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【Objective】 To explore the effects of long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) on the invasion and migration of prostate cancer cells (PC3 cells) by regulating microRNA-181b-5p (miR-181b-5p)/tissue inhibitor of metalloproteinase 3 (TIMP3). 【Methods】 The prostate cancer tissues and adjacent tissues were collected from 20 prostate cancer patients treated in our hospital during Dec.2020 and Dec.2021. The expressions of MEG3 and miR-181b-5p in tissues were detected with quantitative real-time PCR (qRT-PCR). P3 cells were randomly divided into control group (untreated), pcDNA3.1-NC (transfected with pcDNA3.1-NC), pcDNA3.1-MEG3 group (transfected with pcDNA3.1-MEG), pcDNA3.1-MEG3+miR-NC group (pcDNA3.1-MEG3 co-transfected with miR-NC), pcDNA3.1-MEG3+miR-181b-5p mimic group (pcDNA3.1-MEG3 co-transfected with miR-181b-5p mimic). The expressions of MEG3 and miR-181b-5p in PC3 cells were detected with qRT-PCR. The cell viability, invasion and migration ability were determined with MTT assay, Transwell assay and scratch assay. The protein expressions of TIMP3, matrix metalloproteinase (MMP)9 and MMP2 in PC3 cells were detected with Western blot. The targeting relationship of MEG3, miR-181b-5p and TIMP3 was analyzed with dual luciferase assay. 【Results】 The expressions of MEG3 in prostate cancer tissues ( 0.37±0.05 vs. 1.00±0.04) and cells (0.31±0.06 vs. 1.00±0.01) were significantly decreased (P<0.05). Compared with the control group, the pcDNA3.1-MEG3 group had significantly decreased expression of miR-181b-5p (0.26±0.04 vs.1.00±0.02 ), cell survival rate (53.60±5.22 vs.100.00±0.00), number of invasive cells (62.33±9.85 vs.162.34±21.30), cell migration rate (32.85±3.80 vs.75.22±5.96), expressions of MMP9 (0.61±0.08 vs.1.62±0.23) and MMP2 (0.73±0.10 vs.1.20±0.16), but significantly higher expressions of MEG3 (2.31±0.36 vs. 1.00±0.01) and TIMP3 (1.32±0.24 vs. 0.53±0.08) (P<0.05). Overexpression of miR-181b-5p reversed the above changes (P<0.05). MiR-181b-5p had a targeting relationship with MEG3 and TIMP3. 【Conclusion】 Overexpression of lncRNA MEG3 can inhibit miR-181b-5p to promote the expression of TIMP3, thereby inhibiting invasion and migration of PC3 cells.
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Objective: To investigate the main mechanisms of pulmonary fibrosis following silica nanoparticles (SiNPs) exposure through constructing the macrophage-fibroblast model in vitro, which simulated the process of pulmonary fibrosis. Methods: In January 2021, human mononuclear leukemia cells (THP-1) were treated with 0, 25, 50, 100 μg/ml SiNPs for 24 h. The supernatant of THP-1 cells was collected and applied to human embryonic lung fibroblast cells (MRC-5) which divided into control and low, medium and high dose groups at the logarithmic growth stage for 24 h. MRC-5 cell viability was detected by CCK8. The hydroxyproline (Hyp), interleukin 6 (IL-6), interleukin 1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) expression were detected in the supernatants of MRC-5. The changed proteins were detected by liquid-phase mass spectrometry in high dose group. GeneCard database were applied to identity the differential pulmonary fibrosis proteins in high dose group. Gene Ontology (GO) was performed to identity the key biological process in differential pulmonary fibrosis proteins of high dose group. The String database was used to construct the protein-protein interactions (PPI) network of differential pulmonary fibrosis proteins. The APP of CytoHubba was applied to calculate the key protein of differential pulmonary fibrosis proteins in PPI network. Correlation coefficients between key differential pulmonary fibrosis proteins were calculated using Pearson correlation analysis. Western blotting was applied to detect the expression of key proteins of differential pulmonary fibrosis proteins in different groups. Results: CCK8 results showed that MRC-5 cell viability was increasing in low, medium and high dose groups compared with control group (P<0.05). The expression levels of Hyp and IL-1β in different group were increased compared with control group, the expression levels of IL-6 and TNF-α were increased in high dose group compared with control group (P<0.05). GeneCard database identified 26 differential pulmonary fibrosis proteins, which were mainly involved in extracellular matrix hydrolysis, cell inflammatory response, tissue repair, cell proliferation, inflammation response by GO analysis. The APP of CytoHubba was calculated that matrix metalloproteinase 9 (MMP9) and tissue inhibitor metalloproteinase 1 (TIMP1) played an important role in PPI network. The results of correlation analysis showed that MMP9 was correlated with the expression of matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 3 (MMP3), TIMP1 and epidermal growth factor receptor (EGFR) (r=0.97, 0.98, 0.94, 0.93, P<0.05). Western blotting results showed that TIMP1 protein expression was increased in low, medium and high dose groups, while MMP9 protein expression was increased only in high dose group (P<0.05) . Conclusion: Differential expression proteins related with pulmonary fibrosis in MRC-5 cells mainly regulate biological processes of extracellular matrix hydrolysis, tissue repair, and cellular inflammation response following SiNPs exposure. MMP9 and TIMP1 may be the key proteins, which affected the fibrosis process in vitro pulmonary fibrosis model.
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ObjectiveTo investigate the effect and mechanism of pachymic acid （PA） in Poria on the invasion and metastasis of renal carcinoma cells. MethodThe effect of PA （0， 20， 40， 80， 160 μmol·L-1） on cell viability was detected by cell counting kit-8（CCK-8）， and the dose of PA was selected for subsequent experiments. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell proliferation was evaluated by colony formation assay. The effect of PA （0， 20， 40， 80 μmol·L-1） on cell adhesion ability was observed by cell adhesion assay. The effect of PA （0， 20， 40， and 80 μmol·L-1） on cell invasion and metastasis was investigated by Wound healing assay and Transwell invasion assay. The inhibitory effect of PA （0， 20， 40， 80 μmol·L-1） on cell motility was further observed and verified by high-content imaging technology. The effects of PA （0， 20， 40， 80 μmol·L-1） on the expression of matrix metalloproteinase （MMP）/tissue inhibitor of metalloproteinasas （TIMP） related to invasion and metastasis and Smads were detected by Western blot. ResultCCK-8 results showed that compared with the blank group， the PA groups showed decreased cell viability（P<0.01）， with the half-maximal inhibitory concentration （IC50） of ACHN cells of 70.42 μmol·L-1 at 24 h. Colony formation assay showed that the number of cell clonal groups in the PA groups was reduced compared with that in the blank group（P<0.01）. Cell adhesion assay showed that compared with the blank group， the PA groups displayed reduced cell adhesion（P<0.01）. Wound healing assay showed that the wound healing rate of cells in the PA groups was lower than that in the blank group （P<0.05，P<0.01）. Transwell invasion assay showed that compared with the blank group， the number of transmembrane cells in PA groups was reduced（P<0.01）. High-content imaging showed that the cumulative migration distance of cells in the PA groups was shorter than that in the blank group（P<0.01）. The results of Western blot showed that the protein expression of MMP-2 and MMP-9 in the PA groups decreased （P<0.01）， and TIMP-1 protein expression increased （P<0.01） compared with those in the blank group. In addition， compared with the blank group， the PA groups showed decreased protein expression of Smad2 and Smad3 （P<0.01）. ConclusionPA can inhibit the invasion and metastasis of renal carcinoma cells presumably through regulating the homeostasis of MMP/TIMP by Smad2/3.
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AIM: To investigate the effect of the intravitreal injection of vascular endothelial growth factor-A165(VEGF-A165)on the scleral remodeling of guinea pigs with form-deprivation myopia(FDM).METHODS: A total of 120 tricolor guinea pigs, aged three weeks, were randomly divided into 6 groups, with 20 in each group. The blank group did not undergo any intervention. In the FDM group, only the FDM model was established. In the phosphate buffer saline(PBS)group, 2.5 μL of PBS was injected into the vitreous cavity before establishing the FDM model. In the 1ng group, 5ng group, and 10ng group, VEGF-A165 was injected into the vitreous cavity at concentrations of 1, 5 and 10ng, respectively, before the establishment of the FDM model. The FDM model was established by covering the right eyes of guinea pigs with translucent balloons for 14d. The diopter and axial length of the right eyes were measured before and after covering. After 14d, the content of dopamine(DA)in retina was measured by high performance liquid chromatography. Additionally, the mRNA and protein expression levels of matrix metalloproteinase-2(MMP-2), tissue inhibitor of matrix metalloproteinase-2(TIMP-2), transforming growth factor(TGF)-β1, TGF-β2 and α-smooth muscle actin(α-SMA)in sclera were detected by reverse transcription polymerase chain reaction(RT-PCR)and Western blot.RESULTS: Before covering, there were no significant differences in the diopter and axial length of the right eyes of guinea pigs in all groups(P>0.05). After 14d of modeling, when compared with the blank group, FDM group showed an increase in the degree of myopia in the right eye, a prolongation of the axial length, a decrease in the content of DA in the retina, and an increase in the expression of MMP-2, TGF-β2 and α-SMA in the sclera. Conversely, the expression of TIMP-2 and TGF-β1 were decreased(P<0.01). However, in comparison to the FDM group, the degree of myopia in the 1ng, 5ng, and 10ng groups of guinea pigs decreased, the growth trend of axial length slowed, the content of DA in the retina increased, and the expression of MMP-2, TGF-β2 and α-SMA in the sclera decreased. Furthermore, the expression of TIMP-2 and TGF-β1 in the sclera increased(P<0.01). As the concentration of intravitreal injection of VEGF-A165 increased, the degree of myopia in the right eye of guinea pigs gradually increased, and the axial length gradually prolonged. The content of DA in the retina gradually decreased, the expression of MMP-2, TGF-β2, and α-SMA in the sclera gradually increased, while the expression of TIMP-2 and TGF-β1 decreased gradually.CONCLUSION: Intravitreal injection of VEGF-A165 can increase the content of DA in the retina of FDM guinea pigs, affect the expression of MMP-2, TIMP-2, TGF-β1, TGF-β2 and α-SMA in the sclera, and inhibit scleral remodeling of guinea pigs. Notably, the VEGF-A165 at the concentration of 1ng showed the most significant efficacy.
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Objective:To investigate the expressions of tissue inhibitor of matrix metalloproteinase-1 (TIMP1) and fibronectin 1 (FN1) in pregnancy associated breast cancer (PABC) and their correlations with expression of E-cadherin (E-cad).Methods:The clinicopathological data of 55 PABC patients in Binzhou People's Hospital Affiliated to Shandong First Medical University from January 2011 to December 2020 were retrospectively analyzed. Immunohistochemistry was used to detect expressions of TIMP1, FN1 and E-cad in cancer tissues and corresponding paracancerous tissues (>3 cm from the edge of the tumor foci). The expressions of TIMP1 and FN1 proteins in fresh intraoperative frozen cancer tissues and paracancerous tissues of 10 PABC patients were detected by Western blotting. The correlations of TIMP1 and FN1 expressions with clinicopathological characteristics of patients were analyzed by χ2 test, the correlation of TIMP1 and FN1 expressions with E-cad expression was analyzed by Spearman method, and the correlation of TIMP1 and FN1 expressions with survival was analyzed by Kaplan-Meier method. Results:The positive rates of TIMP1 and FN1 in PABC tissues were 72.7% (40/55) and 58.2% (32/55), and 25.5% (14/55) and 18.2% (10/55) in paracancerous tissues, and the differences were statistically significant ( χ2 values were 24.59 and 18.64, both P < 0.001). The results of Western blotting showed that the relative expressions of TIMP1 and FN1 proteins in the fresh cancer tissues of 10 PABC patients was higher than those in the corresponding paracancerous tissues (1.60±0.76 vs. 0.62±0.29, 1.31±0.62 vs. 0.44±0.15), and the differences were statistically significant ( t values were 5.92 and 4.86, both P < 0.001). The expressions of TIMP1 and FN1 in PABC tissues were correlated with estrogen receptor expression, Ki-67 positivity index, TNM stage and lymph node metastasis (all P < 0.05). The expressions of TIMP1 and FN1 were negatively correlated with expression of E-cad in PABC ( r values were -0.471 and -0.432, both P < 0.001). Five cases were lost to follow-up, and the remaining 50 cases had a median follow-up time of 43 months (12-90 months). Among the 50 cases, 36 cases were TMP1-positive and 29 cases were FN1-positive. The overall survival of TIMP1-negative group and FN1-negative group were better than those of the corresponding positive group ( χ2 values were 4.49 and 6.06, both P < 0.05); the median overall survival time of TIMP1-positive group and FN1-positive group were 51 months (95% CI 37-65 months) and 43 months (95% CI 32-53 months), while that of TIMP1-negative group and FN1-negative group were 89 months (95% CI 84-93 months) and 87 months (95% CI 85-92 months). Conclusions:TIMP1 and FN1 expressions are elevated in PABC tissues and negatively correlated with E-cad expression, TIMP1 and FN1 may be involved in PABC invasion through epithelial-mesenchymal transition and affect the prognosis of patients.
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Objective To investigate the structural distribution features and mechanism of elastic fibers and collagen fibers in ventricular interstitium of aged rats. Methods Five young SD rats (24 weeks) and five old SD rats (104 weeks) were used,and their cardiac function was examined by echocardiography. Modified Weigert elastic fiber staining, immunohistochemistry, immunofluorescence and Western blotting techniques were used to detect the expression changes of type I and IH collagen fibers and their proteins, elastic fibers and their proteins, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 2 (TIMP-2), respectively. Results The type I and type IH collagen in the ventricular interstitium of aged rats was very sufficient and wrapped around the cardiomyocytes. Compared with the young rats, the content of collagen protein in the ventricular interstitium of the aged rats significantly increased (P<0. 05). Elastic fibers in the ventricular interstitium of the aged rats were and widely distributed. Compared with the young rats, the number of elastic fibers and the level of elastin in the ventricular interstitium of the aged rats significantly decreased (P<0. 05), and the expression levels of MMP-2 and MMP-9 in ventricular muscle of aged rats increased, and the)' were correlated with the level of elastin. The level of TIMP-2 in ventricular muscle of aged rats decreased with age. Conclusion The number of collagen fibers and elastic fibers in ventricular interstitium of aged rats is fluctuated with each other. With the increase of age, the contents of TIMP-2 and elastic fibers in the ventricular interstitium gradually decreased, and the ratio of collagen fibers to elastic fibers is out of balance.
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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.
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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.
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Objective To explore the effects of miR-221 on tumor cell proliferation, migration and invasion in nonsmall cell lung cancer (NSCLC) xenograft model mice, and to preliminarily analyze its possible mechanism of regulating Akt/ mammalian target of rapamycin(mTOR) signaling pathway by targeting tissue inhibitor of metalloproteinase-2 (TIMP-2) on tumor cells in non-small cell lung cancer (NSCLC) through tumor-bearing nude mice. Methods The A549 cells were divided into control group, mimic group, TIMP-2 group and mimic+TIMP-2 group. The mimic group and TIMP-2 group were transfected with miR-221 mimic and TIMP-2 overexpression plasmids, respectively. The mimic + TIMP-2 group was simultaneously transfected with miR-221 mimic and TIMP-2 overexpression plasmids. The control group was transfected with the same amount of negative control plasmid. After transfection, the cells of each group were injected subcutaneously into the left forelimb to construct the corresponding 4 groups of NSCLC mouse models. The proliferation-related protein (Ki67) was detected by immunohistochemical staining to detected the effect of cell proliferation ability. Matrix metalloproteinase-2 (MMP-2) and N-cadherin proteins in each group were tested by Western blotting to assess and compare the abilities of migration and invasion. The levels of miR-221, TIMP-2 and Akt/ mTOR pathways in bone marrow and tumor tissues were detected by Real-time PCR and Western blotting. Results When co-transfected with wild type(WT)-TIMP-2 and miR-221 mimic, the relative luciferase activity in the cells reduced significantly (P<0. 05). Compared with the control group, the tumor mass, volume, Ki67, MMP-2 and N-cadherin protein expression levels, miR-221 and Akt/ mTOR pathway levels were increased significantly, while the levels of TIMP-2 mRNA and protein were significantly reduced in the mimic group (P<0. 05). Compared with the control group, the levels of TIMP-2 mRNA and protein in the TIMP-2 group increased significantly, while the other indicators decreased significantly (P<0. 05). Tumor tissue mass, volume, Ki67, MMP-2, Ncadherin, miR-221 and Akt/ mTOR pathway levels in mimic+TIMP-2 group were significantly lower than those in the mimic group and significantly higher than those in the TIMP-2 group, while TIMP-2 mRNA and protein levels were significantly higher than those in the mimic group and significantly lower than those in the TIMP-2 group (P<0. 05). Conclusion In the NSCLC transplanted tumor mouse model, miR-221 may mediate the Akt/ mTOR pathway by targeting the expression of TIMP-2 protein to promote cell proliferation, migration and invasion.
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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.
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Objective:To explore the predictive values of tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor binding protein 7 (IGFBP7) in donor sera and lavage fluid on delayed graft function (DGF) in donation after circulatory death (DCD) kidney transplant recipients.Methods:A total of 33 eligible kidney donors and 33 corresponding recipients were recruited. Preoperative serum and renal perfusion fluid samples of donors were collected to determine the levels of TIMP-2 and IGFBP7. Patients were grouped according to whether DGF occurred after kidney transplantation and measured indicators analyzed. Independent sample t test was utilized for comparing the groups with normal distribution measurement data. And χ2 test was employed for comparing the groups with normal distribution counting data and Mann-Whitney test for comparing the groups with non-normal distribution measurement data. Receiver operating characteristic (ROC) curve and area under curve (AUC) were used for evaluating the diagnostic efficacy of indicators. Results:In donor-DGF group, lavage fluid TIMP-2, product of lavage fluid TIMP-2 and IGFBP7 (TIMP-2×IGFBP7), serum IGFBP7 and product of serum TIMP-2 and IGFBP7 (TIMP-2×IGFBP7) were higher than those in donor-non-DGF group ( P<0.05). The AUC of TIMP-2, TIMP-2×IGFBP7, serum IGFBP7 and serum TIMP-2×IGFBP7 in the diagnosis of DGF were 0.753 (95%CI 0.546~0.959), 0.747 (95%CI 0.510~0.984), 0.824 (95%CI 0.615~1.000) and 0.852 (95%CI 0.660~1.000) respectively. Conclusions:Donor serum IGFBP7, donor serum TIMP-2×IGFBP7, lavage fluid TIMP-2 and lavage fluid TIMP-2×IGFBP7 may be used for predicting the occurrence of early DGF after kidney transplantation. Among them, serum TIMP-2×IGFBP7 has the highest diagnostic efficiency and may be an excellent predictor of DGF occurrence.
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Objective:This study is to investigate the predictive value of serum levels of TIMP-2 and insulin-like growth factor-binding protein 7(IGFBP7) in patients with DCD(donation after cardiac death) kidney transplantation.Methods:A prospective research design was used to select DCD kidney transplant patients admitted to the Li Huili Hospital of Ningbo University from January 2018 to October 2020.Inclusion criteria: ①Complete data; ②There were no serious complications affecting the function of the transplanted kidney in the early postoperative period.Exclusion criteria: ①Incomplete data; ②Patients were unable or unwilling to cooperate with the study; ③Severe complications affecting the function of the transplanted kidney occurred early after the operation.The ELASE method was used to quantitatively detect the serum TIMP-2 and IGFBP7 levels at 6, 12, 24, 48, 72 hours and 7 days after renal transplantation, and monitor the serum creatinine values during the same period and 21 days after the operation. According to the occurrence of DGF, the measured values of TIMP-2 and IGFBP7 at different time points and their product's ability to predict the occurrence of DGF after kidney transplantation were analyzed. The receiver operating characteristic (ROC) curve and area under the curve (AUC) were used to evaluate the diagnostic efficacy of TIMP-2 and IGFBP7 for DGF.Results:A total of 33 patients were enrolled, 7 patients (21.2%) in the DGF group and 26 patients (78.8%) in the non-DGF group. Between the two groups, the donor glomerular filtration rate were [98.5(15.8-132.5)ml/(min·1.73m 2) and 79.1(60.6-102.5)ml/(min·1.73m 2)], recipient gender (male/female: 3/4 cases and 10/16 cases), recipient age [48(34-56) Years old and 45(23-61) years old], the recipient's preoperative creatinine [1114.0(731.4-1293.0)μmol/L and 858.4(657.6-1051.9)μmol/L], the recipient's preoperative urea nitrogen [15.0(13.2-19.6)mmol/L and 17.3(13.6-20.9)mmol/L], receptor preoperative albumin [43.5(38.5-45.3)mmol/L and 41.2(37.5-46.1) mmol/L], recipient dialysis method [hemodialysis/peritoneal dialysis: 3/4 cases and 9/17 cases], warm ischemia time [6(5-7) and 5(4-6) min, there was no statistically significant difference] ( P>0.05). The values of serum IGFBP7 and TIMP-2×IGFBP7 in the DGF group were higher than those in the non-DGF group at all time points ( F=15.753, P=0.040; F=13.000, P=0.024), while serum TIMP-2 was not significant between the two groups difference ( F=1.157, P=0.075). For the diagnostic value of DGF, the AUC of serum IGFBP7 at 48 h after surgery was 0.863 (95% CI 0.696-1.000, P=0.004). When 5.97 ng/ml was used as the cut-off value, the sensitivity was 85.7% and the specificity was 80.8 %. The AUC of TIMP-2×IGFBP7 at 48 hours after surgery was 0.819 (95% CI 0.641-0.996, P=0.011). When 62.06(ng/ml) 2 was used as the cutoff value, the sensitivity was 71.4% and the specificity was 80.8%.There was no statistical difference in the area under the curve between the two ( P>0.05). There were differences in the dynamic trend of serum IGFBP7 and creatinine in the DGF group. Serum IGFBP7 at 7 days after surgery was positively correlated with creatinine at 21 days after surgery. Conclusion:Serum IGFBP7 and TIMP-2×IGFBP7 could predict the occurrence of DGF after DCD donor kidney surgery. The predictive value changes with time. Among them, 48h and 7d after surgery are the most valuable. However, serum TIMP-2 has not been found to have predictive value in this study.
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ABSTRACT Introduction: The current study about transition of oral epithelial dysplasia, present in lesions such as leukoplakia, for squamous cell carcinoma (SCC) involves not only the histopathological aspects, but also the analysis of the presence of biomarkers which influence the microenvironment where cells are embedded. Objective: To evaluate the tissue inhibitor of metalloproteinase-1 (TIMP-1) profile in cases of leukoplakia and SCC classified into different degrees of dysplasia and histological grading, respectively. The immunohistochemical findings were confronted with microscopic features adopted in the classification of each lesion. Material and methods: Cases of leukoplakia and SCC were recovered from files of The Oral Pathological Anatomy Service of the Dental School at the Universidade Federal do Espírito Santo (SAPB-UFES), between the years 2004 and 2010. New slides were obtained and submitted to immunohistochemical assay to determine TIMP-1 expression profile. Parenchyma, as well as the different layers of the epithelium and stroma was evaluated. Results: In all cases the presence of TIMP-1 was detected in the stroma and parenchyma. In mild leukoplakia, the basal layer with hyperplasia showed intense immunolabeling, whereas cells with loss of polarity presented weaker expression. In moderate leukoplakia, all epithelium layers, except the cornea, were labeled. Severe leukoplakia had the spinous layer most intensely labeled, with no variation in areas with pleomorphism. Stage I SCC showed the deepest islands with intense labeling in cells with pleomorphism and mitoses. In the tumor islands, less differentiated cells were weakly labeled, and in keratin pearl, labeling was weak or absent in central cells. In stage II SCC, labeling was observed in basal cell with hyperplasia and in cells of the spinous layer, however, the parabasal layer was not labeled. Also, on tumor islands, less differentiated cells did not express the protein and keratin pearls were not labeled. Conclusion: It was possible to detect TIMP-1 immunolabeling in all specimens, ranging in intensity and location. The absence of expression in less differentiated cell suggests that more aggressive lesions present reduced enzyme expression. The microenvironment is important for the various cellular activities, and TIMP is an enzyme that participates in matrix remodeling, therefore changes in its expression can be a valuable tool in the better understanding oral carcinogenesis.
RESUMEN Introducción: El estudio actual de la transición de displasia epitelial oral, presente en lesiones como la leucoplasia, hacia carcinoma epidermoide, implica no solo aspectos histopatológicos, sino también el análisis de la presencia de biomarcadores que influyen en el microambiente en el que se insertan las células. Objetivo: Evaluar el perfil del inhibidor tisular metaloproteinasa-1 (TIMP-1) en casos de leucoplasia y carcinoma de células escamosas (CCE) clasificados en diferentes grados de displasia y grados histopatológicos, respectivamente. Confrontar los hallazgos inmunohistoquímicos con los aspectos microscópicos adoptados en la clasificación de lesiones. Material y métodos: Casos de leucoplasia y CCE fueron recuperados del Servicio de Anatomía Patológica Bucal del Curso de Odontología de la Universidad Federal de Espírito Santo (SAPB-UFES), entre los años 2004-2010. Se obtuvieron nuevos portaobjetos y se los sometieron a ensayos inmunohistoquímicos para determinar el perfil de expresión de TIMP-1. Se evaluó el parénquima, así como las diferentes capas del epitelio y estroma. Resultados: En todos los casos se detectó TIMP-1 en estroma y parénquima. En la leucoplasia leve, la capa basal y con hiperplasia mostró inmunotinción intensa, mientras que las células con pérdida de polaridad tuvieron menos expresión. En la leucoplasia moderada, todas las capas del epitelio, excepto la córnea, mostraron inmunotinción. En la leucoplasia grave la capa espinosa tuvo inmunotinción más intensa, sin variación en áreas con pleomorfismo. El CCE grado I mostró las islas más profundas con tinción intensa en células con pleomorfismo y mitosis. En las islas tumorales, las células menos diferenciadas tuvieron tinción menor, y en las perlas de queratina la tinción fue débil o ausente en las células centrales. En el CCE grado II, se observó tinción en células basales con hiperplasia y, en células de la capa espinosa, la capa parabasal no fue marcada. También en las islas, las células menos diferenciadas no expresaron la proteína y no hubo tinción en las perlas de queratina. Conclusión: Fue posible detectar inmunotinción para TIMP-1 en todos los especímenes, con variación en intensidad y ubicación. La ausencia de expresión en células menos diferenciadas sugiere que las lesiones más agresivas tienen enzima reducida. El microambiente es importante para las diversas actividades celulares, y el TIMP es una enzima que participa en la remodelación de la matriz; por lo tanto, la alteración en su expresión puede ser una herramienta valiosa en el mejor entendimiento de la carcinogénesis de la mucosa oral.
RESUMO Introdução: O estudo atual da transição da displasia epitelial oral, presente em lesões como a leucoplasia, para o carcinoma de células escamosas (CCE), envolve não somente aspectos histopatológicos, como também a análise da presença de biomarcadores, os quais influenciam o microambiente em que as células estão inseridas. Objetivo: Avaliar o perfil da expressão do inibidor tecidual de metaloproteinase 1 (TIMP-1) em casos de leucoplasias e CCE classificados em diferentes graus de displasia e graus histopatológicos, respectivamente, e confrontar os achados imuno-histoquímicos com os aspectos microscópicos adotados na classificação das lesões. Material e métodos: Foram resgatados casos de leucoplasia e CCE do Serviço de Anatomia Patológica Bucal do curso de Odontologia da Universidade Federal do Espírito Santo (SAPB-UFES), entre os anos 2004-2010. Novas lâminas foram obtidas ao serem submetidas ao ensaio imuno-histoquímico para determinação do perfil de expressão de TIMP-1. Foram avaliados parênquima, bem como as diferentes camadas do epitélio e estroma. Resultados: Em todos os casos, foi detectada a presença de TIMP-1 no estroma e no parênquima. Na leucoplasia leve, a camada basal e com hiperplasia apresentou imunomarcação intensa; as células com perda de polaridade tiveram expressão menor. Na leucoplasia moderada, todas as camadas do epitélio, exceto a córnea, apresentaram marcação. A leucoplasia severa teve a camada espinhosa marcada mais intensamente, sem variação em áreas com pleomorfismo. O CCE grau I apresentou as ilhas mais profundas com marcação intensa em células com pleomorfismo e mitoses. Nas ilhas tumorais, células menos diferenciadas tiveram marcação menor, e em pérolas córneas a marcação foi fraca ou ausente nas células centrais. No CCE grau II, foi observada a marcação em células basais com hiperplasia e, em células da camada espinhosa, a camada parabasal não foi marcada. Também nas ilhas, células menos diferenciadas não expressaram a proteína e não houve marcação em pérolas córneas. Conclusão: Foi possível detectar imunomarcação para TIMP-1 em todos os espécimes, com variação em intensidade e localização. A ausência de expressão em células menos diferenciadas sugere que lesões mais agressivas possuem redução da enzima. O microambiente é importante para as diversas atividades celulares, e TIMP é uma enzima que participa da remodelação da matriz. Portanto, alteração na sua expressão pode ser uma valiosa ferramenta para um melhor entendimento da carcinogênese da mucosa bucal.
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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)
Subject(s)
Animals , Male , Rats , Leptin , Matrix Metalloproteinase 2 , Rats, Wistar , Collagen Type I , Myocardium , Obesity/complicationsABSTRACT
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.
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Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.
Subject(s)
Humans , Cloning, Molecular , Escherichia coli/genetics , Recombinant Fusion Proteins/genetics , Recombinant Proteins , Tissue Inhibitor of Metalloproteinase-2/geneticsABSTRACT
Objective: To observe the effect of deuterium depleted water combined with platelet-rich plasma on wound healing of diabetic ulcer in rats, and to explore its possible mechanism. Methods: Male SD rats were randomly divided into two groups, normal control group (group A, n=20) and diabetic group (n=80). Rats in the diabetic group were fed with high-fat diet for 4 weeks, and the rat diabetic ulcer model was replicated by intraperitoneal injection of streptozotocin (STZ) + skin full-thickness resection; then randomly divided into diabetic model group (group B), platelet-rich plasma group (group C), deuterium depleted water group (group D), and deuterium depleted water combined platelet-rich plasma group (group E), with 18 rats for each group. Group A with common feed was fed for 4 weeks after intraperitoneal injection of an equal volume of citric acid-sodium citrate buffer + skin full-thickness resection to replicate the normal ulcer model. The animals were sacrificed after treatment for 3, 7 and 14 d, and the random blood glucose was measured at each corresponding time point. The wound surface and wound margin tissue were taken to observe the wound healing and local histomorphology of each group. The contents of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the wound tissue of each group were detected by enzyme-linked reaction adsorption method. Results: Random blood glucose in group D and group E was lower than that before intervention. The inflammatory response of the wounds in each diabetic group was slower than that in group A. The granulation ripening effect of group E was faster than that of group B, C, and D. The effect was best in each intervention group, and the neovascularization and fibroblasts appeared earlier and in large quantities. The content of TIMP-1 in group A was significantly higher than that in group B, C, D and E (P<0.05). The content of TIMP-1 in group B was significantly lower than that in group C, D and E (P<0.05). The content of TIMP-1 was significantly higher than that of group C and D (P<0.05). The content of MMP-9 in group B was significantly higher than that in group A, C, D and E (P<0.05). The content of MMP-9 in group E was significantly lower than that in group C and D (P<0.05). Conclusion: Deuterium depleted water can promote the healing of diabetic ulcer wounds. Deuterium depleted water combined with platelet-rich plasma can significantly promote the healing of diabetic ulcer wounds, which may be related to the decrease of random blood glucose, the increase of TIMP-1 and the inhibition of MMP-9 expression.
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Matrix metalloproteinase (MMP) is a large class of proteases which can cut or reshape extracellular matrix (ECM) and cell surface proteins. The activity of MMP is regulated by a variety of cytokines, including tissue inhibitor of metalloprotease (TIMP), signal transduction molecules and cell adhesion molecules. The latest research shows that MMP has a role in the pathophysiology process of many acute and chronic kidney diseases. In this article, the classification, expression and distribution in the kidney of MMP and its role in injury related renal transplantation was reviewed.
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BACKGROUND: In the development of osteoarthritis, the mechanism underlying cartilage damage is still unclear. Matrix metalloproteinases have been shown to play important roles in cartilage matric degradation. OBJECTIVE: To observe the correlation between the expression of matrix metalloproteinase 3, tissue inhibitor of metalloproteinase-1 and the pathological degree in knee osteoarthritis. METHODS: The study was approved by the Ethics Committee of Shenyang Orthopedic Hospital. The tibial plateaus of 40 patients with knee osteoarthritis who underwent total knee arthroplasty were collected, and all patients signed the informed consents. The classification of osteoarthritis was evaluated according to Kellgren-Lawrence (KL) scale system: 11 cases of KL grade 2, 15 cases of KL grade 3, and 14 cases of KL grade 4. Control group contained six cases. Samples received hematoxylin-eosin staining. The pathological changes of knee osteoarthritis cartilage were evaluated by Mankin score. Immunohistochemical staining was used to detect the contents of matrix metalloproteinase 3 and tissue inhibitor of metalloproteinase-1 in cartilage. RESULTS AND CONCLUSION: (1) Hematoxylin-eosin staining results showed that in the control group, the layer of cartilage was thick, and there were abundant chondrocytes that arranged regularly. The subcutaneous layer of cartilage in the KL grade 2 group was rugged, and fissure was observed occasionally. Fibrosis of cartilage layer was visible in the KL grade 3 group, and the chondrocytes arranged in disorder. In the KL grade 4 group, the structure of cartilage layer was lost, and there were few chondrocytes that arranged irregularly. (2) Immunohistochemical staining results showed that the expression level of matrix metalloproteinase 3 in the osteoarthritis group was significantly higher than that in the control group. The expression level of matrix metalloproteinase 3 was on a rise in the KL grade 2, 3 and 4 groups (all P < 0.05). The expression level of tissue inhibitor of metalloproteinase-1 in the osteoarthritis group was significantly lower than that in the control group. The expression level of tissue inhibitor of metalloproteinase-1 was on a descent in the KL grade 2, 3 and 4 groups (all P < 0.05). (3) There was significantly positive correlation between matrix metalloproteinase 3 expression level and Mankin score (r=0.899, P < 0.001), and tissue inhibitor of metalloproteinase-1 expression level was negatively correlated with Mankin score (r=-0.903, P < 0.001). There was significantly negative correlation between tissue inhibitor of metalloproteinase-1 expression level and matrix metalloproteinase 3 expression level (f=-0.881, P < 0.001). (4) These results indicate that in the articular cartilage of patients with osteoarthritis, the expression levels of matrix metalloproteinase 3 and tissue inhibitor of metalloproteinase-1 are correlated with the pathological changes, which can be used as an effective index to evaluate the progress of knee osteoarthritis.
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BACKGROUND: Liver fibrosis has higher morbidity and mortality. Activation and proliferation of hepatic stellate cells is a key link in the progression of liver fibrosis. At present, there are still no effective anti-fibrosis agents targeting single links or targets. OBJECTIVE: To analyze the effect of human adipose stem cells derived exosomes on rats with liver fibrosis induced by carbon tetrachloride. METHODS: Human adipose stem cells were obtained from healthy people by enzyme dissolution method. After in vitro culture, human adipose stem cells derived exosomes were obtained by multiple ultrafiltration. Different concentrations of exosomes were used to treat the hepatic stellate cells activated by transforming growth factor β1. The human adipose stem cells activated by transforming growth factor β1 were treated with different concentrations of exosomes. The expression of α-smooth actin in the cells was detected by quantitative PCR, and the growth and apoptosis of activated hepatic stellate cells were detected by CCK-8 and flow cytometry respectively. Rat models of liver fibrosis were established by intraperitoneal injection of carbon tetrachloride and treated by tail vein injection of exosomes. Rat liver function, serum levels of type III procollagen and type IV collagen, and Ishak score were determined. Semi-quantitative analysis of liver fibrosis was performed. The expression levels of tissue inhibitor of matrix metalloproteinase-1, matrix metalloproteinase 9 and α-smooth actin in liver tissue were measured by immunofluorescence method. The study protocol was approved by the Animal Ethics Committee and Medical Ethics Committee, Tongji University, China in January, 2017. RESULTS AND CONCLUSION: Human adipose stem cells derived exosomes inhibited the proliferation of activated hepatic stellate cells. The possible mechanism is to inhibit the proliferation of activated macrophages, reduce the production of collagen fibers, α-smooth actin actin, and tissue inhibitor of matrix metalloproteinase-1, and to increase the expression of matrix metalloproteinase 9. These findings suggest that exosomes can be used to treat carbon tetrachloride induced liver fibrosis.