Evaluation of topical methylene blue nanoemulsion for wound healing in diabetic mice.

CONTEXT: Diabetic wounds (DW) are a complication of diabetes and slow wound healing is the main manifestation. Methylene blue (MB) has been shown to exhibit therapeutic effects on diabetes-related diseases. OBJECTIVE: To investigate the mechanisms of action of MB-nanoemulsion (NE) in the treatment of DW. MATERIALS AND METHODS: The concentration of MB-NE used in the in vivo and in vitro experiments was 0.1 mg/mL. Streptozocin-induced diabetic mice were used as models. The mice were separated into nondiabetic, diabetic, MB-NE treated, and NE-treated groups. Intervention of high glucose-induced human umbilical vein endothelial cells using MB-NE. The mechanism by which MB-NE promotes DW healing is investigated by combining histological analysis, immunofluorescence analysis, TUNEL and ROS assays and western blotting. RESULTS: In diabetic mice, the MB-NE accelerated DW healing (p < 0.05), promoted the expression of endothelial cell markers (α-SMA, CD31 and VEGF) (p < 0.05), and reduced TUNEL levels. In vitro, MB accelerated the migration rate of cells (p < 0.05); promoted the expression of CD31, VEGF, anti-apoptotic protein Bcl2 (p < 0.05) and decreased the expression of the pro-apoptotic proteins cleaved caspase-3 and Bax (p < 0.05). MB upregulated the expression of Nrf2, catalase, HO-1 and SOD2 (p < 0.05). In addition, MB reduced the immunofluorescence intensity of TUNEL and ROS in cells and reduced apoptosis. The therapeutic effect of MB was attenuated after treatment with an Nrf2 inhibitor (ML385). DISCUSSION AND CONCLUSION: This study provides a foundation for the application of MB-NE in the treatment of DW.

Inhibition of canine parvovirus replication in cultured cells by small interfering RNAs expressed from plasmid vectors.

Small interfering RNAs (siRNAs) target complementary mRNA for specific degradation, a mechanism many viruses are susceptible too. Thus, siRNA degradation of target RNAs can be exploited as novel therapeutics. In this report, we show that the vector-based siRNAs (psiSTRIKEs) expressed by a human U6 promoter could efficiently inhibit CPV replication in cell culture. A series of PsiSTRIKE vectors expressing siRNA were constructed that target structural protein genes or nonstructural protein genes of CPV genome. These plasmids were transfected into FK81 cells via lipofectin and the stable transfection clones were selected. The immunostaining, plaque assay, and cell proliferation assay of the cells infected by CPV were performed. The results show that siRNAs against nonstructural protein genes effectively inhibited CPV replication. The inhibition efficiencies detected by immunostaining assay of psiSTRIKE/vp1510, psiSTRIKE/NS160, and psiSTRIKE/NS1939 were 66%, 76% and 78%, respectively at 48h, and 69%, 46% and 67%, respectively at 96h. Plaque assay showed that, comprising to the control, the psiSTRIKE/NS160 reduced the virion production by 100-fold, and psiSTRIKE/NS1939 or psiSTRIKE/VP1510 reduced the virion production 13-fold. When compared to control, the viability of cells transfected psiSTRIKE/NS160 increased 78% and 124%, respectively at 72 and 120h. Our study may provide a potential therapy against CPV infection.