RESUMO
[This retracts the article DOI: 10.3892/etm.2019.7644.].
RESUMO
Hyaluronidase (HAase) can enhance drug diffusion and dissipate edema by degrading hyaluronic acid (HA) in the extracellular matrix into unsaturated HA oligosaccharides in mammalian tissues. Microorganisms are recognized as valuable sources of HAase. In this study, a new hyaluronate lyase (HAaseD) from Bacillus sp. CQMU-D was expressed in Escherichia coli BL21, purified, and characterized. The results showed that HAaseD belonged to the polysaccharide lyase (PL) 8 family and had a molecular weight of 123 kDa. HAaseD could degrade chondroitin sulfate (CS) -A, CS-B, CS-C, and HA, with the highest activity toward HA. The optimum temperature and pH value of HAaseD were 40°C and 7.0, respectively. In addition, HAaseD retained stability in an alkaline environment and displayed higher activity with appropriate concentrations of metal ions. Moreover, HAaseD was an endolytic hyaluronate lyase that could degrade HA to produce unsaturated HA oligosaccharides. Together, our findings indicate that HAaseD from Bacillus sp. CQMU-D is a new hyaluronate lyase and with excellent potential for application in industrial production.
Assuntos
Bacillus , Animais , Bacillus/genética , Polissacarídeo-Liases/metabolismo , Hialuronoglucosaminidase/metabolismo , Ácido Hialurônico/metabolismo , Clonagem Molecular , Oligossacarídeos/metabolismo , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Mamíferos/metabolismoRESUMO
Malignant melanoma (MM) is the most aggressive form of skin cancer originating from melanocytes with increased proliferative and metastatic ability. Previous studies have revealed that microRNA-338-3p (miR-338-3p) functions as a tumor suppressor in several types of cancer, including cervical cancer, renal cell carcinoma and thyroid cancer. However, the function and mechanism underlying the action of miR-383-3p in MM remain unclear. In the study, aberrant downregulation of miR-338-3p was observed in 60 pairs of MM and adjacent non-tumor tissue using quantitative polymerase chain reaction assay. Decreased miR-383-3p expression was associated with advanced clinical stage (P<0.05) and lymph node metastasis (P<0.001). The overexpression of miR-338-3p in A375 and G361 cells suppressed cell proliferation and migration using MTT, colony formation, wound healing and transwell assays. Mechanistically, MACC1 was identified as a direct target for miR-338-3p using bioinformatics prediction and dual-luciferase assays. Furthermore, MACC1 expression was significantly increased and inversely correlated with the levels of miR-338-3p in MM tissues. More importantly, restoration of MACC1 resulted in reversed the inhibitory effects of miR-338-3p overexpression on MM cells by altering the expression levels of PCNA and epithelial-mesenchymal transition (EMT)-associated proteins. These results suggest that miR-338-3p functions as a novel tumor suppressor, at least in part, via targeting MACC1 and suggest that miR-338-3p may serve as a potential target for treatment of MM patients.
RESUMO
OBJECTIVE: To prepare nerve growth factor (NGF)-insulin composite gel and observe the effects of NGF-insulin composite gel on deep second degree scald wound healing in diabetic rats. METHODS: Carbomer 980, NGF (4 000 U), and insulin (800 U) were used to prepare the insulin gel, NGF gel, and NGF-insulin composite gel. The character of NGF-insulin composite gel was observed, and the in vitro drug release was tested. Seventy-five SPF Wistar male rats, weighing 200-250 g, were divided into 5 groups randomly, 15 rats each group: normal control group (group A), diabetes control group (group B), insulin gel treatment group (group C), NGF gel treatment group (group D), and NGF-insulin composite gel treatment group (group E). The type 1 diabetes rat model was established by intraperitoneal injection of Streptozotocin (55 mg/kg) in groups B, C, D, and E, while the rats in group A were injected with the same dose of citric acid and calcium citrate buffer. After modeling success, deep second degree scald wound on the back was made with constant temperature water bath box. Wounds were treated with carbomer blank gel in groups A and B, with insulin composite gel in group C, with NGF gel in group D, and with NGF-insulin composite gel in group E, once a day. At 3, 7, 11, 15, and 21 days after injury, the scald wound healing was observed and healing rate was calculated; the full-thickness skin specimens were harvested from 3 rats of each group for histological and immuohistochemical staining observation. RESULTS: The NGF-insulin composite gel was clear and transparent, and had good moisture retention capacity and adhesion; it was easy to apply and clean up. The drug release in vitro lasted more than 24 hours and maintained for 30 days. No rat died during the experiment. At 3 days after injury, wound area did not reduce in all groups; at 7, 11, 15, and 21 days, group E had the highest wound healing rate, and group B had the lowest; significant differences were found between group E and group B and when compared with the other groups (P < 0.05). HE staining showed that group E surpassed other groups in the growth of granulation tissue and collagen fiber. Immunohistochemical results showed that the CD34 and proliferating cell nuclear antigen (PCNA) expressed at 3 days, and the number of positive cells increased gradually with time; the microvessel density and PCNA expression were highest in group E and were lowest in group B, showing significant differences when compared with the other groups and between group E and group B (P < 0.05). CONCLUSION: NGF-insulin composite gel can improve deep second degree scald wound healing in diabetic rats.
Assuntos
Queimaduras/tratamento farmacológico , Diabetes Mellitus Experimental/fisiopatologia , Insulina/administração & dosagem , Fatores de Crescimento Neural/administração & dosagem , Cicatrização/efeitos dos fármacos , Resinas Acrílicas/química , Administração Tópica , Animais , Queimaduras/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Géis/química , Insulina/química , Insulina/farmacologia , Masculino , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/farmacologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Estreptozocina , Fatores de TempoRESUMO
OBJECTIVE: To explore the possible mechanism of nerve growth factor (NGF) mixed insulin on the angiogenesis of burn wounds and the effect on the expressions of Bcl-2 and Bax in diabetic rats. METHODS: A total of 75 SPF male Wistar rats, weighing 200-220 g, were selected randomly and divided into nomal control (group A, n = 15), the rats with diabetic control (group B, n = 15), insulin treatment (group C, n = 15), NGF treatment (group D, n = 15), NGF and insulin treatment (group E, n = 15) groups. In groups B, C, D, and E, streptozotocin was given by intraperitoneal injection at dose of 10 mg/kg on the 1st day and 50 mg/kg on the 3rd day to prepare the diabetic rat models. In group A, citric acid buffer at the same dose was given. After 1 month of diabetic models, second degree scald was made on the back of the rats, and then wounds were treated with 3-layer normal saline gauze in groups A and B, with 3-layer gauze containing 5 U Novolin 30R and subcutaneous injection of Novolin 30R (4-6 U/kg) everyday in group C, with 3-layer gauze containing 5 mL NGF (25 U/mL) in group D, and with a combination of groups C and D in group E. At 7, 11, 15, and 21 days, the wound healing rate was calculated; at 3, 7, 11, 15, and 21 days, the expressions of Bcl-2, Bax, and CD34 were determined and the microvascular density was measured by immunohistochemistry staining. RESULTS: All rats survived till experiment was finished. The area of wounds became smaller gradually with time. Group E was better than other groups in the wound healing rate (P < 0.05), the skin keratosis, the hair growth, and the granulation tissue and collagen fibers growth. With time, the expressions of CD34 and Bcl-2 increased gradually, reached the peak at 15 days and decreased at 21 days; the expression was stronger in group E than in other groups (P < 0.05). At 3 days, Bax did not express; at 7 days, Bax began to express in new vascular endothelial cells and the expression increased gradually with time; the expression was weaker in group E than in other groups (P < 0.05). CONCLUSION: A combination of NGF and insulin local application can enhance the angiogenesis of the burn wound in diabetic rats and accelerate wound healing by increasing the expression of Bcl-2 and decreasing the expression of Bax and restraining apoptosis of the wounds vascular endothelial cells of diabetic rats.