Correlation between the expression levels of tissue inhibitor of metalloproteinases 1 and drosophila mothers against DDP homolog 4 and postoperative recurrence and malignant transformation in vocal cord precancerous lesions / 中国耳鼻咽喉头颈外科

OBJECTIVE To investigate the correlation between the expression levels of tissue inhibitor of metalloproteinases 1(TIMP-1)and drosophila mothers against DDP homolog 4(Smad4)in vocal cord precancerous lesions and postoperative recurrence and malignant transformation.METHODS The clinical and pathological data of 162 patients with vocal cord precancerous lesions admitted to the First Affiliated Hospital of Zhengzhou University from August 2018 to August 2021 were retrospectively analyzed.The expression of TIMP-1 and Smad4 in the surgically removed precancerous tissues(precancerous lesion group)and adjacent normal mucosal tissues(control group)were detected by immunohistochemical method.The relationship between the positive rate of TIMP-1 and Smad4 and clinicopathological features was analyzed.Kaplan-Meier method and Cox regression analysis were used to analyze the effect on postoperative recurrence and malignant transformation.RESULTS Compared with the normal mucosa of the control group,the positive rate of TIMP-1 was higher and the positive rate of Smad4 was lower in the precancerous lesion group(P<0.05).The positive rates of TIMP-1 and Smad4 in patients with different lesion ranges,anterior commissure involvement and different degree of epithelial dysplasia were different(P<0.05).Postoperative follow-up lasted from 24 to 60 months,with a median follow-up time of 36 months.During the follow-up,6 patients were lost to follow-up,with a follow-up rate of 96.30%(156/162).During the follow-up,35 patients had postoperative recurrence(21.60%)and 16 patients had postoperative malignant transformation(9.88%).Kaplan-Meier survival analysis showed that the postoperative recurrence rate and malignant change rate of TIMP-1 positive patients were higher than those of TIMP-1 negative patients(P<0.05),amd the recurrence rate and malignant change rate of Smad4-negative patients were higher than those of Smad4-positive patients(P<0.05).Multivariate Cox regression analysis showed that laryngeal reflux,lesion scope>1/2,moderate/severe dysplasia,TIMP-1 positive and Smad4 negative were independent risk factors for recurrence(P<0.05),and age>60 years old,anterior union involved,TIMP-1 positive and Smad4 negative were independent risk factors for malignant transformation(P<0.05).CONCLUSION The patients with high expression of TIMP-1 and low expression of Smad4,positive expression of TIMP-1 and negative expression of Smad4 have higher risk of postoperative recurrence and malignant transformation.

Effects of recombinant human collagen supplementation on extracellular matrix remodeling in mouse skeletal muscle after eccentric exercise / 中国组织工程研究

BACKGROUND:Collagen is the most abundant extracellular matrix component,which is closely related to the structure and function of the extracellular matrix of skeletal muscle,but the effect and mechanism of recombinant human collagen(rhC)produced by bioengineering technology on the extracellular matrix of skeletal muscle are unclear. OBJECTIVE:To investigate the effect of rhC supplementation on the remodeling of skeletal muscle extracellular matrix after eccentric exercise,thereby revealing the possible mechanism by which rhC improves the injury of skeletal muscle extracellular matrix and promote the recovery after exercise. METHODS:A total of 104 healthy male C57 mice aged 8 weeks old were randomly divided into control group(normal saline),low-dose rhC group(0.2 g/kg),medium-dose rhC group(1.0 g/kg),and high-dose rhC group(2.0 g/kg).Two mice in each group were selected after continuous 7 days of intragastric intervention,and organs were dissected for hematoxylin-eosin staining to determine inflammatory infiltrates.On the 8th day,the remaining mice were subjected to eccentric exercise.The structural changes of the skeletal muscle extracellular matrix were observed under scanning electron microscopy immediately(0),24,48,and 96 hours after eccentric exercise.Meanwhile,grip strength,creatine kinase activity,and protein levels of matrix metalloproteinases 2,9,14 and tissue inhibitor of metalloproteinase-2 in skeletal muscle were detected by western blot assay. RESULTS AND CONCLUSION:Hematoxylin-eosin staining results indicated that short-term rhC supplementation showed no significant effects on the morphology of the heart,liver,spleen and kidney.After one-time eccentric exercise,the recovery rate of grip strength in the medium-and high-dose rhC groups was significantly increased(P＜0.01).The trend of creatine kinase changes was consistent in all groups and there was no significant difference between groups.The recovery process of the extracellular matrix in the low-dose rhC group was faster than that in the control group,and the muscle tract membrane in the medium-and high-dose rhC groups was more complete.The protein level of matrix metalloproteinase 9 in the high-dose rhC group was significantly decreased(P＜0.05).The protein levels of matrix metalloproteinase 14 in the medium-and high-dose rhC groups were significantly decreased(P＜0.05).The protein levels of matrix metalloproteinase 2 in the medium-and high-dose rhC groups were significantly decreased(P＜0.05).Tissue inhibitor of metalloproteinase-2 protein levels in the medium-and high-dose rhC groups were significantly increased(P＜0.05).The ratio of matrix metalloproteinase 2 to tissue inhibitor of metalloproteinase-2 in each rhC group was significantly decreased(P＜0.05).To conclude,pre-supplementation of 1.0 and 2.0 g/kg rhC for 7 days can inhibit extracellular matrix degradation in skeletal muscle after exercise by modulating matrix metalloproteinases and matrix metalloproteinase inhibitors,thereby promoting recovery of skeletal muscle strength in mice.

Comparative evaluation of skin suture in rats with polyglycaprone 25 and nylon / Avaliação comparativa da sutura de pele com poliglicaprone 25 e nylon em ratos

ABSTRACT Currently, the market offers a wide variety of suture threads, made of materials with different structural and chemical properties. Among many other characteristics, they vary in origin, absorption or degradation, and structure. From this variety, the clinical doubt arises as to which material provides the patient with the best healing quality. Objective: This study aims to comparatively evaluate two different types of suture threads-Monocryl® (polyglycaprone 25) and Ethilon® (nylon)-regarding their ability to aid in tissue regeneration by a histological and immunohistochemical analysis of the skin of rats sutured with the aforementioned materials. Methods: This basic experimental study used 12 adult Wistar rats, randomly divided into three groups with four animals each and subjected to four longitudinal incisions under anesthesia. Each group corresponded to a postsurgical evaluation date (one, seven, and 14 days). Results: At 14 postoperative days, the studied groups had no histological difference. However, the use of nylon thread showed greater evidence of earlier fibrotic union. Conclusion: This study found no histological difference in healing 14 days after surgery among the techniques and the types of suture threads. Level of Evidence II, Therapeutic Studies.

RESUMO Atualmente, encontra-se disponível no mercado uma grande variedade de fios de sutura, compostos de materiais com diferentes propriedades estruturais e químicas, que variam quanto à origem, absorção ou degradação e estrutura, entre outras características. A partir dessa disponibilidade, emerge a dúvida clínica quanto ao material que propicia a melhor qualidade de cicatrização ao paciente. Objetivo: Avaliar comparativamente dois tipos de fios - Monocryl ® (poliglicaprone 25) e Ethilon ® (nylon) - quanto à sua capacidade de auxílio na regeneração tecidual, por meio da análise histológica e imuno-histoquímica da pele de ratos submetidos a suturas com esses materiais. Métodos: Neste estudo básico experimental, foram utilizados 12 ratos adultos da linhagem Wistar, randomicamente divididos em três grupos com quatros animais cada, que foram submetidos a quatro incisões longitudinais sob anestesia. Cada grupo correspondeu a uma data de avaliação pós-cirúrgica (1, 7 e 14 dias). Resultados: Passados 14 dias após a operação, não houve diferença histológica em relação aos grupos estudados. No entanto, o uso de fio de nylon apresentou evidência de união fibrótica mais precoce. Conclusão: Não há diferença histológica de cicatrização após 14 dias pós-operatórios entre as técnicas e os tipos de fio de sutura. Nível de Evidência II, Estudos Terapêuticos.

Regulation of liver fibrosis by matrix metalloproteinase/tissue inhibitor of metalloproteinase and research advances in related therapeutic drugs / 临床肝胆病杂志

Liver fibrosis is the common consequence of various chronic liver injuries and is mainly characterized by the imbalance between the production and degradation of extracellular matrix, which leads to the accumulation of interstitial collagen and other matrix components. Matrix metalloproteinases (MMPs) and their specific inhibitors, i.e., tissue inhibitors of metalloproteinases (TIMPs), play a crucial role in collagen synthesis and lysis. Through a literature review, this article reviews the experimental studies of liver fibrosis based on MMPs/TIMPs, summarizes the components that may exert an anti-liver fibrosis effect by affecting the expression or activity of MMPs/TIMPs, and attempts to clarify the mechanism of MMPs/TIMPs in regulating collagen homeostasis, so as to provide support for the development of anti-liver fibrosis drugs.

Research progress of urine TIMP-2×IGFBP-7 in early diagnosis, risk stratification and prognosis of acute kidney injury / 中华检验医学杂志

Acute kidney injury (AKI) is a common complication in critically ill patients with complex etiology and high morbidity, which is closely related to the patient's mortality rate, hospital stay and long-term poor outcomes. Therefore, timely detection of AKI in the early reversible stage is particularly important to prevent its progression to renal failure and initiating renal replacement therapy. Therefore, exploring the relevant biomarkers in the occurrence and development of acute kidney injury has important clinical significance for the diagnosis and treatment of the disease. Tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor-binding protein-7 (IGFBP-7) are used as cell cycle arrest proteins, which has shown certain advantages in the early diagnosis, risk stratification, prognosis judgment, and treatment effect of acute kidney injury. Cell cycle arrest protein [TIMP-2]×[IGFBP-7] plays a role in acute kidney injury caused by various reasons and can be used as a reference index for disease prognosis.

A Redução do Colágeno Tipo I está Associada ao Aumento da Atividade da Metaloproteinase-2 e da Expressão Proteica de Leptina no Miocárdio de Ratos Obesos / Decreased Collagen Type I is Associated with Increased Metalloproteinase-2 Activity and Protein Expression of Leptin in the Myocardium of Obese Rats

Resumo Fundamento A obesidade é um fator de risco para complicações médicas, incluindo o sistema cardiovascular. Há informações limitadas sobre o colágeno no coração obeso. Nosso estudo anterior demonstrou uma redução dos níveis proteicos de colágeno miocárdico tipo I em ratos obesos alimentados com uma dieta com alto teor de gordura durante 34 semanas. No entanto, os mecanismos responsáveis pelos níveis baixos não estão completamente elucidados. Objetivo O objetivo deste estudo foi testar a hipótese de que a redução do colágeno tipo I está associada ao aumento da atividade da metaloproteinase-2 (MMP-2), a qual está ligada à elevação de leptina no miocárdio de ratos obesos. Métodos Ratos Wistar machos com 30 dias de idade foram randomizados em dois grupos: controle (dieta padrão) e obeso (dieta com alto teor de gordura), e alimentados durante 34 semanas. Foram avaliados as características gerais dos animais e os perfis metabólicos e endócrinos. Foram avaliados as expressões proteicas miocárdicas de colágeno tipo I, leptina e inibidores teciduais de metaloproteinases (TIMP), bem como a atividade da MMP-2. O teste de correlação de Pearson foi aplicado para determinar as associações entre variáveis. O nível de significância foi de 5%. Resultados Os animais obesos apresentaram índice de adiposidade mais elevado em comparação ao controle. Foram observadas comorbidades como intolerância à glicose, hiperinsulinemia, resistência à insulina, hiperleptinemia e hipertensão nos ratos obesos. A obesidade reduziu o colágeno tipo I, TIMP-1 e TIMP-2, e aumentou a leptina e a MMP-2 no miocárdio. Houve uma correlação negativa entre o colágeno tipo I e a MMP-2 e uma correlação positiva entre a leptina e a MMP-2. Conclusão Foi confirmada a hipótese de que a redução do colágeno tipo I está associada ao aumento da atividade da MMP-2 e da expressão de leptina no miocárdio de ratos obesos. (Arq Bras Cardiol. 2020; 115(1):61-70)

Abstract Background Obesity is a risk factor for medical complications, including the cardiovascular system. There is limited information on collagen in the heart in obesity. Our previous study showed decreased protein levels of myocardial collagen type I in obese rats fed a high-fat diet for 34 weeks. However, the mechanisms responsible for low levels are not fully elucidated. Objective The purpose of this study was to test the hypothesis that the reduction in collagen type I is associated with increased metalloproteinase-2 (MMP-2) activity, which is linked to elevated leptin in the myocardium of obese rats. Methods Thirty-day-old male Wistar rats were randomized into two groups, control (standard diet) and obese (high-fat diet), and fed for 34 weeks. The general animal characteristics and metabolic and endocrine profiles were evaluated. Myocardial protein expressions of collagen I, leptin, tissue inhibitors of metalloproteinases (TIMP), and MMP-2 activity were assessed. Pearson correlation was employed to determine the associations between variables. The level of significance was 5%. Results The obese animals had increased adiposity index compared to control. Comorbidities such as glucose intolerance, hyperinsulinemia, insulin resistance, hyperleptinemia, and hypertension were observed in obese rats. Obesity reduced collagen I, TIMP-1, and TIMP-2, and it increased leptin and MMP-2 in the myocardium. There was a negative correlation between collagen I and MMP-2 and a positive correlation between leptin and MMP-2. Conclusion The hypothesis was confirmed; the reduction in collagen type I is associated with increased MMP-2 activity and leptin expression in the myocardium of obese rats. (Arq Bras Cardiol. 2020; 115(1):61-70)

Effects of quercetin on the dentin resistance to erosion / 中华口腔医学杂志

Objective@#To evaluate the effects of quercetin on dentin resistance to erosion and provide evidence-based recommendations for the prevention and therapy of dental erosion.@*Methods@#One hundred and twenty-eight dentin samples were prepared from 50 extracted human wisdom teeth (collected from Department of Oral Surgery, School and Hospital of Stomatology). Ninety-six samples were randomly divided into 8 groups using the following different soaking solutions: deionized water, ethanol (control groups), 12.300 mg/L sodium fluoride, 0.120 mg/L chlorhexidine, 0.183 mg/L epigallocatechin gallate (EGCG), and 0.075, 0.150 and 0.300 mg/L quercetin. In each group, twelve specimen was prepared. Before daily acid challenge, the samples were immersed in the respective solutions for 2 min, rinsed with deionized water, and immersed in artificial saliva for 2 h. The samples were then subjected to 4 cycles of in vitro acid challenges. This protocol was applied for 7 d. The surface microhardness (SMH) and surface profiles were measured before and after erosion using the surface microhardness tester and contact profilometry, respectively. The change in surface profiles and reduction in SMH were used to calculate the substance loss and reduction percentage of SMH (SMH%) respectively. Scanning electron microscope (SEM) images were taken to observe the surface morphology of the samples. Additionally, another thirty two samples were divided into 8 groups (n=4) as mentioned above. The specimens were treated with 10% phosphoric acid and desiccated, immersed in the respective solutions for 2 min, rinsed, and immersed in the artificial saliva at 37 ℃ for 7 d. The content of cross-linked carboxyterminal telopeptide of type Ⅰ collagen (ICTP) in the soaking solutions were measure quantitatively.@*Results@#Compared with the control groups, the application of chlorhexidine, quercetin, and EGCG were effective in preventing the surface softening and substance loss of human dentin after erosion. More specifically, the specimens treated with 0.300 mg/L quercetin exhibited the lowest SMH% [(8.75±4.95)%], the lowest surface substance loss [(2.26±1.16) μm], and the lowest contents of ICTP in the soaking solution [(5.72±0.88) ng], showing significant differences to the chlorhexidine and EGCG treated samples (P<0.05). No significant differences were found in the substance loss and ICTP contents in the three soaking solutions with different concentrations of quercetin(P>0.05). However, the specimens treated with 0.300 mg/L quercetin exhibited significantly lower SMH% than those treated with the other two concentrations of quercetin (P<0.05).@*Conclusions@#Within the limitations of the current study, immersion in the quercetin solution is effective in improving the dentin resistance to erosion by inhibiting the dentinal MMP. Among all the concentrations tested, 0.300 mg/L quercetin showed the best performance.

Clinical value of matrix metalloproteinase-3 in rheumatic immune disease / 中华检验医学杂志

Rheumatic immune disease is a kind of disease caused by the abnormal of autoimmune system, the pathogenesis is complex and affected by many factors. Matrix metalloproteinases are a kind of proteolytic enzyme that can degrade extracellular matrix components, while the matrix metalloproteinase3 is the main enzyme that promote their degradation. In recent years, studies have found that matrix metalloproteinase-3 participates in the development of rheumatic immune disease. It is a potential marker not only can reflect the activity of some diseases, but also to evaluate therapeutic effect.

Effect of Anluohuaxianwan on the expression of matrix metalloproteinases and their inhibitors in rat liver with fibrosis / 中华肝脏病杂志

Objective@#To investigate the effect of anluohuaxianwan (ALHXW) using rat model of carbon tetrachloride (CCl4) induced liver fibrosis on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).@*Methods@#Thirty-six male Wistar rats were randomly assigned into control, model and treatment groups. Rats in the model and treatment groups were injected intraperitoneally with 40% CCl4 (2 ml/kg), and the control group were given isotonic saline twice a week for six weeks. Meanwhile, the treatment group were gavaged with ALHXW solution daily (concentration 0.15 g/ml, 9.9 ml/kg) for 6 weeks, while the control and model groups were given isotonic saline once a day for 6 weeks. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured at the end of third and sixth week. At the end of six weeks, liver tissues were harvested for histopathological evaluation and the detection of mRNA and protein expression levels of MMP-2/13 and TIMP-1/2. According to different data, LSD method, parametric (one-way ANOVA) and non-parametric tests (Kruskal-Wallis H-test and Mann-Whitney U test) were used for statistical analysis.@*Results@#Compared with the model group, ALHXW markedly alleviated liver injury in the treatment group, and thereby improved the general state of rats, liver and spleen morphological characteristics, and ALT and AST levels. Histopathological examination demonstrated that the extent of liver fibrosis was improved (2.75 ± 0.75 vs. 3.55 ± 0.69, P = 0.015) in the treatment group as compared with the model group. The mRNA and protein expression levels of MMP-13 in the treatment group were significantly higher than that of the model group (mRNA: 10.50 ± 7.64 vs. 4.40 ± 2.97, P = 0.029. Protein: 1.15 ± 0.09 vs. 0.78 ± 0.21, P = 0.016), whereas the mRNA and protein expression levels of MMP-2, TIMP-1/2 in the treatment group were significantly lower than that of the model group (mRNA: 4.55 ± 3.29 vs. 7.83 ± 4.19, P = 0.048; 1.66 ± 0.73 vs. 3.69 ± 2.78, P = 0.023; 2.25 ± 1.16 vs. 3.41 ± 1.51, P = 0.049; respectively. Protein: 0.44 ± 0.11 vs. 0.65 ± 0.05, P = 0.03; 0.69 ± 0.06 vs. 1.07 ± 0.21, P = 0.016; 0.46 ± 0.09 vs. 0.81 ± 0.13, P = 0.003; respectively).@*Conclusion@#ALHXW exerts anti-liver fibrosis effects mainly by improving liver function, inhibiting the activation of hepatic stellate cells, enhancing the expression of MMP-13, and inhibiting the expression of MMP-2 and TIMP-1/2.

Clinical value of matrix metalloproteinase-3 in rheumatic immune disease / 中华检验医学杂志

Rheumatic immune disease is a kind of disease caused by the abnormal of autoimmune system, the pathogenesis is complex and affected by many factors. Matrix metalloproteinases are a kind of proteolytic enzyme that can degrade extracellular matrix components, while the matrix metalloproteinase3 is the main enzyme that promote their degradation. In recent years, studies have found that matrix metalloproteinase-3 participates in the development of rheumatic immune disease. It is a potential marker not only can reflect the activity of some diseases, but also to evaluate therapeutic effect.

Expressions of TOLL-like receptor 4 (TLR-4) and matrix metalloproteinase 9 (MMP-9)/Tissue inhibitor of metalloproteinase 1 (TIMP-1) in pulmonary blood vessels with chronic obstructive pulmonary diseases and their relationships with pulmonary vascular remodelling

SUMMARY OBJECTIVE: This study aims at investigating the expressions of TOLL-like receptor 4 (TLR-4) and matrix metalloproteinase 9 (MMP-9)/ tissue inhibitor of metalloproteinase 1 (TIMP-1) in pulmonary blood vessels with chronic obstructive pulmonary disease (COPD) and their relationships with pulmonary vascular remodelling (PVR). METHODS: 60 para-tumour tissues were divided into the COPD group and the control group (n=30); the inflammations, pulmonary artery wall area/total artery area (WA%), and wall thickness/vascular outer diameter (WT%) were compared. The expressions of TLR-4, MMP-9/TIMP-1, and PCNA in pulmonary vascular smooth muscle cells were detected, and their relationships with PVR were then analysed. RESULTS: The inflammations (1.6±0.8), WA% (44.0±6.4), and WT% (27.3±3.3) in the COPD group were higher than in the control group (0.3±0.5, 26.1±2.8, 15.6±1.8), and the expressions of TLR-4 (31.4±147) and MMP-9/TIMP-1 (2.2±2.6) were increased compared to the control group (4.7±4.5, 1.9±12). Correlation analysis: TLR-4 and MMP-9/TIMP-1 were positively correlated with the inflammations (r=0.18, P<0.01), WA% (r=0.68, P<0.01), and WT% (r=0.73, P<0.01), as well as positively correlated with the expression of PCNA (r=0.44, P<0.01); the upregulation of TLR-4 was positively correlated with the expressions of MMP-9 and TIMP-1. CONCLUSIONS: The upregulation of TLR-4 in the pulmonary arterial smooth muscle cells of COPD patients could promote the inflammations and the MMP-9 expression, thus causing abnormal degradation of extracellular matrix, so it played an important role in the process of PVR.

RESUMO OBJETIVO: Este estudo tem como objetivo investigar as expressões de TOLL-like receptor 4 (TLR-4) e metaloproteinase 9 da matriz (MMP-9)/inibidor de tecido da metaloproteinase 1 (TIMP-1) em vasos sanguíneos pulmonares com doença pulmonar obstrutiva crônica (DPOC) e suas relações com o remodelamento vascular pulmonar (PVR). MÉTODOS: Sessenta tecidos paratumorais foram divididos em grupo COPD e o grupo controle (n = 30). Foram comparadas as inflamações, área da parede da artéria pulmonar/área da artéria total (WA%) e espessura da parede/diâmetro externo vascular (WT%). As expressões de TLR-4, MMP-9/TIMP-1 e PCNA em células de músculo liso vascular pulmonar foram detectadas, e suas relações com PVR foram então analisadas. RESULTADOS: As inflamações (1,6 ± 0,8), WA% (44,0 ± 6,4) e WT% (27,3 ± 3,3) no grupo COPD foram maiores que no grupo controle (0,3 ± 0,5; 26,1 ± 2,8; 15,6 ± 1,8). E as expressões de TLR-4 (31,4 ± 14,7) e MMP-9/TIMP-1 (2,2 ± 2,6) foram aumentadas em relação ao grupo controle (4,7 ± 4,5, 1,9 ± 1,2). Na análise de correlação, TLR-4 e MMP-9/TIMP-1 foram positivamente correlacionadas com as inflamações (r = 0,18; P <0,01), WA% (r = 0,68; P <0,01) e WT% (r = 0,73; P <0,01), bem como correlacionadas positivamente com a expressão de PCNA (r = 0,44; P <0,01). A elevação da TLR-4 foi correlacionada positivamente com as expressões de MMP-9 e TIMP-1. CONCLUSÕES: A regulação positiva do TLR-4 nas células do músculo liso arterial pulmonar de pacientes com DPOC poderia promover as inflamações e a expressão de MMP-9, causando assim uma degradação anormal da matriz extracelular, por isso desempenhou um papel importante no processo de PVR.

Effect of magnesium-calcium alloy extract on matrix metalloproteinase-9 and matrix metalloproteinase inhibitor-3 in human colonic epithelial cells / 新乡医学院学报

Objective To investigate the effect of different concentrations of magnesium-calcium alloy extract on the expression of matrix metalloproteinase-9 (MMP9) and tissue inhibitor of metalloproteinase-3 (TIMP3) in human colonic epithelial NCM460 cells.Methods The different concentrations of extracts (the volume fraction was 10％,50％ and 100％ respectively) were made with magnesium-calcium alloy.The 5 × 106 L-1 NCM460 suspension was randomly divided into control group,experimental group 1,experimental group 2 and experimental group 3.The cells in the control group were cultured by 2 000 μL high glucose Dulbecco's modified Eagle's medium (containing 10％ volume fraction of fetal bovine serum).The cells in the experimental group 1,2 and 3 were cultured by 2 000 μL magnesium-calcium alloy extract with volume fraction of 10％,50％ and 100％ respectively.The expressions of MMP9 and TIMP3 mRNA in NCM460 cells was detected by real-time fluorescence quantitative polymerase chain reaction,and the expression of MMP9 and TIMP3 protein in NCM460 cells was detected by Western blot at after one,three and five days of cultivation respectively.Results The expression of MMP9 mRNA and TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly lower than that in the control group after one day of cultivation (P ＜ 0.05).After three and five days of cultivation,the expression of MMP9 mRNA in NCM460 cells of the experimental group 1 was significantly lower than that in the control group (P ＜ 0.05),but the expression of MMP9 mRNA in the NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the control group and the experimental group 1 (P ＜ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 3 was significantly higher than that in the experimental group 2 after five days of cultivation (P ＜ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 1,2 and 3 after three and five days of cultivation was significantly higher than that after one day of cultivation(P ＜ 0.05).There was no significant difference in the expression of MMP9 mRNA in NCM460 cells of the experimental group 1 between three and five days of cultivation (P ＞ 0.05).The expression of MMP9 mRNA in NCM460 cells of the experimental group 2 and 3 after five days of cultivation was significantly higher than that after three days of cultivation(P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the experimental group 1 after one day of euhivation (P ＜ 0.05).After three days of cultivation,the expression of TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly lower than that in the control group (P ＜ 0.05);the expression of TIMP3 mRNA in NCM460 cells of the experimental group 2 was significantly lower than that in the experimental group 1 and 3 (P ＜ 0.05).After five days of cultivation,the expression of TIMP3 mRNA in NCM460 cells of the experimental group 1,2 and 3 was significantly higher than that in the control group (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after three and five days of cultivation was significantly higher than that after one day of cultivation (P ＜ 0.05),and the expression of TIMP3 after five days of cultivation was significantly higher than that after three days of cultivation in the experimental group 1 (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after three days of cultivation was significantly lower than that after one day of cultivation (P ＜ 0.05),and the expression of TIMP3 after five days of cultivation was significantly higher than that after one and three days of cultivation in the experimental group 2 (P ＜ 0.05).The expression of TIMP3 mRNA in NCM460 cells after five days of cultivation was significantly higher than that after one and three days of cultivation in the experimental group 3 (P ＜ 0.05).After five days of cultivation,there was no significant difference in the expression of MMP9 protein in NCM460 cells between the experimental group 1 and control group (P ＞ 0.05),the expression of MMP9 protein in NCM460 cells of the experimental group 2 and 3 was significantly higher than that in the control group and the experimental group 1 (P ＜ 0.05),but there was no significant difference in the expression of MMP9 protein in NCM460 cells between the experimental group 2 and 3 (P ＞ 0.05).After five days of cultivation,the expression of TIMP3 protein in NCM460 cells of the experimental group 1,2 and 3 was significantly higher than that in the control group (P ＜0.05);but there was no significant difference in the expression of TIMP3 protein in NCM460 cells among the experimental group 1,2and 3 (P ＞ 0.05).Conclusions The high concentration of magnesium-calcium alloy extract has certain influence on the expression of MMP9 and TIMP3 gene in NCM460 cells,which may lead to the early inflammatory reaction,and the mechanism may be related to the calcium ion concentration in the extract.

Effect of tissue inhibitor of metalloproteinases-1 siRNA on endothelial cells injury induced by septic serum / 中华急诊医学杂志

Objective To investigate the effect and possible mechanism of tissue inhibitor of Metalloproteinases-l(TIMP-1) siRNA on human umbilical vein endothelial cells injury induced by serum of septic patient.Methods Serum samples were separately collected from septic patients and healthy controls.Human umbilical vein endothelial cells (HUVECs) were randomly divided into blank group (normal culture cells),control group (culture medium with 10％ control serum),septic group (culture medium with 10％ septic serum),negative control group (negative siRNA + 10％ septic serum),and TIMP-l siRNA group (TIMP-1 siRNA + 10％ septic serum).The survival rate of endothelial cells was detected by MTT assay.The levels of matrix Metalloproteinase-9 (MMP-9) and TIMP-1 in supernatant of culture medium were measured by enzyme-linked immunosorbent assay (ELISA).The levels of MMP-9,TIMP-1 and Thrombomoduline (TM) in endothelial cells were examined by Western blot.Results Compared with control group,the cell survival rate of septic group decreased 12 hours after the addition of serum (P＜0.05) and reached minimum 48 hours later.The levels of MMP-9 and TIMP-1 in supernatant of culture medium of septic group significantly increased (P＜0.01).The levels of MMP-9 and TIMP-1 increased in the septic group (P＜0.01),while the level of TM reduced at the same time in septic group (P＜0.01).Compared with septic group,the cell survival rate ofTIMP-1 siRNA group decreased (P＜0.05).The level of MMP-9 in supematant of culture medium of TIMP-1 siRNA group increased (P＜0.05),while the level of TIMP-1 decreased (P＜0.05).The level of MMP-9 increased in TIMP-1 siRNA group (P＜0.01),whereas the levels of TIMP-1 and TM reduced in TIMP-1 siRNA group (P＜0.01).Conclusions TIMP-1 plays a protective role in endothelial cells injury induced by septic serum.

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro / 中国病理生理杂志

AIM:To explore the effects of chloroquine (CQ) on collagen Ⅰ and collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS:Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h.The cells were divided into 5 groups as follows:control group,TGF-β1 group,TGF-β1 + CQ (15 μmol/L) group,TGF-β1 + CQ (30 μ mol/L) group and TGF-β1 + CQ (60 μmol/L) group.Western blot was used to determine the expression of LC3-Ⅱ/LC3-Ⅰ,P62 and o-SMA in activated HSC-T6 cells.The expression of collagen Ⅰ and collagen Ⅲ was detected by immunocytochemical staining,Western blot and RT-qPCR.Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13),tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS:The ratio of LC3-Ⅱ / LC3-Ⅰ and P62 expression were increased after CQ intervention.Moreover,they were significantly higher in the TGF-β1 + CQ groups than those in TGF-β1 group (P ＜ 0.01).The expression of collagen Ⅰ and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1 + CQ groups as compared with TGF-β1 group (P ＜0.01),and it was markedly increased among TGF-β1 + CQ groups in a dose-dependent manner.The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1 + CQ groups as compared with TGF-β1 group (P ＜0.05).CONCLUSION:Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen Ⅰ and collagen Ⅲ in a dose-dependent way,probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro / 中国病理生理杂志

AIM:To explore the effects of chloroquine (CQ) on collagen Ⅰ and collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS:Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h.The cells were divided into 5 groups as follows:control group,TGF-β1 group,TGF-β1 + CQ (15 μmol/L) group,TGF-β1 + CQ (30 μ mol/L) group and TGF-β1 + CQ (60 μmol/L) group.Western blot was used to determine the expression of LC3-Ⅱ/LC3-Ⅰ,P62 and o-SMA in activated HSC-T6 cells.The expression of collagen Ⅰ and collagen Ⅲ was detected by immunocytochemical staining,Western blot and RT-qPCR.Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13),tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS:The ratio of LC3-Ⅱ / LC3-Ⅰ and P62 expression were increased after CQ intervention.Moreover,they were significantly higher in the TGF-β1 + CQ groups than those in TGF-β1 group (P ＜ 0.01).The expression of collagen Ⅰ and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1 + CQ groups as compared with TGF-β1 group (P ＜0.01),and it was markedly increased among TGF-β1 + CQ groups in a dose-dependent manner.The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1 + CQ groups as compared with TGF-β1 group (P ＜0.05).CONCLUSION:Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen Ⅰ and collagen Ⅲ in a dose-dependent way,probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.

Effects of estrogen on the expression of MMP-2, TIMP-2 and TGF-β1 in human corneal stromal cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the effects of estrogen on the expression of matrix metalloproteinases-2(MMP-2), tissue inhibitor of metalloproteinases-2(TIMP-2) and transforming growth factor-β1(TGF-β1) in cultured human corneal stromal cells.METHODS: Inflammatory environments of human corneal stromal cells were simulated by using 1.5ng/mL IL-1β.The cells were then treated with or without different concentrations of estrogen(0, 1×10-4, 1×10-6, 1×10-8, 1×10-10mol/L estradiol)in vitro.Cell viability was evaluated by MTT.Expression levels of MMP-2, TIMP-2 and TGF-β1 proteins were measured by enzyme-linked immunosorbent assay(ELISA).RESULTS:Estrogen did not affect the viability of human corneal stromal cells.Compared with the control group, expression levels of MMP-2 and TGF-β1 proteins in E2 treatment group significantly decreased after being treated with estrogen, while the expression level of TIMP-2 significantly increased.CONCLUSION: Estrogen could, to some extent, down-regulate the expression of MMP-2 and TGF-β1 and up-regulate the expression of TIMP-2, which might contribute to protecting human cornea.

Relationship between matrix metalloproteinases and the occurrence and development of ovarian cancer

Ovarian cancer is one of the most malignant genital cancers, with a high mortality rate. Many researchers have suggested that matrix metalloproteinases (MMPs) have remarkably high expression in ovarian cancer tissues. MMPs are considered to be related to the occurrence, development, invasion and metastasis of ovarian cancer. Moreover, some studies have discovered that the unbalance between MMPs and tissue inhibitor of metalloproteinases (TIMPs) are associated with the malignant phenotype of tumors. This review summarizes the latest research progress of MMPs in ovarian cancer. The investigation of MMP mechanism in ovarian cancer will facilitate the development of effective anti-tumor drugs, and thereby improve the survival rate of patients with ovarian cancer.

Study on the carvacrol induced apoptosis in human non-small cell lung cancer cell line H1299 / 天津医药

Objective To study whether carvacrol can cause apoptosis in non-small cell lung cancer (NSCLC) cell line NCI-H1299, and explore its possible molecular mechanism. Methods NCI-H1299 cells were treated with different concentrations of carvacrol (20, 40, 60 and 80μmol/L) for 24 or 48 h. The viability of cells was evaluated by MTT assay, apoptosis was analyzed by flow cytometry (FCM) and the effect of carvacrol on metastasis of NCI-H 1299 was analyzed by Transwell assay. The expression level of caspase-9, MMP-9 and TIMP-1 were detected by quantitative realtime-PCR and Western blot assay. Results After treatment with carvacrol, the viability of NCI-H1299 cells was suppressed dramatically (P0.05). After being incubated with carvacrol for 24 h, FCM analysis indicated that carvacrol effectively induced apoptosis in NCI-H1299 cells (P0.05). The ability of invasion was decreased (40.67±3.63 vs. 76.00±5.78). Carvacrol inhibited the protein and mRNA expression levels of caspase-9, but increased the expression of MMP-9 and TIMP-1 (P<0.05). Conclusion Carvacrol can induce apoptosis of NCI-H1299 cells and inhibit their invasion, which may be associated with up-regulation of caspase-9 expression and down-regulation of MMP-9 expression.

TIMP-1 and Ang-1 gene-modified BMSCs transplantation to improve cardiac function of rats with myocardial infarction / 中华器官移植杂志

Objective To evaluate the effects of TIMP-1 and Ang-1 gene-modified BMSCs transplantation on the left ventricular function of rats with myocardial infarction.Methods The rat BMSCs were.transfected with eukaryotic expression plasmid encoding TIMP-1 or/and Ang-1 gene by liposome.Acute myocardial infarction was made in male rats by ligation of the left anterior descending (LAD) coronary artery.BMSCs carrying TIMP-1 or/and Ang-1 gene were injected into the ischemic myocardium after LAD ligatior.Four weeks after the administration,cardiac function was assessed by echocardiography and the hearts were harvested and sectioned for immunohistochemistry to examine the apoptosis,the collagen content and angiogenesis density.Results TIMP-1 and Ang-1 genemodified BMSCs transplantation significantly improved the cardiac function,myocardial apoptosis was alleviated,collagen content decreased and the angiogenesis density in border-zone was increased significantly (P＜0.05).Conclusions The results suggest that the combination of TIMP-1 and Ang-1-gene modified BMSCs transplantation can improve the cardiac function of rats with myocardial infarction.The increase of the blood supply,the alleviation of myocardial apoptosis and ventricle remolding after myocardial infarction possibly play important roles in the mechanism.

Role of metalloproteinases and their inhibitors in intervertebral disc degeneration: Research progress / 第二军医大学学报

Intervertebral disc degeneration (IVDD) is characterized by the excessive degradation of extracellular matrix (ECM), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs) and a disintegrin metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation. This review summarizes the current literatures on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVDD. Reports have showen that specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) are upregulated in human degenerated intervertebral discs. Tissue inhibitor of metalloproteinase-3 is downregulated and TIMP-1 is upregulated in human degenerated intervertebral discs relative to nondegenerated intervertebral discs. Regulation of the MMP and ADAMTS expression is affected by many factors including mechanical, inflammatory, oxidative stress, and so on. Genetic predisposition also plays an important role in expression of MMP-1, -2, -3, and -9. Upregulation of MMP and ADAMTS expression is implicated in ECM destruction, which can lead to the development of IVDD. Future IVDD therapeutics may target specific MMPs and ADAMTSs which is essential in the pathological proteolysis of ECM.