mTOR inhibitor INK128 promotes diabetic wound healing by regulating MDSCs

Background: Skin wounds in diabetic patients are hardly to recover. Accumulating evidence has shown that mammalian target of rapamycin (mTOR) pathway and myeloid-derived suppressor cells (MDSCs) are involved in inammation-related response. INK128 is a novel mTOR kinase inhibitor in clinical development. However, the exact roles of MDSCs and INK128 in healing wound of diabetic patients are unclear. Methods: Mice models of normal, diabetic, and diabetic+INK128 were constructed. Bone marrow (BM)-derived macrophages and RAW264.7 cell line co-cultured with MDSCs, which were induced at different conditions. Flow cytometry, western blot, quantitative real-time PCR, and immunohistochemical analysis were performed. Results: Diabetic mice (DM) had a slower recovery rate, thinner epidermal and dermal, and less blood vessels than those of normal mice. MDSCs were abnormally accumulated in DM and mTOR was activated in MDSCs from DM or treated with high glucose. Moreover, mTOR signaling inhibitor INK128 could promoted wound healing through reducing the MDSCs. MDSCs function was disordered in DM and high glucose environments, while INK128 could help retrieve their function. Furthermore, high glucose and other factors in DM could promote M-MDSCs differentiation to M1 pro-inammatory macrophage cells, thus inhibiting wound healing. The differentiation, which was dependent on mTOR signaling, could be reversed by INK128. Conclusion: INK128 is potential to be developed as a clinical strategy to promote wound healing of diabetic patients.

Moreover, the pbFGF-loaded electrospun brous was reported to have the function of accelerating skin healing of diabetic patients [4]. Despite this, more therapies were demanded for diabetic patients to promote wound healing.
Wound healing is a multifaceted and dynamic process included four overlapping phases of coagulation, in ammation, proliferation and remodeling, which requires a well-orchestrated integration of the complex biological and molecular events of cells and cell signaling [5]. Usually, diabetic wound healing is characterized by delayed acute and chronic wound unveiling impaired healing due to a postponed, incomplete or uncoordinated healing process. Especially, diabetic wound exhibits a persistent in ammatory response, making wound di cult to transiton to the proliferation phase [6]. As well known, myeloid cells suh as macrophages and neutrophils are of vital importance in the process of wound healing mainly by triggering and regulating the in ammatory response [7,8]. Recently, it is found that myeloid-derived suppressor cells (MDSCs) are a group of immature and heterogeneous myeloid cell populations. Increased numbers of MDSCs have been observed under pathological conditions associated with in ammation such as tumors, autoimmune diseases, infection and obesity [9][10][11][12][13]. MDSCs can be characterized by the expression of CD11b and Gr-1 in mice [14]. They can be further divided into granulocyte-like MDSCs (G-MDSCs) and monocyte-like MDSCs (M-MDSCs) [11]. Studies have shown that the proportion of MDSCs in peripheral blood of type I diabetic patients is signi cantly higher than those of normal people, while the proportion of M-MDSCs decreases [15]. The frequency of CD33 + HLA -DR -/low MDSC-like cells is also higher in patients with type 2 diabetic mellitus (DM2) comparing with non-DM2 individuals [16]. Similar conclusion was drawn by Whit eld-Larry et al. [17], namely, MDSCs are unexpectedly enriched in peripheral blood of both mice and patients with autoimmune diabetes. In contrast, some studies reported that the immunosuppressive function of native T1D MDSCs was impaired. Arctigenin was able to ameliorate in ammation through accumulating G-MDSCs, and enhancing the immunosuppressive function of MDSCs [14]. This provides us with ideas that the changes both in number and function of MDSCs may be crucial for the development of in ammation state of diabetic mellitus and maladjustment of the wound healing process. Nonetheless, it is still not clear whether accumulation of MDSCs leads to impaired wound healing of diabetes, and which factors contribute to the changes of MDSCs in diabetic mellitus.
The mammalian target of rapamycin (mTOR) signaling pathway has been widely recognized to control cell survival, metabolism and proliferation [18]. mTOR forms the catalytic subunit of two distinct protein complexes, known as mTOR Complex 1 (mTORC1) and 2 (mTORC2) [19]. Studies suggest that mTOR activity mediates MDSCs accumulation in tumor and in ammatory diseases, thereby affecting the outcome of the diseases. Wu et al. reported that the allosteric mTORC1 inhibitor, rapamycin, inhibited MDSCs accumulation in tumor and skin allografts [20]. Welte et al. [21] showed that mTOR signaling in cancer cells dictated a mammary tumor's ability to stimulate MDSCs accumulation through regulating G-CSF. Of note, it is reported that the second generation mTOR inhibitor INK128 is an oral, highly effective and selective adenosine triphosphate (ATP) competitor that inhibits both mTORC1 and mTORC2 [22,23].
The rst generation mTOR inhibitor mainly inhibit the complex mTORC1, which may cause negative feedback on the PI3K signaling to be affected, thereby, enhancing the phosphorylation activity of AKT and making patients prone to drug resistence. Theoretically, INK128 may overcome the limitation of rapamycin, which represents the rst generation of mTOR signaling. According to our previous studies [24,25], INK128 had a good therapeutic action on lupus and colitis development by regulating MDSCs. A few studies suggest that the effects of mTOR on diabetes may be complexed with the anti-and prodiabetic effects. The activation of mTOR in β cells stimulated their proliferation, while the mTOR activation in immune cells exacerbated β cells dysfunction thus aggravating diabetes [26]. Accordingly, we propose hypothesis that INK128 may affect the course of wound healing in diabetic mice by adjusting MDSCs.
To verify the role of mTOR in MDSCs on regulating the diabetic skin wound healing, we both constructed streptozotocin (STZ)-induced diabetic mice and used different concentration of glucose to mimic the MDSCs living environment. By focusing on mTOR signaling, we found that high glucose environment activated mTOR signaling in MDSCs, which resulting in aberrant accumulation and differentiation of MDSCs. Moreover, we explored mechanism on treatment with INK128 in accelerating the wound closure of diabatic mice in vivo and in vitro. Our data indicate that INK128 is potential to promote wound healing of diabetic patients via regulating MDSCs.

Mice model construction
Male C57/B6 mice (6-8 weeks old) were obtained from the Model Animal Research Center of Nanjing University. They were kept under pathogene-free conditions in 12 h:12 h light and dark cycle. All procedures involving mice were approved by the Medical School for Animal Use and Care Committee of Nanjing University in accordance with guidelines of the US NIH. Diabetic mice were induced by low-dose injections of STZ. Mice were fasted for 5 h and then injected with vehicle or STZ (intra-peritoneal (i.p.) injection, 50mg/kg per day, pH4, dissolved in 0.1 M sodium citrate buffer) for 5 consecutive days. After blood glucose level keeps steadily over 16.6 mM for 3 weeks, two full-thickness wounds of 5 mm in diameter were made on the dorsal surface of mice. To evaluate the effects of INK128, one month after STZ injection, diabetic mice were received daily intraperitoneal injection of 1mg/kg INK128 for another 45 days, then two full-thickness wounds of 5 mm in diameter were made on the dorsal surface. Photos of wound area at 1d, 3d, 5d, 7d, 9d, 11d were taken and wound area was quanti ed by using ImageJ software (National Institutes of Health). 2-mm skin region surrounding the wound site was collected at 3d and 7d for further HE staning, immunostaning and immuno uorescence. After HE staining, the thickness of epidemis and dermis were measured by ImageJ software (National Institutes of Health).
Macrophage differentiation assay BM cells were cultured in the presence of 40ng/ml murine IL-6 and 40ng/ml GM-CSF and added 5mM glucose, 30mM glucose, 30mM glucose+50nM INK128 for 4 days. After the different incubation periods, MDSCs were collected and co-cultured with BMDM and RAW264.7 seperately. phenotypes of BMDM and RAW264.7 were determined by ow cytometry analysis.
Flow cytometry analysis BM cells, splenocytes and PBMCs from mice were prepared as single-cell suspensions. To detect mouse MDSC subsets, cells were pre-incubated with FITC-conjugated anti-mouse CD11b mAb and APCconjugated anti-mouse Gr-1 mAb, then they were stained for 20 min at room temperature in the dark. For the detection of macrophages, cells were labeled with FITC-conjugated anti-mouse CD11b mAb and APCconjugated anti-mouse F4/80 mAb, and then incubated for 20 min at room temperature in the dark. For the detection of M-MDSC and G-MDSC subsets, cells were labeled with FITC-conjugated anti-mouse CD11b mAb, APC-conjugated anti-mouse Ly6C mAb and PE-conjugated anti-mouse Ly6G mAb, then cells were incubated for 20 min at room temperature in the dark. Flow cytometry was performed on a FACSCalibur ow cytometer (BD Biosciences). The data was analyzed by the FlowJo software.

Western blot analysis
The protein expression levels of p-S6, S6, p-4EBP-1, 4EBP-1, on MDSCs were evaluated. β-Tubulin was used as an internal control in our study. Proteins were extracted on a normal way [28], and the western blot analysis was performed according to Wang et al. [29]. Protein bands were visualized using ECL Plus Western blotting detection reagents (Millipore, Bedford, MA, USA).

RNA extraction and quantitative real-time PCR
Total RNA of MDSCs were isolated with Trizol Reagent according to the manufacturer's instructions. Quantitative real-time PCR experiment was performed using SYBR green dye on Step One sequence detection system (Applied Biosystems, Waltham, MA, USA). Relative expression of genes was calculated using 2 −ΔΔCT method, with GAPDH as internal control. Primers sequences are as shown in Table 1.

Histologic and immunohistochemical analyses
Epidermal and dermal and blood vessels of wound skin tissue were obtained from para n-embedded tissue, xed in formalin and stained with HE, Masson, and CD31, as well as DAPI.

Statistics analysis
Results were expressed as mean±SEM of three independent experiments and each experiment were tripled. Data between two groups were statistically evaluated by Student's t-test. P <0.05 was presents as statistically signi cant difference.

Results
Skin wound recovery of diabetic mice is slow after injury To evaluate the wound closure rate of control mice and diabetic mice, a 5mm full-thickness round cut on the back was made, which could be normally healed within 11 days. As shown in the images in Figure 1A and 1B, the wound closure of diabetic mice was continuously slower than that of control group. The wound area of DM was signi cantly larger than that of CON on days of 3 (P < 0.05), 5 (P < 0.01), 7 (P < 0.05), 9 (P < 0.05), and 11 (P < 0.01). On the 11 th day of healing, the wound of CON was completely healed, while the DM still had obvious wounds. Then, the thicknesses of epidermal and dermal were assessed. The epidermal thickness of DM exhibited a signi cant reduction over 60% than CM wound (P < 0.01, Figure 1B). DM group displayed an ~35% reduction in dermal thickness compared with CON group (P < 0.05, Figure 1C). Moreover, we also calculated the number of endothelium blood vessels in DM and CON. Figure 1D showed that the number of endothelium blood vessels in DM was also signi cantly less than that of normal mice (P < 0.01) at 7-days after injury. Taken together, the DM exibited a slower recovery rate, thinner epidermis and dermis, and less regeneration of blood vessels than normal mice. mTOR inhibitor INK128 promotes skin wound healing and angiogenesis in diabetic mice The wound recovery rate of DM and DM+INK128 was assessed. As shown in Figure 2A that the wound healing rate in DM+INK128 did signi cantly increased than DM. The wound area of DM was signi cantly larger than that of DM+INK128 on days of 5 (P < 0.05), 7 (P < 0.01), 9 (P < 0.05), and 11 (P < 0.05). The epidermal thickness of DM was similar with that of DM+INK128 (P > 0.05, Figure 2B), whereas DM displayed a signi cant reduction in dermal thickness compared to DM+INK128 (P < 0.05, Figure 2C). Figure 2D showed that the number of endothelium blood vessels in DM was also signi cantly less than that of DM+INK128 (P < 0.01) at day 7, the proliferative phase after injury. These results suggest that INK128 may promote diabetic skin wound healing.
The percentage of MDSCs is increased in high glucose environment The key for proper wound healing is whether the various reactions in the in ammatory can transition to proliferative phases appropriately [30]. As reported that the MDSCs in peripheral blood mononuclear cell (PBMC) of type I diabetic patients are signi cantly accumulated than healthy human [15], we doubted that whether the slow recovery of wound related to the accumulation of MDSCs. Thus, the amount of MDSCs at in ammatory (3 days after injury) and proliferative (7 days after injury) phases in BM, spleen, and PBMC were detected.
We found that the percentage of MDSCs in BM, PBMC and spleen were signi cantly higher in DM than that in control group both at in ammatory and proliferative phases after injury (Figure 3A-F; P < 0.05). In diabetic mice, the in ltration of MDSCs cells in the skin tissue around the wound increased on in ammatory and proliferative phases after injury through immuno uorescence staining Gr-1 of skin tissue ( Figure 3G, H). These results indicated the persistence of in ammation during wound healing process in diabetic mice.
To investigate whether the increased glucose concentration in DM contributed to the accumulation of MDSCs, the BM cells were isolated, treated with IL-6, GM-CSF and graded glucose of 5mM, 10mM, 20mM, 30mM, 60mM, and 120mM for 4 days to generate MDSCs. Under the gradient addition of glucose in BM cells, the percentage of MDSCs exhibited an increasing trend ( Figure 3I). Then we chose 5mM and 30mM glucose concentration for further in vitro experiments corresponding to control mice and diabetic mice, for 25-35mM is commonly recognized range in hyperglycemic study [31][32][33][34]. Taken together, the results showed that MDSCs were abnormally accumulated in DM and high glucose promoted the increase of MDSCs proportion.

High glucose promotes the activation of mTOR signaling in MDSCs
To con rm the role of mTOR signaling on accumulation of MDSCs in high glucose. The activation of mTOR pathway were assessed by evaluating the protein expression of downstream signaling molecules phosphorylated mammalian target of rapamycin (p-4EBP1), 4EBP1, phosphorylated ribosomal protein S6 (p-S6) and S6, with β-Tublin as the reference. Our results showed that p-4EBP1 and p-S6 were signi cantly highly expressed in MDSCs isolated from diabetic mice than control mice ( Figure 4A). Moreover, the protein expression of p-4EBP1 and p-S6 in BM-derived MDSCs under the presence of 5mM and 30mM glucose were also evaluated. The results showed that p-4EBP1 and p-S6 were highly expressed in 30mM than that in 5mM ( Figure 4B). These results indicated that mTOR signaling in MDSCs was activated in high glucose microenvironment.

mTOR inhibitor INK128 inhibits the accumulation of MDSCs in high glucose
To explore the role of mTOR on MDSCs expansion, gradient doses (0nM, 25nM, 50nM, and 100nM) of INK128 was added to BM in the process of generating MDSCs. Figure 5A showed that the percentage of MDSCs under addition of 25mM, 50mM and 100mM INK128 was signi cantly decreased compared to control, which demonstrated that INK128 could suppress BM cells differentiate into MDSCs. BM cells under treatments of 5mM glucose, 30mM glucose, and 30mM glucose + 50nM INK128 showed that high glucose promoted MDSCs expansion, which can be inhibited by INK128 ( Figure 5B).
To further con rm INK128 inhibit the accumulation of glusoce-induced MDSCs in vivo, STZ-induced diabetic mice were treated with vehicle and 1mg/kg INK128 for 45 days. The percentage of MDSCs in BM, PBMC and spleen was signi cantly lower in DM+INK128 group than that in DM group both at in ammatory and proliferative phases after injury (Figure 5C-I; P < 0.05). In INK128 treated diabetic mice, the in ltration of MDSCs in the skin tissue around the wound signi cantly decreased at in ammatory and proliferative phases after injury through immuno uorescence staining Gr-1 of skin tissue ( Figure 5J, K). Taken together, these results suggested high glucose caused the accumulation of MDSCs on mTORdependent manner and INK128 inhibited the expansion of MDSCs in vitro and in vivo.

INK128 suppresses functional gene expression of high glucose-induced MDSCs
To examine whether the function of MDSC cells was changed in diabetic mice, the MDSCs were isolated from spleens and the expression of several functional molecules including p47phox, gp91phox, arginase-1 (Arg-1), and inducible nitric oxide synthase (iNOS) were detected. The results showed that the expression levels of them were signi cantly higher in diabetic mice than that in control mice (P < 0.05; Figure 6A-C). Moreover, the effect of INK128 on MDSCs function was also evaluated in vivo and in vitro. A reduction of gene expression of p47phox, gp91phox, arginase-1 (Arg-1), and inducible nitric oxide synthase (iNOS) was detected in INK128 treated diabetic mice compared with diabetic mice. The gene expression levels of Arg-1, iNOS, and IL-6 were assessed in MDSCs supplemented with glucose of 5mM, 30mM, and 30mM+INK128. These genes presented a higher expression level in 30mM glucose than that of 5mM and INK128 suppressed the elevation of gene expression in 30mM group (P < 0.05; Figure 6D-F).
Together, the results demonstrated that the MDSCs function were disordered in DM and high glucose environments, which could explain for the slow wound healing. Moreover, INK128 could help retrieve their function, thus promote wound healing.

INK128 inhibits high glucose-induced differentiation of MDSCs into macrophages
Macrophages are considered as the primary effector cells in regulating wound healing, unregulated macrophage activation represent a source of excessive in ammation, leading to aberrant wound healing [8,35,36]. MDSCs have the potential to differentiate to macrophages in chronic in ammation [37]. Diabetes presents a systemic in ammatory state. It is unclear whether high glucose promotes macrophage development and mTOR signaling is involved. Therefore, the amount of macrophage in BM and spleen of CON, DM, DM+INK128 were detected. Figure 7A-D showed that the percentage of CD11b + F4/80 + macrophages increased in diabetic mice compared with control group and INK128 reduced macrophages in DM (P < 0.05). Moreover, in the skin tissue around the wound of diabetic mice showed massive macrophage in ltration which was mitigated by INK128 treatment (Figure 7E, F).
To explore whether mTOR signaling was involved in differentiation of MDSC cells into macrophages in high glucose in vitro, BM cells were incubated with IL-6, GM-CSF as well as 5mM glucose, 30mM glucose, and 30mM glucose + INK128 for 4 days. The MDSCs treated with 30mM glucose displayed a higher macrophage amount than that of 5mM glucose and 30mM glucose + INK128 ( Figure 7G), which demonstrated that high glucose could promote MDSCs differentiated into macrophage, which was on a mTOR-dependent manner. Some studies have demonstrated that S100A8 and S100A9 proteins are directly involved in inhibiting MDSCs maturation [37]. Our result showed that INK128 signi cantly increased the expression levels of S100A8 and S100A9 (P < 0.05; Figure 7H). Taken together, high glucose promoted MDSCs to differentiate into macrophage, and INK128 suppressed the differentiation.

INK128 reduces differentiation of M-MDSCs into pro-in ammatory macrophages under induction of high glucose
The phenotype of mice MDSCs is CD11b + Gr-1 + , which can be further divided into two subtypes, including G-MDSCs and M-MDSCs. It was reported that M-MDSCs is the subtype which can differentiate into macrophages, therefore, we detected the percentage of M-MDSCs. Figure 8A-D demonstrated that the percentage of M-MDSCs in DM was signi cantly higher than that in CON and INK128 reduces the M-MDSC in DM (P < 0.05). In vitro, the percentage of M-MDSCs decreased within the increace of INK128 ( Figure 8E). Moreover, the percentage of M-MDSCs in 30mM glucose group was signi cantly higher than that in 5mM glucose and 30mM glucose + INK128 (P < 0.01; Figure 8F). In summary, the results suggested the percentage of M-MDSCs increased in diabetes and high glucose environment, which can be inhibited by INK128.
Macrophages can be divided into pro-in ammatory (M1) and anti-in ammatory (M2) types. In the early stage of wound formation, M1 macrophages in ltrate the periwound tissue, swallow pathogens and necrotic tissue, and play a cleaning role. In the later stage, M2 macrophages cells perform repair functions. The continuous presence of M1 type causes persistence of in ammatory state poor wound healing. [38] In our study, in ammatory cell model BMDM and RAW264.7 were co-cultured with MDSCs pretreated with glucose 5mM, 30mM, and 30mM+INK128 ( Figure 8G). Then, the expression of M1 macrophage markers, i.e., IL-6, and iNOS in BMDM cells, IL-6 and IL-1β in RAW264.7 cells were detected. The expression of M2 macrophage markers, i.e., CD206, IL-10 in BMDM cells, CD206 and IGF-1 in RAW264.7 cells were detected. The result showed that the relative expression levels of CD206 and IL-10 were signi cantly lower, and IL-6 and iNOS were signi cantly higher under 30mM glucose treatment in BMDM than that under 5mM glucose and 30mM glucose+INK128 ( Figure 8H). The relative expression levels of CD206 and IGF-1 were signi cantly lower, and IL-6 and IL-1β were signi cantly higher under 30mM glucose treatment in RAW164.7 than that under 5mM glucose and 30mM glucose+INK128 ( Figure  8I). These results indicated that MDSCs from high glucose environment promoted macrophages differentiate towards M1 type and INK128 suppressed the effect of high glucose. Taken together, these ndings demonstrated that high glucose caused M-MDSCs to differentiate into M1 type which can be inhibited by INK128.

Discussion
The skin of diabetic patients is easily damaged and di cult to cure, which troubles diabetic patients and affects their lives and health. Zhang et al. [39] reported that MDSCs ameliorated acute kidney injury and the protective effect was enhanced by mTOR signal inhibition. As MDSCs was signi cantly increased in type I diabetic patients [15,40], we wonder whether the MDSCs could promote wound recovery of diabetic patients through mTOR-dependency way. In the present study, we obtained the following conclusions: 1) MDSCs were abnormally accumulated in diabetic mice and high glucose environment; 2) mTOR signaling pathway promotes abnormal accumulation of MDSCs, moreover, the mTOR inhibitor INK128 can alleviate wound healing by regulating the accumulation of MDSCs; 3) the dysfunction of MDSCs in diabetic mice and high glucose environment leading to di cult wound healing; 4) It was the high glucose environment that promoted M-MDSCs to differentiate into pro-in ammatory macrophages, resulting in di cult wound healing. In the present study, the therapeutic action of mTOR inhibitor INK128 for healing of diabetic wound was covered for the rst time. Moreover, these ndings provide important theoretical basis for treating diabetic patients with di cult wound healing.
In the study, we found that the diabetic mice had a slower recovery rate, thinner epidermal and dermal, and less blood vessels than comtrol mice. In the previous studies, the healing methods is mainly around increasing angiogenesis, and proliferation of endothelial cells [2,3]. While, in the present study, we investigated the connection of wound healing with MDSCs, and tried to nd another molecular method to enhance wound recovery. The key for wound healing is whether the various reactions in the in ammatory and proliferative phases can be completed on time and appropriately [30], and the MDSCs was detected to be highly accumulated at in ammatory and proliferative phases in diabetic mice and cells in high glucose. The result was consistent with that in diabetic patients [40]. Moreover, we found that the MDSCs were increased in multiple organs (bone marrow, PBMC, and spleen) of the diabetic mice. Furthermore, we found that the MDSCs amount were correlated positively with the supplementary of glucose, which might indicate that high glucose in diabetic mice is the main factor responsible for MDSCs accumulation.
The increased mTOR activity is related to insulin resistance and short-term treatment with rapamycin can led to an increase of insulin sentitivity, thus ameliorate diabetic mellitus. Therefore, we speculated that mTOR inhibitor treaiment could promote diabetic wound healing by regulating MDSCs. However longterm and chronic mTOR inhibiton by rapamycin or other rapalogs has been associated with glucose intolerance [41]. Since the drug failure of rapamycin may due to incomplete mTOR suppression, INK128 was selected in our study for its ability to more effectively inhibit mTORC1, and to inhibit mTORC2 additionally [42]. As expected, the results in our present study con rmed that mTOR signaling could promote the MDSCs. When applied the mTOR inhibitor INK128 to diabetic mice and cells in high glucose, the amount of MDSCs was reduce, and the wound healing rate was improved. we demonstrated that the INK128 promoting wound healing through two ways in diabetic mice. Firstly, the function of MDSCs disordered in diabetic mellitus. We found that the level of ROS (up-regulated with P47 and GP91) produced by MDSCs in diabetic mice was signi cantly higher. The expression of Arg-1 and iNOS in MDSCs also increased signi cantly. Moreover, the gene expression levels of Arg-1, iNOS, and IL-6 in cells supplemented with high glucose in vitro was also signi cantly highly expressed. Under proin ammatory conditions, human islets produce and release IL-1, resulting in inhibition of β-cell function [43]. Besides, stimulation of mTOR in immune cells, such as NKs and CD8+T cells, ampli es their functions, potentially exacerbating immune-mediated -cell damage and dysfunction [18,44]. Elevated number of CD8+ T cells are implicated in the pathogenesis of TIDM. In humans and experimental animals with T2DM, migration to and in ltration of pancreatic islets with immune cells, especially macrophages, can be elevated. Secondly, mouse macrophages readily express iNOS in response to LPS and IFN-γ, and for this reason, it is recognized as an M1 macrophage marker in mice [45]. In the present study, the expression of iNOS was signi cantly higher in high glucose cells. M1 macrophages express CD86, and produce high levels of ROS and pro-in ammatory cytokines, including IL-6 [46], which was highly expressed in high glucose environment. Moreover, the M2 macrophages markers, such as CD206, IL-10, and IGF-1, with their expression signi cantly decreased in high glucose environment [47]. In the wound healing process, M1 pro-in ammatory macrophages was supposed to conversed to M2 anti-in ammatory macrophage. While, in diabetic mice, their function is improperly regulated, caused a prolonged M1 macrophage presence and ine cient transition to the M2 phenotype, with diabetic mice retained pro-in ammatory characteristics at day 10 after injury [48]. Notably, as the gene expression in 30mM+INK128 group have the consistent level with low glucose group, we suspected that the INK128 might promete macrophages into M2 proin ammatory phenotype, moreover, it might promote the transition from M1 to M2 macrophage, thus promoting wound healing. Moreover, mTOR suppression in uenced the differentiation of MDSCs, which further con rmed that mTOR-dependency way of MDSCs to regulete wound healing of diabetic mice. Some studies have shown that S100A8 and S100A9 proteins are directly involved in inhibiting the maturation of MDSCs [24], thus our study indicated that mTOR inhibitor INK128 have obvious effect on suppressing MDSCs to M1 macrophages. Together, the result revealed that injury caused M-MDSCs accumulation in in diabetic mice, which differentiated in M1 macrophages, thus suppressing wound healing. Conversely, mTOR inhibitor INK128 could decrease the percentage of M-MDSCs, recover the function of MDSCs, and inhibit M-MDSCs differentiated to M1 macrophage thus achieving the purpose of promoting wound healing of diabetic mice.

Conclusion
In summary, we demonstrated that the number and function of MDSCs in diabetic mice was disorder.
MDSCs in high glucose were abnormally accumulated and tend to differentiate into M1 macrophages, thus inhibited wound healing in an mTOR-dependent way. mTOR inhibitor INK128 could reverse high glucose induced changes of MDSCs, thus accelerate wound healing process. Taken together, these ndings highlight that INK128 is a potential therapeutic strategy to promote diabetic wound healing.