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Objective:To investigate the impact of matrix metalloproteinase 13 (MMP13) and low-density lipoprotein receptor-related protein 1 (LRP1) on autophagy of articular chondrocytes in patients with Kashin-Beck disease (KBD).Methods:Human articular cartilage samples obtained from 4 KBD patients and 4 control subjects were collected from Shaanxi Institute for Endemic Disease Prevention and Control, and the expression levels of MMP13 and LRP1 in cartilage tissue were determined using immunohistochemistry (IHC). Chondrocytes were extracted and cultured in vitro, the mRNA and protein expression levels of LRP1 and the autophagy related genes [Beclin 1 (BECN1), microtubule associated protein 1 light chain 3 (LC3)], cartilage injury related genes [MMP13, caspase-3 (CASP3)], chondrocyte differentiation related genes [collagen type Ⅱ alpha 1 chain (COL2A1), and SRY-box transcription factor 9 (SOX9)] were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot (WB), respectively. Chondrocytes from 3 KBD patients were extracted, and MMP13 gene silencing experiment was performed by RNA interference (RNAi) technology, the mRNA and protein expression levels of the above genes were detected by qRT-PCR and WB, respectively. In addition, the antagonist receptor associated protein (RAP) of LRP1 was used to block the LRP1 of human normal chondrocytes (C28/I2 cells), and qRT-PCR and WB were used to detect the mRNA and protein expression levels of LRP1, chondrocyte autophagy, differentiation and cartilage injury related genes, respectively. Results:The IHC results showed that the expression levels of MMP13 (1.67 ± 0.21, 0.59 ± 0.15, 0.51 ± 0.12) in the surface, middle, and deep layers of cartilage tissue of KBD patients were significantly higher than those of control subjects (0.25 ± 0.03, 0.26 ± 0.04, 0.06 ± 0.01), and the differences were statistically significant ( t = - 11.38, P < 0.001; t = - 3.82, - 6.26, P = 0.019, 0.003). The expression levels of LRP1 (0.10 ± 0.02, 0.03 ± 0.01, 0.17 ± 0.03) were significantly lower than those of control subjects (1.63 ± 0.40, 0.44 ± 0.12, 0.34 ± 0.08), and the differences were statistically significant ( t = 6.61, 5.61, 3.64, P = 0.003, 0.005, 0.022). The mRNA and protein expression levels of MMP13, CASP3, SOX9 in chondrocytes of KBD patients were significantly higher than those of control subjects, and the differences were statistically significant ( P < 0.05). The mRNA expression levels of LRP1, LC3, COL2A1 were significantly lower than those of control subjects, and the differences were statistically significant ( P < 0.05). After silencing the MMP13 gene in chondrocytes of KBD patients, there were no significant differences in the mRNA and protein expression levels of LRP1, BECN1, LC3, CASP3, COL2A1, and SOX9 ( P > 0.05). After blocking LRP1 with RAP, the protein expression levels of LRP1, BECN1, LC3, MMP13, COL2A1 and SOX9 in chondrocytes were significantly lower than those in control group ( P < 0.05). Conclusions:There is no direct correlation between MMP13 and abnormal autophagy of articular chondrocytes in KBD patients. After blocking LRP1, the expression of the autophagy related genes BECN1 and LC3 in chondrocytes is decreased.
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Objective@#To explore the biological effects of electromagnetic pulse (EMP) with different high intensities on condylar cartilage in rats. @*Methods@#SD rats were randomly divided into a sham group (Sham) and an irradiation group (EMP1: 500 kV/m, 10 Hz; EMP2: 270 kV/m, 10 Hz). Then, they were sacrificed at 1 h, 3 h, 12 h, 24 h and 3 d after irradiation. The degree of cartilage degeneration was evaluated by HE, safranine O-fast green, type Ⅱ collagen immunohistochemistry and TUNEL staining. Immunohistochemistry and western blot were performed to detect the expression of the matrix degradation factors: matrix metalloproteinase-13 (MMP-13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-5) and the apoptosis key factor cleaved-cysteinyl aspartate specific proteinase (cleaved-Caspase3) in condylar cartilage. @*Results @#HE staining showed that, compared with the Sham group, a small amount of exfoliation was found on the fibrous surface layer of the cartilage after irradiation in the EMP1 and EMP2 groups. Compared with the Sham group, the percentage of safranine O-fast green-positive area decreased significantly at 12 h and 24 h (both P<0.01) in the EMP1 group and 12 h and 24 h in the EMP2 group (both P<0.05); the percentage of type Ⅱ collagen-positive area decreased significantly at 3 h and 12 h (P<0.05, P<0.001) in the EMP1 group. In addition, the number of TUNEL-positive apoptotic cells increased significantly at 1 h, 3 h, 12 h, and 24 h in the EMP1 group and 1 h, 3 h, and 12 h in the EMP2 group (P<0.05). Moreover, at different timepoints (except at 3 d) in the EMP1 group and EMP2 group, the percentage of MMP-13, ADAMTS-5- and cleaved Caspase3-positive chondrocytes and their protein levels in condylar cartilage increased significantly after irradiation (P<0.05). @* Conclusion@# EMP with a certain degree of high-intensity can induce early transient damage to condylar cartilage. This effect is dose-and time-dependent.
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BACKGROUND: The establishment of coculture system combined with physical factors and scaffold materials and the Induction of cytokines have become the focus of chondrogenlc differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To observe the effect of bone morphogenetic protein 7 combined with porous tantalum on chondrogenlc differentiation of bone marrow mesenchymal stem cells. METHODS: Bone marrow mesenchymal stem cells of Sprague-Dawley rats (provided by Beijing Huafukang Biology) were Isolated and cultured. Group Intervention: (1) in the experimental group, porous tantalum tablet was added, while In the control group, porous tantalum tablet was not added. At 5 days after culture, cell growth on the surface of porous tantalum tablet was observed by phalloidin staining. At 1, 3,5, and 7 days after culture, CCK-8 method was used to detect cell proliferation. (2) Group A was added with chondrocyte Inducer; group B with chondrocyte Inducer and bone morphogenetic protein 7; group C with domestic porous tantalum material and chondrocyte Inducer; group D with domestic porous tantalum material and chondrocyte Inducer and bone morphogenetic protein 7. At 7,14 and 21 days after culture, the levels of type II collagen, SRY type high mobility group protein and matrix metalloproteinase-13 secreted by cells In each group was detected by ELISA. Western blot assay was used to detect the expression of type II collagen, SRY type high mobility group protein and matrix metalloproteinase-13. This study was approved by the Animal Experimental Ethics Committee of North China University of Science and Technology. RESULTS AND CONCLUSION: (1) The phalloidin staining results showed that bone marrow mesenchymal stem cells grew well on and around the porous tantalum surface. (2) At 3 and 5 days after culture, the proliferation of bone marrow mesenchymal stem cells was slower In the experimental group than in the control group (P 0.05). (3) At 7,14 and 21 days, the expression of type II collagen and SRY high mobility group protein Increased gradually among groups A, B, C and D (P 0.05). At 21 days, there was no significant difference among groups A, B, C and D (P > 0.05). (4) Western blot assay showed that at 7,14 and 21 days after culture, the expression level of type II collagen and SRY high mobility group protein Increased gradually In groups A, B, C and D (P 0.05). (5) The results showed that bone morphogenetic proteln-7 combined with domestic porous tantalum could Induce cartilage differentiation of bone marrow mesenchymal stem cells, facilitate the expression of type II collagen and SRY high mobility group protein, and Inhibit the expression of matrix metalloproteinase-13.
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OBJECTIVE@#To investigate the effect of Modified Xiaochaihu Decoction (MXD, ) on collagen degradation in rats with chronic pancreatitis (CP).@*METHODS@#Rats were injected dibutyltin dichloride (DBTC, 7 mg/kg of body weight) into the right caudal vein to induce CP model. Thirty heallhy male Wistar rats were randomly divided into three groups by a random number table: the control, the model and the treatment groups. Rats of treatment group were administered MXD (10 g/kg of body weight) orally once daily starting from the day post-model establishment. Pancreatic tissues were harvested after 28-day feeding and fibrosis was evaluated by picro-sirius red staining. The contents of collagen type I and III were detected using enzymelinked immunosorbent assay (ELISA), the expression of matrix metalloproteinase 13 (MMP13) and tissue inhibitor of metalloproteinase 1 (TIMP1) was analyzed by Western blot and real-time polymerase chain reaction (PCR).@*RESULTS@#The fibrosis scoring of pancreatic tissues, the concentrations of collagen type I and III, the expression levels of MMP13 and TIMP1 proteins and mRNA in the model group were all increased compared with the control group (P0.05).@*CONCLUSIONS@#MXD could promote collagen degradation and reverse pancreatic fibrosis in CP rats via a mechanism involve up-regulation of MMP13 expression.
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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.
Subject(s)
Animals , Rats , Antirheumatic Agents , Chemistry , Pharmacology , Therapeutic Uses , Arthritis, Experimental , Drug Therapy , Pathology , Cell Movement , Cell Nucleus , Metabolism , Cells, Cultured , Gene Expression Regulation, Enzymologic , Interleukin-1beta , Pharmacology , Matrix Metalloproteinase 13 , Genetics , NF-kappa B , Genetics , Metabolism , Phosphorylation , Protein Transport , Signal Transduction , Synoviocytes , Metabolism , Transcriptional Activation , Triterpenes , Chemistry , Pharmacology , Therapeutic UsesABSTRACT
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.
Subject(s)
Animals , Rats , Antirheumatic Agents , Chemistry , Pharmacology , Therapeutic Uses , Arthritis, Experimental , Drug Therapy , Pathology , Cell Movement , Cell Nucleus , Metabolism , Cells, Cultured , Gene Expression Regulation, Enzymologic , Interleukin-1beta , Pharmacology , Matrix Metalloproteinase 13 , Genetics , NF-kappa B , Genetics , Metabolism , Phosphorylation , Protein Transport , Signal Transduction , Synoviocytes , Metabolism , Transcriptional Activation , Triterpenes , Chemistry , Pharmacology , Therapeutic UsesABSTRACT
Objective To preliminarily optimize the energy and density of laser radiation for the early treatment of hyperplastic scars in a rabbit ear model,and to explore possible therapeutic mechanisms.Methods Sixty-one hyperplastic scars were successfully established on the ears of 10 healthy New Zealand white rabbits with large ears,and randomly divided into 2 groups:1-week group (30 scars) and 3-week group (31 scars).These 2 groups were separately divided into 5 subgroups:group A treated with laser at a density of 100 PPA and an energy of 10 mJ,group B with laser at a density of 100 PPA and an energy of 50 mJ,group C with laser at a density of 169 PPA and an energy of 10 mJ,group D with laser at a density of 169 PPA and an energy of 50 mJ,and group E receiving no treatment.There were 6 scars in each group,except the group E in the 3-week group.Two healthy New Zealand white rabbits with large ears were not subjected to modeling,and served as group F (blank control group).Immunohistochemical study was performed to determine the expression of matrix metalloproteinase (MMP)-13 in the skin tissues from the rabbit ears 1 week after the treatment.Three weeks after the treatment,the skin tissues from the rabbit ears were subjected to hematoxylin-eosin (HE) staining and Masson staining.Then,the structure of scars was observed,and scar elevation index was calculated.Statistical analysis was carried out by Kruskal-Wallis H test for the comparison of scar elevation index,and by one-way analysis of variance (ANOVA) for the comparison of the average absorbance value of MMP-13.Results As HE staining revealed,the groups A,B,C and D all showed thicker dermis and increased number of collagen fibers compared with the group F (normal skin tissues),but showed thinner dermis,decreased number and more ordered arrangement of collagen fibers compared with the group E (untreated scar tissues).No obvious difference was observed in the thickness of the dermis among the groups A,B,C and D.The scar elevation index significantly differed among the 6 groups (H =22.757,P ＜ 0.05).Multiple comparisons showed that the scar elevation index was significantly lower in the groups B,C and D (2.597 ± 0.344,2.850 ± 0.282,2.658 ± 0.134,respectively)than in the group E (3.460 ± 0.583,all P ＜ 0.05).As Masson staining revealed,the groups A,B,C and D all showed thinner dermis and more irregular arrangement of collagen fibers compared with the group E.However,no obvious differences were observed in the dermal thickness or number of collagen fibers among the groups A,B,C and D.Immunohistochemical study showed that the expression of MMP-13 was significantly higher in the high-energy (50 mJ) laser groups than in the low-energy (10 mJ) laser groups (P ＜ 0.05) at the same laser density.With the same laser energy,the expression of MMP-13 was significantly higher in the group A than in the group C (P ＜ 0.01),but there was no significant difference between the group B and D (P ＞ 0.05).Conclusions Non-ablative fractional laser is effective for the treatment of early-stage hyperplastic scars.At the same laser density,50-mJ laser was superior to 10-mJ laser for the treatment of hyperplastic scars,likely because high-energy laser can stimulate the recombination of extracellular matrices and up-regulated MMP-13 expression to a greater extent.
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Objective:To investigate whether endogenous hydrogen sulfide (H2 S)was involved in the pathogenesis of osteoarthritis (OA)and its underlying mechanism,to detect H2 S and its synthases ex-pression in knee cartilage in patients diagnosed with different severity of OA,and to explore the transcrip-tion and expression of gene MMP-13 in chondrocytes treated with IL-1βor H2S.Methods:Synovial fluids of the in-patients with different severity of OA hospitalized in Peking University First Hospital were collected for measurement of H2 S content using methylene blue assay.Articular cartilages of the patients who underwent knee arthroplasty were collected for the cell culture of relatively normal chondrocytes.The chondrocytes were cultured to the P3 generation and H2 S molecular probes were used for detection of endogenous H2 S generation in the chondrocytes.Immunocytochemistry was used to detect the localization of H2 S synthases including cystathionine β-synthase (CBS),cystathionine-γ-lyase (CSE),and mercap-topyruvate sulfurtransferase (MPST)in OA chondrocytes.Western blot was used to quantify the protein expressions of CSE,MPST,and CBS in cartilage tissues of the patients who were diagnosed with OA and underwent knee arthroplasty.The relatively normal human chondrocytes were cultured to passage 3 and then divided into 4 groups for different treatments:(1 )the normal control group,no reagent was added;(2)the IL-1βgroup,5 μg/L of IL-1βwas added;(3)the IL-1β+H2S group,200 μmol/L of NaHS was added 30 min before adding 5 μg/L of IL-1β;(4)the H2 S group,200 μmol/L of NaHS was added. The transcription and expression of gene MMP-13 in chondrocytes of each group were determined with Real-time PCR and Western blot,respectively.And the total NF-κB p65 and phosphorylated NF-κB p65 in chondrocytes were detected with Western blot.Results:The content of H2 S in the synovial fluid of degenerative knee was (14.3 ±3.3)μmol/L.Expressions of endogenous H2 S and its synthases including CBS,CSE and MPST were present in the cytoplasm of chondrocytes.CSE protein expression in Grade 3 (defined by outerbridge grading)cartilage tissues was significantly increased as compared with that of Grade 1 cartilage tissues (1.67 ±0.09 vs.1.26 ±0.11,P<0.05).However,no significant difference of CBS or MPST expression among the different groups was observed.The expression of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (1 .87 ±0.67 vs.0.22 ± 0.10,P<0.05 ),and that in the IL-1β+H2 S group was significantly decreased than that in the IL-1βgroup (0.55 ±0.11 vs.1.87 ±0.67,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.The transcription of MMP-13 protein in the IL-1βgroup was significantly higher than that in the normal chondrocytes (31.40 ±0.31 vs.1.00 ±0.00,P<0.05), and that in the IL-1β+H2 S group was significantly decreased than that in the IL-1βgroup (24.41 ± 1.28 vs.31.40 ±0.31,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.The total NF-κB p65 in the IL-1βgroup was significantly higher than that in the normal chondrocytes (2.13 ±0.08 vs.0.73 ±0.08,P<0.05),and that in the IL-1β+H2S group was significantly decreased than that in the IL-1βgroup (1 .24 ±0.13 vs.2.13 ±0.08,P<0.05 ),and that in the H2 S group had no significant difference compared with that in the normal control group.The phosphorylated NF-κB p65 in IL-1βgroup was significantly higher than that in the normal chondrocytes (1.30 ±0.13 vs.0.19 ±0.04,P<0.05),and that in IL-1β+H2S group was significantly decreased than that in the IL-1βgroup (0.92 ±0.26 vs.1.30 ±0.13,P<0.05),and that in the H2S group had no significant difference compared with that in the normal control group.Conclusion:H2 S affected the cartilage degeneration by partly inhibiting the degradation of extracellular matrix.
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Objective To observe the effect of intra-articular injection of berberine on the expression of matrix metalloproteinase (MMP)-13,ADAMTS-5,Aggrecan and Collagen Ⅱ in the cartilage of rabbits with osteoarthritis.Methods Thirty male New Zealand White rabbits underwent bilateral anterior cruciate ligament transection (ACLT).On the basis of randomization one knee of each rabbit was treated with 0.3 ml 100 μmol/L berberine resolved in the normal saline (NS) (the experimental group) and the other knee was treated under the same schedule using NS (the placebo group) 4 weeks after transection,once a week for five weeks.Nine weeks after ACLT,all rabbits were killed and the knee joints were evaluated by histology and biochemistry.The mRNA expression of MMP-13,ADAMTS-5,Aggrecan and Collagen Ⅱ in the cartilage was analyzed using reverse transcription polymerase chain reaction (RT-PCR).All data were analyzed using Student's t test.Results Mankin histological evaluation showed that the extent and grade of cartilage damage in the experimental group (6.9±1.9) were less severe than the placebo group (9.3±1.2)(t=3.394,P＜0.01).The mRNA expression of MMP-13 and ADAMTS-5 decreased (160±54;166±47) (t=3.311,P＜0.01;t=2.651,P＜0.05) while that of Aggrecan (261±50) and Collagen Ⅱ (335±64) increased in cartilage compared to the placebo group (233±45;234±67;186±64;254±69),the differences were significant (t=2.941,P＜0.01;t=2.743,P＜0.05).The content of hydroxyproline (Hyp) and glycosaminoglycan (GAG) in cartilage [(23.5±2.8) μg/mg;(30±3) μg/mg] increased significantly in the experimental group (t=2.941,P＜0.01;t=2.743,P＜0.05) compared to the placebo group [(19.9±2.8) μg/mg;(27.4±2.9) μg/mg].Conclusion Berberine protects against cartilage degradation and inhibits the progression of osteoarthritis by suppressing MMP-13 and ADAMTS-5 expression.
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[ ABSTRACT] AIM:To investigate the effect of tripterygium hypoglaucum Hutch ( THH) on collagen-induced ar-thritis ( CIA) in rats and its possible mechanism.METHODS:SD rats were randomly divided into normal group and CIA group.The rat model of type II CIA was successfully established and the model rats were randomly divided into 4 different groups:model group, dexamethasone group, THH (200 mg/kg) group, and THH (400 mg/kg) group.The contents of IL-12, IL-23 and IL-37 in the serum and foot paws of the CIA rats were detected by ELISA.The histopathological changes of the skin of the food paws were observed by HE staining.The protein expression of MMP-13 was determined by Western blot.The MMP-13 activity in the foot paws was detected by fluorescence labeling method.RESULTS:Compared with CIA group, THH at dose of 400 mg/kg significantly reduced the weight loss in typeⅡCIA rats ( P<0.01 ) .THH at dose of 400 mg/kg obviously decreased the contents of IL-12 by 28.31%, IL-23 by 41.57%in the serum and IL-12 by 30.78%, IL-23 by 39.46%in the foot paws, while IL-37 was significantly increased by 79.43% in the serum and 75.78% in the foot paws ( P<0.01) .The pathological changes of the subcutaneous tissues were improved by treating with THH (400 mg/kg).The protein expression of MMP-13 was significantly decreased by 31.82%(P<0.01), and the MMP-13 activity was also reduced.THH at dose of 200 mg/kg had no obvious improvement on the above indexes.CONCLUSION:THH has significant inhibitory effect on rat CIA by reducing the content of proinflammatory cytokines IL-12 and IL-23, increasing the content of anti-inflammatory factor IL-37, inhibiting inflammatory cell infiltration and vascular proliferation, and attenuating the protein expression of MMP-13 and MMP-13 activity in rats.
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To investigate the effect of Tinospora crispa (T. crispa) extract on matrix metalloproteinase 13 (MMP-13) expression and cell migration. Methods: The cytotoxicity of T. crispa extract was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on head and neck squamous cell carcinoma (HNSCC) cell lines. The effect on expression of MMP-13 was analysed by RT-PCR and ELISA. The migration was assessed by wound healing assay. Results: MMP-13 mRNA was highly expressed in the metastatic human HNSCC cell lines, HN22 and HSC-3. T. crispa extract at a concentration of 100.0 μg/mL caused about 50% reduction of cell survival. T. crispa extract at a non-toxic concentration of 12.5, 25.0 and 50.0 μg/mL significantly suppressed MMP-13 mRNA expression and secreted MMP-13 in both HN22 and HSC-3. The expression of tissue inhibitors of metalloprotease by HSC-3 cells was attenuated by 25.0 and 50.0 μg/mL of T. crispa extract. Addition of the extract to cells in a wound healing assay showed inhibition of cell migration by HN22 cells. Conclusions: These data suggest that T. crispa could be considered as a potential therapeutic drug to prevent metastasis of HNSCC.
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Objective: To investigate the effect of Tinospora crispa (T. crispa) extract on matrix metalloproteinase-13 (MMP-13) expression and cell migration. Methods: The cytotoxicity of T. crispa extract was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on head and neck squamous cell carcinoma (HNSCC) cell lines. The effect on expression of MMP-13 was analysed by RT-PCR and ELISA. The migration was assessed by wound healing assay. Results: MMP-13 mRNA was highly expressed in the metastatic human HNSCC cell lines, HN22 and HSC-3. T. crispa extract at a concentration of 100.0 μg/mL caused about 50% reduction of cell survival. T. crispa extract at a non-toxic concentration of 12.5, 25.0 and 50.0 μg/mL significantly suppressed MMP-13 mRNA expression and secreted MMP-13 in both HN22 and HSC-3. The expression of tissue inhibitors of metalloproteinase-2 (TIMP-2) by HSC-3 cells was attenuated by 25.0 and 50.0 μg/mL of T. crispa extract. Addition of the extract to cells in a wound healing assay showed inhibition of cell migration by HN22 cells. Conclusions: These data suggest that T. crispa could be considered as a potential therapeutic drug to prevent metastasis of HNSCC.
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Background It is estimated that discoidin domain receptor 2 (DDR2) and matrix metalloproteinase-13 (MMP-13) play an important role in the development of tumor angiogenesis.However,their effects on choroidal neovascularizaiton (CNV) have not been clarified yet.Objective This study was to observe the expression pattern of DDR2 and MMP-13 in CNV and to further investigate the regulation role of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in CNV.Methods CNV models were established in 78 Brown Norway (BN) rats by retinal photocoagulation with 532 nm laser.Then the animals were randomly divided into the normal control group (n =7),the model control group (n =39),PD98059 (MEK1 inhibitor) group (n =16) and LY294002 (PI3K inhibitor) group (n =16),and 5 mmol/L PD98059 or 5 mmol/L LY294002 3 μl was intravitreally injected 1 day and 7 days after photocoagulation in the PD98059 group or LY294002 group.The expression of DDR2 and MMP-13 mRNA and proteins in the CNV area were detected by using reverse transcription PCR (RT-PCR),and the expression levels of p-ERK/ERK and p-Akt/Akt protein were detected by Western blot assay.CNV thickness was measured by pathological examination 14 days after photocoagulation,and the changes of CNV thickness,the expression levels of DDR2 and MMP-13 in CNV were compared among the model control group,PD98059 group and LY294002 group.Results Three days after photocoagulation,the cells within the lasered lesions proliferated,then CNV formed 7 days after photocoagulation and became stable 14 days after photocoagulation.Immunohistochemistry staining indicated that DDR2 was weakly expressed in the cells of ganglion cell layer,inner nuclear layer and vascular endothelial cells;while MMP-13 was strongly expressed in the cells of the inner limiting membrane layer,photoreceptor layer and sclera layer.Both DDR2 and MMP-13 were strongly expressed in CNV area.Double immunofluorescence staining revealed that MMP-13 and DDR2 co-expression in CNV area.RT-PCR revealed that the relative DDR2 mRNA levels at 1 day,3 days,7 days and 14 days after photocoagulation were 55.22±4.03,47.74±2.23,14.82±4.56 and 5.59±2.47 respectively,while the relative MMP-13 mRNA levels were 25.54±3.83,43.51±4.36,10.90±4.00 and 5.23±3.23 respectively.Compared with the normal control group,the expression of DDR2 and MMP-13 were significantly increased (all at P＜0.05).Immunofluorescence staining showed that the relative fluorescence unit (RFU) values at 1 day,3 days,7 days and 14 days after photocoagulation were 2.73±0.53,5.21±0.31 and 3.22±0.33 for DDR2 and 1.66±0.17,3.57±0.44,2.67±0.21 for MMP-13,respectively.The RFU values in the PD98059 group and LY294002 group 14 days after photocoagulation were 1.14±0.19,1.03±0.14 for DDR2 and 1.37±0.25,1.24±0.20 for MMP-13,respectively.Compared with the model control group,the differences were statistically significant (both at P＜0.05).Western blot results showed that,compared to the normal control group,the expression levels of p-ERK and p-Akt pretein increased at day 7 after photocoagulation (both at P＜0.05),and returned back to the baseline at day 14 after photocoagulation (both at P＞0.05).Both PD98059 and LY294002 treatment were able to attenuate the thickness of CNV to 57.21％ and 50.34％ at day 14 after photocoagulation and further decrease the expression levels of DDR2 and MMP-13 in CNV (all at P＜0.05).Conclusions The expressions of DDR2 and MMP-13 up-regulate in laser-induced CNV.MEK/ERK and PI3K/Akt pathways suppress the development of CNV by regulating the expression of DDR2 and MMP-13.
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Objective To observe the effect of interleukin-1β(IL-1β) on expression of Matrix Metalloproteinases 13 (MMP-13) in rat chondrocytes and its regulation of miR-27b. Methods Chondrocytes were extracted from 7 Wistar male rats. Expression of MMP-13 were examined by Western blot at 0 h, 24 h, 48 h after IL-1βstimulation. Differential miRNAs expression profiles were examined by miRNAs microarray. The most obviously down-regulated miRNAs were confirmed by quantitative Real-time PCR. Targeted regulation relationship between miR-27b and MMP-13 was set up by Luciferase re?porter gene experiments. Results Expression of MMP-13 in rat chondrocytes was increased at a timely dependent manner upon IL-1βstimulation(P<0.05);Microarray revealed 36 miRNAs whose expression changed, among which 6(miR-27b, miR-31, miR-26a, miR-26b, miR-23, miR-204)were especially obvious. Real-time PCR confirmed that miR-27b was the one whose expression level were most down-regulated. Transient co-transfection of miR-27b mimics with luciferase expres?sion plasmids resulted in significant repression of luciferase activity in rat chondrocytes (P < 0.05). Conclusion IL-1βstimulation result in down-regulation of miR-27b and up-regulation of MMP-13 expression. MiR-27b and MMP-13 show targeted regulation relationship.
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PURPOSE: The degradation of the extracellular matrix has been shown to play an important role in the treatment of hepatic cirrhosis. In this study, the effect of thalidomide on the degradation of extracellular matrix was evaluated in a rat model of hepatic cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in Wistar rats by intraperitoneal injection of carbon tetrachloride (CCl4) three times weekly for 8 weeks. Then CCl4 was discontinued and thalidomide (100 mg/kg) or its vehicle was administered daily by gavage for 6 weeks. Serum hyaluronic acid, laminin, procollagen type III, and collagen type IV were examined by using a radioimmunoassay. Matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1), and alpha-smooth muscle actin (alpha-SMA) protein in the liver, transforming growth factor beta1 (TGF-beta1) protein in cytoplasm by using immunohistochemistry and Western blot analysis, and MMP-13, TIMP-1, and TGF-beta1 mRNA levels in the liver were studied using reverse transcriptase polymerase chain reaction. RESULTS: Liver histopathology was significantly better in rats given thalidomide than in the untreated model group. The levels of TIMP-1 and TGF-beta1 mRNA and protein expressions were decreased significantly and MMP-13 mRNA and protein in the liver were significantly elevated in the thalidomide-treated group. CONCLUSION: Thalidomide may exert its effects on the regulation of MMP-13 and TIMP-1 via inhibition of the TGF-beta1 signaling pathway, which enhances the degradation of extracellular matrix and accelerates the regression of hepatic cirrhosis in rats.
Subject(s)
Animals , Male , Rats , Actins , Carbon Tetrachloride/toxicity , Collagen Type III/metabolism , Down-Regulation , Extracellular Matrix/metabolism , Immunohistochemistry , Immunosuppressive Agents/pharmacology , Liver Cirrhosis, Experimental/chemically induced , RNA, Messenger/analysis , Rats, Wistar , Thalidomide/pharmacology , Tissue Inhibitor of Metalloproteinase-1/biosynthesis , Transcription Factor RelA/biosynthesis , Transforming Growth Factor beta1/biosynthesis , Transforming Growth Factors/metabolismABSTRACT
Objective: To observe miRNA-140 expression in chondrocytes of patients with early osteoarthritis (OA)and the effect of transfecting double-stranded miR-140 (d-miRNA-140) on chondrocyte function. Methods: Normal and OA chondrocytes (4 weeks and 8 weeks) were collected from rabbit model of early OA (A, B and C group, respectively). Quantitative polymerase chain reaction was used to examine miRNA-140 expression and Western blotting analysis was used to examine Col2a1 and MMP-13 protein expression in each group. Col2a1 and MMP-13 protein expression was also observed in chondrocytes transfected with ds-miR-140. Results: Compared with group A, miR-140 expressions in group B and C were reduced to 63% and 57%, respectively (P0.05). Compared with group A, Col2a1 mRNA expressions in group B and C were reduced by 52% and 63%, respectively (P<0. 01); while MMP-13 mRNA expressions were up-regulated by 3. 01 and 4. 15 folds, respectively (P< 0. 01). Transfection with ds-miR-140 increased Col2a1 mRNA by 60% and 127% in group B and C, respectively(P<0. 01), and the expressions of MMP-13 mRNA in group B and C were reduced to 54. 53% and 42. 61%, respectively(P<0. 01). The changes of Col2a1 and MMP-13 protein expression were the same as that of mPNA. Conclusion: miR-140 expression is reduced in early OA cartilage, and transfection with ds-miR-140 may increase Col2a1 expression and reduce MMP-13 expression.
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Objective:To study the effects of rat interleukin-10 (rIL-10) gene treatment on the expression of collagen , matrix metalloproteinase 13(MMP13) and their specific inhibitors the tissue inhibitor of metalloproteinase 1(TIMP1) in porcine serum in-duced liver fibrosis rats then to explore the anti-fibrotic effect of rL-10.Methods:Thirty SD rats were divided into normal control and fibrosis model group.Normal control group (group C) was intraperitoneally injected with 0.5 ml normal sodium twice a week for 8 week, while the fibrosis model group was injected with equal volume of pig serum for 8 week.At the beginning of the 5th week, fibrosis model group was further randomly divided into a fibrosis model subgroup ( group M ) , rIL-10 gene treatment subgroup ( group I ) and empty vector control subgroup(group P).Rats in group C and M were injected with Ringer’s solution as a reagent control via the tail vein weekly, rats in group I were injected with the rIL-10 plasmid pcDNA3-rIL-10, and rats in group P were injected with empty vector pcDNA3.All rats were sacrificed at the end of 8th week, and the liver tissue samples were collected to observe deposition of collegan in liver tissue by sirius red staining and detected the expression of MMP 13 and TIMP1 in the liver tissue by SP immunohistochemistry .Re-sults:Sirius red staining showed that the area of the collegan deposition was dramatically increased in fibrosis model subgroup and emp -ty vector control subgroup compared with the normal control group , and the area of the collagen deposition was dramatically decreased in rIL-10 gene treatment subgroup compared with the fibrosis model and empty vector control subgroup .Immunohistochemistry analysis showed that the expression of MMP 13 and TIMP1 in fibrosis model subgroup and empty vector control subgroup was significantly higher than the normal control group , but compared with normal control group , expression of MMP13 was significantly increased and expres-sion of TIMP1 was significantly decreased in rIL-10 gene treatment subgroup .Compared with fibrosis model subgroup and empty vector control subgroup, the expression of MMP13 and TIMP1 was dramatically decreased in rIL-10 gene treatment subgroup.Conclusion:rIL-10 gene treatment attenuates the area of collagen deposition in liver fibrosis rats associated with downregulation of TIMP 1.
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Objective To investigate the effects of miR-10a expression on migration and invasion of human pancreatic cancer cells AsPC-1.Methods Small interfering RNA targeting at miR-10a (miR-10a-siRNA) was constructed,then it was transfected into pancreatic cancer AsPC-1 cells,and nonsense siRNA (Nc-siRNA) group and blank control group was established.Real time PCR assay was used to detect the expression of miR-10a in the 3 groups,and wound healing assay and Transwell assay were used to determine the migration and invasion abilities of cancer cells.The amount of matrix metalloproteinase-13 (MMP-13) in supernatant of cancer cell culture of each group was examined by ELISA assay.Results The miR-10a levels in control group,NC-siRNA group and miR-10a-siRNA group were 1.05 ±0.08,1.03 ±0.06,0.02 ±0.01 ; and the number of transmembrane cell were (150 ± 2.6),(145 ± 2.2),(62 ± 1.8),the levels of MMP-13 in the supernatant were (108.5 ± 2.8),(107.8 ± 2.5),(35.8 ± 1.5) pg/ml.The values were significantly lower in miR-10a-siRNA group than those in control group and NC-siRNA group (P ＜ 0.01).The distance of cultured clone in miR-10a treated cancer cells (736± 18 μm) was significantly longer than those in the controls (385 ±5 μm) and NC-siRNA group (395± 13 μm,P＜0.01).Conclusions Down-regulation of miR-10a by siRNA may inhibit migration and invasion of pancreatic cancer AsPC-1 cells,and the downregulated expression of MMP-13 may be one of the important mechanisms.
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Objective To find out the correlation between MMP-13 and clinicopathological parameters of breast cancer and identify clinical significance of MMP-13 overexpression on overall survival of breast cancer.Methods Immunohistochemistry was performed on paraffin-embedded tissue microarray containing 159 tissue dots from breast cancer patients.The intensity and the extent of IHC were scored by pathologists blind to clinicopathological parameters of the specimens.Different expression profiles of MMP-13 in breat cancer tissues and paraneoplastic tissues,and correlation between MMP-13 and breast cancer clinicopathological parameters were analyzed for statistical significance respectively.The impact of MMP-13 overexpression on overall survival of breast cancer.Results MMP13 expression were significantly higher in breast cancer tissues(54.4％) than in their corresponding paraneoplastic tissues(27.5％)(P =0.003).Expression of MMP-13 in breast cancer positively correlated with lymphma node metastasis(r =0.257,P =0.006),clinical TNM classification (r =0.310,P =0.001),HER2 expression (r =0.192,P =0.041).However,no significant correlation were oberserved between MMP-13 expression and tumor size,MMP-13 expression and tumor grade,MMP-13 expression and ER expression,MMP-13 expression and PR expression respectively.Conclusions Overexpress of MMP-13 is more common in breast cancer tissues than in their corresponding paraneoplastic tissues,and is an independent prognosis indicator of breast cancer.
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A 78-year-old male patient presented with double vision, painless palpable mass under the right superolateral orbital rim, downward displacement and restricted adduction of the right eye. His visual acuity was 20/50 OD and 20/20 OS. Hertel exophthalmometry was 21 mm OD and 17 mm OS. Computed tomographic scans showed an infiltrative orbital mass with ill-defined, irregular margins, involving the lacrimal gland and the lateral rectus muscle. The patient underwent an anterior transcutaneous transseptal orbitotomy with incisional biopsy and surgical debulking. Histopathologic evaluation revealed primary ductal adenocarcinoma of the lacrimal gland. Following the metastatic work up, he underwent an eyelid-sparing orbital exenteration. Microscopically, the tumor elements were characterized by large polygonal cells with vesicular nuclei, prominent nucleoli and amphophilic cytoplasm. The tumor components comprised duct-type structures with papillary and cribriform patterns, surrounded by prominent basement membrane. The tumor cells were positive for cytokeratin-7, matrix metalloproteinase (MMP)-2, MMP-9, MMP-13 and proto-oncogene Her-2/neu, but negative for cytokeratin-5, cytokeratin-20, p63, prostate-specific antigen, S-100 protein and thyroid transcription factor. These histopathologic findings were compatible with poorly differentiated ductal adenocarcinoma of the lacrimal gland, T3N0M0. Twenty-four months after orbital exenteration, the patient was diagnosed with ipsilateral parotid gland and cervical lymph node metastases and died of disease.
Paciente do sexo masculino e com 78 anos de idade apresentou diplopia, massa palpável abaixo da margem orbitária direita, deslocamento inferior do bulbo ocular direito e limitação da adução do olho direito. A acuidade visual foi 20/50 OD e 20/20 OE. A exoftalmometria de Hertel foi 21 mm OD e 17 mm OE. Tomografia computadorizada mostrou uma massa orbitária, infiltrativa e com margens irregulares, envolvendo a glândula lacrimal e o músculo reto lateral. O paciente foi submetido a uma orbitotomia anterior com biópsia incisional. O exame histopatológico revelou adenocarcinoma ductal primário da glândula lacrimal. Em seguida, o paciente foi submetido a uma exenteração orbitária com preservação das pálpebras. Microscopicamente, os elementos tumorais foram caracterizados por células poligonais grandes com citoplasma anfofílico, núcleo vesicular e nucléolo proeminente. Os componentes tumorais incluíram estruturas ductais com padrões cribriforme e papilífero e cercadas por membrana basal proeminente. As células tumorais foram positivas para citoqueratina 7, metaloproteinase 2 da matriz, metaloproteinase 9 da matriz, metaloproteinase 13 da matriz e Her-2/neu, mas negativas para citoqueratina 5, citoqueratina 20, p63, antígeno prostático específico, proteína S-100 e fator de transcrição da tiroide. Estes achados histopatológicos foram compatíveis com o diagnóstico de adenocarcinoma ductal pouco diferenciado da glândula lacrimal, T3N0M0. Vinte e quatro meses após a exenteração orbitária, o paciente foi diagnosticado com metástases nos linfonodos cervicais ipsilaterais e na glândula parótida ipsilateral e faleceu.