells differentiation trend in the liver.@*CONCLUSIONS@#The Rougan Huaqian granules combined with hMSC transplantation can alleviate liver fibrosis in cirrhosis rats.

SKI306X inhibition of glycosaminoglycan degradation in human cartilage involves down-regulation of cytokine-induced catabolic genes

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.

Effect of Ermiao Recipe with medicinal guide Angelicae Pubescentis Radix on promoting the homing of bone marrow stem cells to treat cartilage damage in osteoarthritis rats / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Ermiao Recipe (, EMR) with medicinal guide Angelicae Pubescentis Radix (APR) on the homing of bone marrow stem cells (BMSCs) to focal zone in osteoarthritis (OA) rats.</p><p><b>METHODS</b>Forty-eight Sprague-Dawley rats were randomly assigned to the sham-operated, model, EMR, and EMR plus APR groups (12 rats in each group). The OA rat model was induced by anterior cruciate ligament transection and medial meniscus resection. All rats were injected with recombinant human granulocyte colonystimulating factor [rhG-CSF, 30 μg/(kg·d) for continuous 7 days], and rats in the EMR and EMR plus APR groups were treated with EMR or EMR plus APR at 1.6 or 1.9 g/(kg·d) for 3 or 6 weeks, respectively. Cartilage histopathologic changes were observed by hematoxylin and eosin staining. Chondrocytes apoptosis and cartilage matrix components were tested by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay and special staining. Interleukin-1β (IL-1 β), tumor necrosis factor α (TNF-α), bone morphogenetic protein 2 (BMP-2), and transforming growth factor beta-1 (TGF-β1) in serum were detected by enzyme-linked immunosorbent assay or radioimmunoassay assay. Matrix metalloproteinase (MMP)-13, tissue inhibitors of metalloproteinase (TIMP)-1, bromodeoxyuridine (BrdU), cluster of differentiation 34 (CD34), and stromal cell derived factor 1 (SDF-1) were measured by immunohistochemistry assay.</p><p><b>RESULTS</b>EMR and EMR plus APR significantly inhibited articular cartilage damage and synovium inflammation in OA rats at 3 or 6 weeks of treatment, the most obvious changes in these parameters were found in the EMR plus APR group. At 6 weeks, compared with EMR treatment, EMR plus APR remarkably inhibited chondrocytes apoptosis and the release of IL-1β and TNF-α, obviously decreased MMP-13 expression, and significantly increased expressions of proteoglycan, collagen, type II collagen and TIMP-1, serum levels of BMP-2 and TGF-β1 as well as expressions of BrdU, CD34 and SDF-1 in cartilage articular (P<0.01 or P<0.05).</p><p><b>CONCLUSION</b>The medicinal guide APR improved the therapeutic effects of EMR on OA rats by promoting directional homing of BMSCs to focal zone.</p>