Simvastatin combined with bone marrow mesenchymal stromal cells (BMSCs) improve burn wound healing by ameliorating angiogenesis through SDF-1α/CXCR4 pathway.

OBJECTIVES: Chemokines are wound mediators that promote angiogenesis during wound healing. We hypothesized that Simvastatin in combination with the bone marrow mesenchymal stromal cells (BMSCs) improve burn wound healing by ameliorating angiogenesis via SDF-1α/CXCR4 pathway. MATERIALS AND METHODS: Under general anesthesia, deep partial-thickness burns were created on the inter-scapular area of 48 male rats. Study groups were administrated with petroleum jelly (Simvastatin Vehicle), a single dose of intradermal BMSCs (1×106), topical Simvastatin (0.5 mg/kg) daily and combination of BMSCs and Simvastatin for 14 days. In this study, we used MTT assay, in vivo and in vitro wound closure, H&E and Trichorome staining, immunohistochemistry (IHC), real- time PCR, Western blot and tube formation assay. RESULTS: A significant improvement in wound closure percentage, epithelial thickness, collagen remodeling, and up-regulation of stromal cell-derived factor 1 alpha (SDF1α), C-X-C chemokine receptor type 4 (CXCR4), protein kinase B (AKT), and phosphatidylinositol 3- kinase (PI3K), as well as CD31 and vascular endothelial growth factor (VEGF) expression were observed after treatment with simvastatin, BMSCs and combination of them compared to the vehicle group. However, the co-treatment group revealed considerable superiority in examined factors. BMSCs treated with Simvastatin showed the highest viability in the concentration of 0.5 and 1 Nanomolar (nM). Increment in proliferation and capillary vessels formation of BMSCs was observed in the 0.5 nM and 1 nM concentrations of Simvastatin in vitro. CONCLUSION: Treatment of deep partial-thickness of burns with co-treatment of BMSCs and Simvastatin resulted in improved burn wound healing through up-regulating of SDF-1α/CXCR4 pathway.

Bone Marrow-Derived Mesenchymal Stem Cells Combined With Simvastatin Accelerates Burn Wound Healing by Activation of the Akt/mTOR Pathway.

Burn wound healing is one of the most important problems in the field of medical science. Promising results have recently been reported by researchers who used bone marrow mesenchymal stem cells (BMSCs) to treat burn wounds. In this study, we investigated the effects of BMSC therapy in combination with simvastatin (SMV) on angiogenesis as well as on the activity of the Akt/mTOR signaling pathway during burn wound healing in rats. After creating second-degree burn wounds, 40 adult male Wistar rats were randomly divided into four treatment groups: the control, SMV, BMSCs, and the combination therapy group (BMSCs+SMV). Animals were killed 14 days after treatment initiation, and the wounds were removed for histological and molecular analyses. All in all, combination therapy produced better outcomes than individual therapy in terms of the wound closure area, epidermal regeneration level, collagen deposition intensity, and reepithelialization rate. In addition, the elevations of expression levels of Akt and mTOR genes, at both mRNA and protein levels, were more pronounced in the BMSCs+SMV group (P < .05, at least, for both qRT-PCR and western blot assessments). qRT-PCR findings also demonstrated that the wounds treated with the combination of BMSCs and SMV had the highest expression levels of CD31 and VEGF genes (P < .01 for all comparisons). These data suggest that the combined administration of BMSCs transplantation and topical SMV has a great potential in burn wound healing. According to the findings, the beneficial effects of the combination therapy are caused, at least in part, through stimulating Akt/mTOR signaling pathway.

Effects of Hair Follicle Stem Cells on Partial-Thickness Burn Wound Healing and Tensile Strength

Background: The recent improvements in wound healing have led to new strategies in regenerative medicine. Burn wound healing is an important issue in skin regeneration and has multiple indications for stem cell therapy. Hair follicle stem cells (HFSCs) are a highly promising source of stem cells for healing use, as these cells are accessible, active and pluripotent adult stem cells. Methods: HFSCs of the rat whisker were isolated, cultured, and labeled with DiI. Flow cytometry method was used to detect special markers of HFSCs. Deep partial-thickness burn wound was created, and labeled HFSCs were injected around the wound bed. Wound closure was recorded via digital photographs. The inflicted rats were sacrificed at 3, 7, or 14 days post burn and used for subsequent histological and tensiometry analysis. Results: Our results indicated that HFSCs were positive for Nestin and CD34 markers, but negative for Kr15. Morphological and histological photographs revealed that wound closure rate was accelerated in stem cell-treated group compared with other groups. In addition, faster re-epithelialization and collagen deposition were observed. The immunohistochemical analysis suggested that CD31 expression and vascular density enhanced in the stem cell-treated group. Further, tissue tensile strength increased in HFSCs-treated rats in comparison to the control group. Conclusion: The present study demonstrates that HFSC could accelerate burn wound healing as well as tensile strength in rats.

In vitro Differentiation of Hair Follicle Stem Cell into Keratinocyte by Simvastatin

Background: Hair follicle stem cells (HFSCs) located in the bulge area has shown to be highly proliferative and could differentiate into neurons, glia, smooth muscle cell, and melanocytes in vitro. Simvastatin is an HMG-CoA reductase inhibitor that exerts pleiotropic effects beyond simple low-density lipoprotein lowering and has a similar impact on the differentiation of bone marrow stromal cells and peripheral blood mononuclear cells. The present study examined the hypothesis that the application of simvastatin would induce the HFSCs differentiation into keratinocyte. Methods: The bulge of the hair follicle was anatomized, and HFSCs were cultivated. The flow cytometry and immunocytochemical staining for detection of nestin, CD34, and Kr15 biomarkers were performed before differentiation. In order to hasten the HFSCs differentiation to keratinocyte, HFSCs were treated with 1 µM, 2 µM, and 5 µM of simvastatin daily for a week. After differentiation, the flow cytometry and immunocytochemical staining were performed with Kr15 and Kr10 biomarkers, and the MTT assay was carried out as an index of cell viability and cell growth. Results: Our results showed that bulge of HFSCs were nestin and CD34 positive and Kr15 negative. Simvastatin significantly increased the viability of HFSCs (p < 0.05) at the concentration of 5 µM. In addition, the percentages of keratinocyte-differentiated cells treated with 5 µM of simvastatin showed a significant increase compared to all other treated groups (p < 0.05). Conclusion: Our findings demonstrate that 5 µM of simvastatin could induce HFSCs differentiation into keratinocyte.

Synergistic effect of rapamycin and metformin against germ cell apoptosis and oxidative stress after testicular torsion/detorsion-induced ischemia/reperfusion in rats.

The aim of this study was to investigate the effects of rapamycin (rapa) and metformin (met), combined administration on testicular torsion-detorsion (T/D) injury. A total of 108 male rats were divided randomly into six groups (n = 18), control, sham-operated, T/D, T/D + met (100 mg/kg), T/D + rapa (0.25 mg/kg) and T/D + met (100 mg/kg)+rapa (0.25 mg/kg). Except for the control and sham groups, torsion was created by rotating the right testis 720° in a clockwise direction for 1 h. Treatment groups received drug intraperitoneally, 30 min before detorsion. The right testis of 6 animals from each group was excised 4 h after detorsion for the measurement of lipid peroxidation, caspase-3, and antioxidant enzyme activities. Histopathological changes and germ cell apoptosis were determined by measuring mean of seminiferous tubules diameters (MSTD) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) test in rest of animals, 24 h after detorsion. In T/D group tissue malondialdehyde (MDA) level and caspase-3 activity increased and the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) decreased in comparison with the control group after detorsion. Met and rapa separately pre-treatment reduced MDA and caspase-3 levels, normalized antioxidant enzyme activities, reduced germ cell apoptosis and improved the MSTD in comparison with T/D group. However combined administration of met and rapa indicated a significant augmented effect as compared to the individual drug interventions on the reversal of T/D induced oxidative stress, apoptosis, and histologic changes, suggesting a synergistic response. Thus, this study shows that rapa and met combination have significant synergistic effects against oxidative stress and apoptosis and opens up further possibilities for the design of new combinatorial therapies to prevent tissue damage after ischemia-reperfusion (I/R).