A low dose of simvastatin enhanced the therapeutic efficacy of mesenchymal stem cell (MSC) transplantation in skin wound healing in diabetic mice associated with increases in pAkt, SDF-1, and angiogenesis

Purpose We aimed to determine the possible mechanisms of underlying the effects of low dose simvastatin on enhancing the therapeutic efficacy of MSC transplantation in diabetic wound healing. Methods Balb/c nude mice were divided into five groups:- control mice (CON), diabetic mice (DM), diabetic mice pretreated with low-dose simvastatin (DM+SIM), diabetic mice implanted with MSCs (DM+MSCs) and diabetic mice pretreated with low-dose simvastatin and implanted with MSCs (DM+MSCs+SIM). Seven days before wound induction, low dose simvastatin was orally administered to the DM+SIM and DM+MSCs+SIM groups. Eleven weeks after the induction of diabetes, all mice were given bilateral full-thickness excisional back skin wounds. Results By comparing the DM+MSCs+SIM and DM+MSCs groups, the results showed that on day 14; the wound closure (%WC) and capillary vascularity (%CV) in the DM+MSCs+SIM group were significantly increased compared to those in the DM+MSCs group. In addition, by using immunohistochemical techniques, it was also shown that the expression of SDF-1, a chemotactic factor regulating the migration of stem cells, in the DM+SIM+MSCs group was increased compared with that in the DM+MSCs group. Furthermore, using phospho-Akt (S473) Pan Specific DuoSet IC ELISA (R&D Systems, USA) kits, the increased tissue Akt levels were found in the DM+SIM+MSCs group but not in the other groups. Conclusions Our study suggests that a low dose of simvastatin enhanced the therapeutic efficacy of MSCs in diabetic wound healing, and this effect was associated with increases in pAkt levels, SDF-1 levels, and angiogenesis, and improved wound closure.


Introduction
In diabetes, hyperglycemia-induced oxidative stress resulting from ROS 51 production has been observed in several cell types [1]. Studies have shown that 52 hyperglycemia-induced oxidative stress could lead to impaired wound healing via 53 inducing a prolonged chronic inflammatory state. This prolonged inflammatory 54 phase could in turn disturb collagen metabolism, resulting in poor blood supply, 55 the reduced production of growth factors, and reduced angiogenesis, all of which 56 contribute to delayed diabetic wound healing [2][3][4]. Nearby 2% to 3% of DM   In addition, accumulating evidence has revealed that the therapeutic 84 benefits of MSCs are largely dependent on their capacity to act as a trophic factor 85 pool. After MSCs home into the damaged tissue sites for repair, they will closely 86 interact with the local stimuli, such as inflammatory cytokines, the ligands of 87 By considering these previous findings, we hypothesized that the anti-104 oxidative stress and anti-inflammatory effects of low-dose simvastatin 105 supplementation will provide the optimal microenvironment for improving the    The animals were randomly divided into five groups, as follows: group 1 137 is the control group (CON; n=12); group 2 was the untreated diabetic group (DM; 138 n=12); group 3 was the diabetic group that received daily oral treatment with  The diabetic mice were induced by intraperitoneal injection of 145 streptozotocin (STZ; Sigma Chemical Co., USA, 45 mg/kg BW, daily for 5 days).

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Streptozotocin was freshly dissolved in citrate buffer, pH 4.5 (Sigma Chemical 147 Co., USA). The same volume of citrate buffer, pH 4.5, was injected via the same 148 route into nondiabetic control animals. Glucose levels were measured by a 149 glucometer in tail vein blood samples obtaineds 2 weeks after diabetic induction.

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Diabetic conditions were defined as a plasma glucose concentration equal to or 151 greater than 200 mg/dL [28].   Table 1.  In the 11th week after STZ induction, a cutaneous wound was generated.    The results were expressed as the means ± standard errors of mean (SEM).

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The differences between groups were determined by one-way analysis of  The increased in the tissue malondialdehyde levels (MDA, an ROS 287 indicator), in the wound area; confirmed that oxidative stress contributed to poor 288 diabetic wound healing (shown in Fig.1A). The consequent pathogenesis of 289 wounds under diabetes induced oxidative stress, which was confirmed by high 290 numbers of local infiltrating neutrophils, and high IL-6 levels, (Fig.1B, 1C), could   DM+MSCs+SIM groups than in the DM group (Fig 2A). However, on day 14, 339 the results showed no significant difference among all groups.

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The phosphatidylinositol 3-kinases (PI3Ks) and their downstream target 341 AKT are considered crucial signal transduction enzymes that have been 342 investigated extensively for their effects on cell proliferation, and angiogenesis 343 in wound healing. Therefore, we conducted further investigation by using both 344 ELISA and immunohistochemistry techniques to determine the effects of 345 simvastatin on VEGF, pAkt and SDF-1 levels in the wound area (Fig 2A-C). On 346 day 7 and day 14, pAkt and SDF-1 levels were significantly higher in the 347 DM+MSCs+SIM than that in the DM group.

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On day 14, a significant decreased %CV was observed in wounds in mice 349 in the DM compared to that in wounds in the control mice group. Interestingly, 350 on the day 14, the %CVs of all treatment-groups, including DM+SIM, 351 DM+MSCs, and DM+MSCs+SIM, were significantly higher than that in the DM 352 group (Fig 2D). (hMSCs-RFP) was observed by using confocal microscopy, (Fig.3A-D). In 361 addition, the area of wound neovascularization (expressing green fluorescent 362 FITC (Fig.3E) was also observed. By using Image Pro II software, the number

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In the present study, we aimed to identify the therapeutic agent that could   function and angiogenesis [49][50][51]. Therefore, it is notable that the cytokine