The serine/threonine kinase Akt (also known as protein kinase B or PKB), which was first identified as a proto-oncogene, has gained widespread attention due to its important roles in regulating a variety of cellular activities, including cell cycle, apoptosis, angiogenesis and glucose metabolism ^[1][59]. Phosphatidylinositide 3-Kinase (PI3K) is an intracellular phosphatidylinositide kinase that binds to Akt upon activation. Once activated, PI3K can be recruited to the cell membrane, causing the phosphorylation of phosphatidylinositol 4,5-diphosphate (PIP2) to generate phosphatidylinositol 3,4,5-triphosphate (PIP3), which then promotes the phosphorylation of Akt ^[2][60]. It has been shown that some of the factors involved in the PI3K/Akt signaling pathway, such as mammalian target of rapamycin (mTOR) and glycogen syntheses kinase-3 (GSK-3), contribute also to glucose metabolism and wound healing processes in diabetes mellitus ^[2][60].

Various exosomes derived from different types of stem cells hold great promise for diabetic wound healing through PI3K/Akt-modulated pathways. Zhang et al. showed that adipose-tissue-derived stem cells (ADSCs) promoted wound healing by releasing exosomes in terms of promoting fibroblast proliferation and migration and optimizing collagen deposition through PI3K/Akt signaling ^[3][32]. Chen et al. demonstrated that highly expressed miRNA-21 in ADSC-derived exosomes (ADSC-exos) promoted the migration and proliferation of human immortalized keratinocytes (HaCaTs) by up-regulation of matrix metalloproteinase-9 (MMP-9) expression through the PI3K/Akt pathway, which further accelerated wound healing ^[4][35]. In a study by Wang et al., it was found that the hypoxic adipose stem-cell (HypADSCs)-derived exosomes (HypADSC-exos) exhibited down-regulated miRNA-99b and miRNA-146-a genes and up-regulated miRNA-21-3p, miRNA-126-5p and miRNA-31-5p compared with ADSC-derived exosomes ^[5][36]. Such miRNAs induced the proliferation and migration of fibroblasts and enhanced the secretion of vascular endothelial growth factor (VEGF) and extracellular matrix by activating the PI3K/AKT signaling pathway and thus improved the quality of diabetic wounds ^[5][36]. Furthermore, in studies by Zhang et al., exosomes derived from miRNA-126-over-expressing human bone marrow mesenchymal stem cells (HBMMSC-exos) promoted human umbilical vein endothelial cell (HUVEC) proliferation, migration and angiogenesis by regulating the PI3K/Akt signaling pathway ^[6][37]. In addition, numbers of newly formed capillaries were significantly increased and wound healing processes were accelerated in vivo ^[6][37]. MSC-derived exosomes contain long non-coding RNA (lncRNA) cRNA H19 in diabetic foot ulcers (DFUs)DFUs, which promoted the proliferation and migration of fibroblasts and inhibited cell apoptosis and inflammation by activating the PI3K/Akt signaling pathway and thus promoted wound healing in DFU mice ^[7][38]. Similarly, exosomes secreted by oral squamous cell carcinoma cells (OSCC-exos) up-regulated miRNA-210-3p expression and down-regulated adrenaline A3 expression in HUVECs, thereby promoting vascular formation through the PI3K/Akt signaling pathway ^[8][39]. Moreover, human amniotic epithelial-cell-derived exosomes (HAEC-exos) promoted angiogenesis and activated fibroblast function by activating the PI3K-Akt-MTOR pathway, representing a novel diabetic wound-healing strategy ^[9][40].

The Wingless/Integrated (Wnt) signaling pathway exists widely in animals and is highly evolutionarily conserved. The Wnt signaling pathway plays an important role in early embryo development, organogenesis, tissue regeneration and other physiological processes. In particular, it is also involved in the regulation of skin development, angiogenesis and epithelial remodeling processes, which are closely related to diabetic wound healing ^[10][61]. The Wnt signaling pathway is a complex regulatory network, which consists of three branches: the classical Wnt signaling pathway, the Wnt/planner cell polarity (PCP) pathway and the Wnt/Ca²⁺ pathway ^[11][62]. Among them, the most closely related to diabetic wound healing is the classical Wnt signaling pathway, which is activated via β-catenin, through the Wnt/β-catenin pathway. Numerous studies have confirmed the involvement of the Wnt/β-catenin pathway in promoting angiogenesis and epithelial remodeling, as well as the proliferation, differentiation and migration of skin cells ^[12][63].

Chronic hyperglycemia leads to epithelial dysfunction, resulting in decreased angiogenic signaling and chronic wounds that have difficulty in healing. In the study by Xiong et al., significant up-regulation of MiRNA-20b-5p was observed in exosomes isolated from patients with type 2 diabetes mellitus (T2DM), which inhibited angiogenesis by regulating Wnt9b/β-catenin signaling. When MiRNA-20b-5p was knocked out, angiogenesis and wound healing were dramatically improved in diabetic mice ^[13][42]. Furthermore, Malat-1 was one of the earliest lncRNAs identified as being associated with human disease and has been reported to be involved in micro-vascular dysfunction caused by diabetes ^[14][64]. It was further confirmed that such protective effects could be attributed to the targeting of miRNA-124 and activation of the Wnt/β-catenin pathway, resulting in a positive role in cutaneous wound healing ^[15][65].

Nuclear factor-kappa B (NF-κB) is an important intracellular nuclear transcription factor, which participates in many physiological and pathological processes, such as inflammatory response, immune response, cell survival and apoptosis ^[16][66]. It is now believed the NF-κB pathway is the most typical pro-inflammatory signaling pathway based on its roles in the expression of pro-inflammatory genes, including cytokines, chemokines and adhesion molecules ^[17][67]. Extensive and persistent chronic inflammation is harmful for diabetic wound healing. However, a slow launch of inflammation and inadequate healing ability in wounds will also delay healing processes ^[18][68]. Therefore, the modulation of the NF-κB pathway by exosomes either through the inhibition of excessive inflammation or the activation of healing processes will conduce to favorable outcomes for diabetic wounds.

Hyperglycemia can trigger oxidative stress and promote the production of pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which can result in the activation of the NF-kB pathway and further promote the synthesis of inflammatory factors, disrupting diabetic wound healing ^[19][69]. Fan et al. showed that HBMMSCS-derived exosomes (HBMMSCS-exos) significantly inhibited the expression of pro-apoptotic proteins and pro-inflammatory factors, while the expression of anti-apoptotic proteins and anti-inflammatory factors was up-regulated in lipopolysaccharide (LPS)-induced PC12 cell apoptosis ^[20][70]. On the other hand, menstrual-blood mesenchymal stem-cell-derived exosomes (MenSC-exos) were shown to resolve inflammation via induced M1-M2 macrophage polarization and enhance angiogenesis through up-regulation of VEGF and re-epithelialization in diabetic mice. These effects were most likely achieved through up-regulation of NF-κB p65 subunit expression to activate the NF-κB signaling pathway ^[21][44]. Furthermore, Zhang et al. reported that Circ_0075932, a circular exosome secreted by adipocytes, activated inflammation and induced apoptosis of human dermal keratinocytes by directly binding to Pumilio 2 (PUM2) and promoting the PUM2-mediated NF-κB pathway ^[22][45]. Therefore, the silencing of PUM2 or blockade of NF-κB might be a useful tool to reduce inflammation and promote the healing of wounds. Moreover, a study by Wu et al. showed that ADSC-exos could enhance angiogenesis as well as migration and tube formation in HUVECs upon LPS stimulation ^[23][71]. The mechanistic study revealed that these effects were due to the activation of the NF-κB pathway. Therefore, both activation and inhibition of the NF-κB pathway mediated by exosomes could be beneficial for the healing of diabetic wounds, depending on the specific circumstances.

Mitogen-activated protein kinases (MAPKs) are a group of evolutionarily conserved serine/threonine protein kinases involved in various biological processes, such as cell growth, apoptosis, hormone signaling, immune response and inflammation ^[24][72]. It has been confirmed that MAPK genes can be divided into three main subfamilies, namely, extracellular signal regulated kinases (ERKs), Jun N-terminal kinases (JNKs) and the p38 MAPKs ^[24][72]. Impaired keratinocyte migration caused by hyperglycemic states is one of the important factors leading to the delay of wound healing in diabetics ^[25][73]. It has been suggested that the P38/MAPK pathway controls autophagy and regulates keratinocyte migration in wound healing. Li et al. found that the P38/MAPK pathway was down-regulated and accompanied by autophagy inactivation, which inhibited keratinocyte migration under high-glucose environments ^[25][73]. Moreover, it has been proved that negative-pressure wound therapy can inhibit inflammation and promote wound healing via down-regulation of the MAPK/JNK signaling pathway in diabetic foot patients ^[26][74], illustrating the important roles of the MAPK pathway in diabetic wound healing.

It is believed that sustained and chronic inflammation is also one of the key factors impairing the healing of diabetic wounds. In recent years, exosomes have emerged as new intercellular communication mediators and play important roles in regulating the inflammatory immune micro-environments of diabetic wounds. Chen et al. reported that exosomes from MSCs (MSC-exos) can protect β cells from hypoxia-induced apoptosis by carrying miRNA-21, alleviating endoplasmic reticulum (ER) stress and inhibiting P38 MAPK signaling ^[27][34], suggesting great potential in promoting diabetic wound healing. Additionally, exosomes secreted by umbilical cord mesenchymal stem cells (UC-MSCs) reduce the deposition of fibronectin and collagen I by inhibiting the cell proliferation mediated by the MAPK signaling pathway and reduce fibrosis ^[28][46]. It was also found that inhibition of miRNA-155 expression in exosomes derived from hypertrophic cardiomyocytes (HC-exos) can reduce inflammation and attenuate the responses of P38, JNK and ERK ^[29][47], revealing the potent modulatory abilities of exosomes in inflammatory responses and diabetic wound healing. Furthermore, angiogenesis is one of the processes required for proper healing of diabetic foot ulcers. Scar formation after wound healing is also an urgent problem to be solved. Studies have shown that ADSC-exos can increase the expression of MMP3 in fibroblasts by activating the ERK/MAPK pathway, regulate the proportion of type III collagen and facilitate the remodeling of ECM, which illustrates an innovative research direction for reducing scar formation in wound healing ^[30][48]. In addition, Li et al. found that exosomes from MiRNA-126-3p over-expressed synovial mesenchymal stem cells (SMSCs) promote the re-epithelialization of wound surfaces, accelerate angiogenesis and enhance collagen maturation by activating the MAPK/ERK signaling pathway and promote diabetic chronic wound healing ^[31][49].

The Notch gene was first discovered by Morgan and colleagues in 1917 in mutant fruit flies and exists widely in vertebrates and invertebrates. The Notch signaling pathway is composed of Notch receptors, Notch ligands, C-repeat binding transcription factor-1 (CBF-1), DNA binding protein and Notch regulatory molecules and has important roles in wound healing through the regulation of angiogenesis, cell migration and inflammatory responses ^[32][75]. Accumulating evidence has revealed that macrophage-modulated consistent and chronic inflammation is one of the key factors resulting in impaired healing in DFUs. It has been reported that Notch1 signaling can be activated by hyperglycemia in diabetic skin and inhibit diabetic wound healing ^[33][76]. Furthermore, Chen et al. found that the activation of Notch signaling could regulate the differentiation of macrophages into the M1 phenotype and promote inflammation, while blocking of Notch signaling could polarize macrophages into the M2 phenotype and inhibit inflammation ^[33][76]. Similarly, Andrew et al. confirmed the important roles of the Notch signaling pathway in directing macrophage function in wound repair, suggesting that Notch could be a translational target for the treatment of refractory diabetic wounds ^[34][77].

MSCs have been shown to promote angiogenesis through a hypoxia-enhanced mechanism. It was found that the exosomes released by MSCs with over-expressing hypoxia-inducible factor-1α (HIF-1α) generated an increased angiogenic capacity partly via the Notch pathway as a candidate mediator of exosome communication ^[35][50]. Similarly, Li et al. found that exosomes from human adipose-derived mesenchymal stem cells (ADMSC-exos) inhibited the production of extracellular matrix in keloid fibroblasts by partially down-regulating Notch-1 by preventing transforming growth factor-β2 expression ^[36][51]. In addition, recent studies have demonstrated that embryonic stem cells (ESCs) can stimulate Notch ligand Jagged1 (Jag1) over-expression through the Notch signaling pathway to accelerate diabetic wound healing ^[11][62]. The results showed that fetal dermal mesenchymal stem-cell-derived exos (FDMSC-exos) could activate the migration and proliferation of adult dermal fibroblasts through the Notch signaling pathway, promoting the deposition of extracellular matrix and re-epithelialization in the wound area to accelerate skin wound healing ^[37][52]. These results demonstrated that the use of FDMSC-exos, through down-regulation of the Notch signaling pathway, might be a promising strategy for the treatment of diabetic skin wounds.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) belongs to the Cap-n-Collar family of basic leucine zipper proteins and is best known as an important anti-oxidative stress signaling pathway ^[38][78]. An imbalance of free radicals and antioxidants in the body may lead to the overproduction of reactive oxygen species (ROS), resulting in tissue damage and refractory wound healing outcomes in DFU patients ^[39][79]. After sensing the redox status of the cell, Nrf2 can bind to antioxidant response elements (AREs) and activate various antioxidant genes. Under physiological conditions, kelch-like ECH-associated protein 1 (Keap1) is ubiquitous and degrades Nrf2. However, Keap1 and Nrf2 could be more easily dissociated under oxidative status ^[40][80]. Therefore, reducing reactive oxygen species (ROS) levels through the antioxidant Nrf2 signaling pathway can reduce oxidative-stress-induced injury and might promote diabetic wound healing ^[39][79].

Exosomal Nrf2 and exosomal Nrf2-mediated products have been shown to modulate oxidative hemostasis in target cells to induce regenerative wound repair, including diabetic foot ulcer repair ^[41][81]. For example, human embryonic stem-cell-derived exosomes (HESC-exos) increased the efficiency of ulcer healing by inducing angiogenesis through the Nrf2 signaling pathway in pressure ulcer healing in aged mice ^[42][82]. In addition, hyperglycemia may also lead to the premature senescence of endothelial progenitor cells (EPCs) and inflammation due to increased levels of ROS. Researchers found that ADSC-exos had the ability to promote EPC proliferation and angiogenesis in a high-glucose environment. More interestingly, the over-expression of Nrf2 can enhance, while the down-regulation of Nrf2 can inhibit, these effects ^[43][8]. Treatment with exosomes from Nrf2-overexpressing ADSCs significantly reduced ulcers in diabetic rat foot wounds, with increased granulation tissue formation, enhanced angiogenesis, as well as increased expression of growth factors. By contrast, the inflammatory and oxidative-stress-related proteins were significantly decreased ^[43][8]. Furthermore, in the study by Wang et al., BMSC-derived exosomes (BMSC-exos) promoted, while Nrf2-knockdown inhibited, EPC tube formation, re-epithelialization, collagen deposition and neovascularization ^[44][53]. Moreover, these effects were enhanced when treated with BMSC-derived exosomes combined with a small-molecule activator of Nrf2, namely, tert-butylhydroquinone (tBHQ). These results suggested that the combination of BMSC-exos and small-molecule Nrf2 activators could be a new option for the treatment of chronic diabetic wounds.

Hypoxia-inducible factor-1α (HIF-1α) has been identified as a key regulator of the response upon ischemic injury. Duscher et al. reported that deletion of HIF-1α in fibroblasts resulted in impaired wound vascularity and delayed wound healing ^[45][83]. This is because HIF-1α can further modulate the expression of factors involved in glucose metabolism and angiogenesis, such as VEGF and erythropoietin, thereby altering the ischemic state ^[45][83]. On the other hand, angiogenesis disorder is one of the well-known reasons for the delayed healing of diabetic wounds. The VEGF pathway has been established as one of the key regulators for angiogenesis. The binding of VEGF receptors to ligands could activate the downstream signaling cascades that promote endothelial cell proliferation, migration and survival ^[46][84]. Zhu et al. showed that roxadustat, a pharmaceutical component that uses hypoxia-inducible factors to increase erythropoietin expression, promoted angiogenesis by activating the HIF-1α/VEGF/VEGFR2 pathway and thus accelerated diabetic wound healing ^[47][85].

Plasma exosomes (P-exos) have been suggested to possess significant therapeutic efficacy in promoting diabetic wound healing ^[48][54]. The results showed that P-exos-loaded carboxymethyl chitosan (CMC) hydrogels promoted local wound healing processes and enhanced angiogenesis in type 1 diabetes patients by activating the angiogenesis-related pathways mediated by VEGF ^[48][54]. In another study, researchers investigated the roles of ADSC-exos over-expressing miRNA-21 in promoting endothelial angiogenesis. The results showed that ADSC-exos that over-expressed miRNA-21 significantly promoted the angiogenesis of HUVEC cells through the HIF-1α/VEGF pathway ^[49][55]. In addition, it was shown that ADSC-exos up-regulated the phosphorylation of Akt and the expression of HIF-1α in keratinocytes, confirming that the effects of ADSC-exos are based on the activation of the Akt/HIF-1α signaling pathway ^[50][56].

Transforming growth factor-β (TGF-β) is considered a pleiotropic signaling pathway that is involved in numerous processes, such as cell growth, differentiation, apoptosis, epithelial–mesenchymal transition and the production of extracellular matrix in both mature organisms and developing embryos ^[51][86]. Smad proteins are downstream of TGF-β family receptors and transmit the signals generated by the binding of TGF-β and its receptors from the cytoplasm to the nucleus. The TGF-β/Smad signaling pathway has also been confirmed to play a critical role in the regulation of extracellular matrix remodeling and wound healing ^[52][87].

Jiang et al. investigated the effects and the underlying mechanism of HBMMSC-derived exosomes (HBMMSC-exos) on cutaneous wound healing ^[53][88]. It was found that the HBMMSC-exos treatment effectively promoted skin cell growth in vitro and accelerated wound healing in vivo through inhibition of the TGF-β/Smad signal pathway. Interestingly, the HBMMSC-exos showed an improved effect relative to HBMMSC treatment, revealing that HBMMSC-exos could be a promising source for cell-free therapy and skin regeneration. In addition, wound healing after skin injury will inescapably result in the formation of scars, which is assumed to occur via the recruitment and maintained differentiation of myofibroblasts through the activation of TGF-β ^[54][89]. Therefore, the inhibition of the TGF-β signaling pathway has been considered an effective strategy for reducing scar formation. Recent studies have confirmed the effects of umbilical cord blood MSC-derived exos (UCBMSC-exos) in stimulating regenerative wound healing, inhibition of scar formation and myofibroblast accumulation through interference with the miRNA-21-5p- and miRNA-125b-5p-mediated TGF-β2/SMAD2 pathway ^[55][57]. These results suggest that the use of UCBMSC-exos might represent a novel strategy to prevent scar formation and modulate more appropriate wound healing in the clinical treatment of diabetic wounds.

Except for the exosome-mediated diabetic wound healing processes that occur through the separate signaling pathways discussed above, there are also interactions between the different signaling pathways. For example, melatonin (MT)-pretreated MSC-derived exosomes (MT-exos) exhibited significant suppression of the pro-inflammatory factors IL-1β and TNF-α as well as promotion of the anti-inflammatory factor IL-10 and thereby accelerated the healing of diabetic wounds ^[56][90]. It was shown that these inflammation-inhibitory activities were achieved through inhibition of the AKT signaling pathway, indicating cross-talk between AKT and inflammatory signaling pathways. Hu et al. found that exosomes derived from MSCs pretreated with pioglitazone (PGZ) significantly promoted the cell viability and proliferation of HUVECs damaged by high glucose through the activation of the PI3K/AKT/eNOS pathway, resulting in sufficient blood vessels in diabetic wound healing ^[57][91]. In addition, human adipose stem-cell-derived exosomes can stimulate the activation of the AKT and ERK signaling pathways in HUVECs, HaCATs and other cells and significantly increase epithelial regeneration and neovascularization, which accelerate diabetic wound closure ^[58][92].