Exploring microRNAs in diabetic chronic cutaneous ulcers: Regulatory mechanisms and therapeutic potential

4.1. Role of miRNAs in regulation of inflammation and angiogenesis

The inflammatory response is the first stage of wound healing. After skin damage, a large number of inflammatory cells and inflammatory factors migrate to the wound site to repair damage and prevent microbial invasion. In human and mouse DCU tissue specimens, there is an increase in the expression of the cytokine IL‐1β, which further induces changes in macrophage phenotypes and promotes wound healing through inhibition of the IL‐1β signalling pathway (Mirza, Fang, Ennis, & Koh, 2013).

On the contrary, in human serum, high levels of IL‐1β, CCL2, and TNF‐α failed to promote the healing of ulcer wounds (Mirza, Fang, Weinheimer‐Haus, Ennis, & Koh, 2014). In the wound, macrophages are the main source of inflammatory factors, and they are a highly plastic cell that can form a range of different phenotypic cells, as the environment changes. For instance, at the inflammatory stage or the beginning of tissue formation, primarily due to a shift in macrophage populations, the macrophage phenotype at the wound changes from the inflammatory type to the anti‐inflammatory type. In addition to macrophages, the neutrophils that turn into the neutrophil extracellular traps (NETs) enhanced the wound's inflammatory response and had an adverse effect on the healing of DCU (Menegazzo et al., 2015). Moreover, in the continuous inflammatory process of a DCU wound, the expression of matrix metalloproteinases (MMPs: MMP‐1, MMP‐8, and MMP‐9) are increased, and the expression of MMP inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) is decreased. Down‐regulation of TIMPs decreased the remodelling of ECM, thus slowing the wound healing process (Gibson & Schultz, 2013). This unbalanced expression of MMPs and TIMPs not only increases the degradation of ECM but also prevents the migration of cells and the synthesis of collagen. Therefore, abnormal regulation of the inflammatory reaction can lead to wound infection, healing failure, or scar formation (Eming, Wynn, & Martin, 2017). While cytokines and protein kinases participate in the process of inflammation, a large number of studies have revealed that miRNAs and mRNAs also have essential roles in the process of chronic wound healing (Figure 1).

miRNAs, such as miR‐142, 155, 132, 21, and 191, are key regulators of the inflammatory response and tissue repair and act through the translation process of target mRNAs. One example is miR‐142 mediated inhibition of healing of wounds induced by Staphylococcus aureus infection. The complex of miR‐142‐Ago2 binds with the target genes Rac1 and RhoA and then regulates polarity, migration distance, and migration rate of neutrophils via inhibiting the transcription of GTPase, eventually promoting the accumulation of neutrophils at the wound site (Tanaka et al., 2017).

Another example is provided by miR‐146a, which binds to target genes IRAK1 and TRAF6, which promote the NF‐κB signalling pathway to induce inflammation‐mediated by inflammatory factors such as IL‐6, IL‐8, and the chemokine CXCL2. Thus, in whole, un‐injured skin, miR‐146a is highly expressed to inhibit inflammation, but once injury occurs and a wound is created, miR‐146a would be inhibited, allowing inflammatory processes to remove necrotic tissue and bacteria (Meisgen et al., 2014). In patients with DCU, miR‐146a was dysregulated, blocking the wound healing process (Xu et al., 2012).

Another miRNA which is elevated in patients with DCU, is miR‐155, a multifunctional miRNA that has significant roles in the development and function of immune cells. The expression of miR‐155 target genes, BCL6, RhoA, SHIP1, and FIZZ1 are increased in wounds. A reduction in miR‐155 expression can reduce the concentration of inflammatory cells in the wound site, reduce the inflammatory response, improve regeneration of the tissue structure, and accelerate wound closure (van Solingen, Araldi, Chamorro‐Jorganes, Fernández‐Hernando, & Suárez, 2014).

The anti‐inflammatory effect of miR‐132 has been demonstrated in numerous cells that are crucial for wound healing. For example, miR‐132 inhibits the production of a large number of pro‐inflammatory cytokines by Toll‐like receptors (TLR) induction in monocytes and macrophages (Liu et al., 2015). In neutrophils, the expression of miR‐132 is up‐regulated when cells migrate to the wound site (Larsen et al., 2013). In NK cells, IL‐12 signalling up‐regulates the expression of miR‐132, thus inhibiting the expression of STAT4 and IFN‐γ (Huang et al., 2011). In leukocytes, the inflammatory response induces the expression of miR‐132, which increases the level of the anti‐inflammatory transmitter ACh by regulating the targeting of acetylcholinesterase (Shaked et al., 2009).

Interestingly, recent observations have also demonstrated that miR‐132 binds to the target gene HBEGF, which reduces chemokines and reduces the ability of keratinocytes to attract white blood cells. Consistent with the anti‐inflammatory function of miR‐132, this miR is up‐regulated during the process of wound repair from inflammation to cell proliferation stages (Li et al., 2015).

Utilizing both pharmacological and genetic approaches, miR‐21 was also identified to be important for the resolution of inflammation. In macrophages, the expression of miR‐21 is induced by eliminating endogenous lipid mediators produced during the process of inflammatory regression (Recchiuti, Krishnamoorthy, Fredman, Chiang, & Serhan, 2011). At the same time, during metastasis of inflammation, which is a key process in tissue repair, macrophages ingest apoptotic neutrophils, and miR‐21 expression increases. In macrophages, miR‐21 induces an inflammatory reaction by silencing the LPS targets, PTEN and GSK‐3β. The combination of miR‐21 and the target gene PDCD4 enhances the activity of c‐Jun‐AP‐1, inducing macrophages to produce a large number of IL‐10 anti‐inflammatory cytokines, thus promoting wound healing (Herter & Xu Landén, 2017).

One of the important roles of inflammatory cytokines in the wound site is to regulate angiogenesis. Endothelial cell migration and capillary formation are the main steps in this stage. The entry of capillaries into the wound bed is the key in supporting regenerative tissue, so the inhibition of angiogenesis in the wound may be critical in healing of chronic wounds (Chan & Loscalzo, 2010). The first evidence that miRNAs are involved in angiogenesis comes from the observation that endothelial capillary germination, migration, and tubule formation can be inhibited by knockout of Dicer and Drosha (Kuehbacher, Urbich, Zeiher, & Dimmeler, 2007; Shilo, Roy, Khanna, & Sen, 2008). Embryonic angiogenesis in mice also needs Dicer, and the loss of endothelial miRNAs caused by the inactivation of Dicer will affect the angiogenic response to various stimuli after birth, including VEGF, tumours, limb ischaemia, and wound healing (Suárez et al., 2008). Recent research shows that the miR15b and miR200b are negative modulators in diabetic wound angiogenesis, and anti‐miR15b and anti‐miR200b treatment improved the wound healing process (Chan, Roy, Khanna, & Sen, 2012; Pizzino et al., 2018). Generally, existing research on miRNAs has mainly focused on the in vitro experiments. However, they may provide important clues for further direct study on the role of miRNAs in angiogenesis and wound healing.

Alternatively, studies have shown that, in the vascular endothelial cells of an ulcer, the inflammatory response stimulates the secretion of miR‐191. The miR‐191 then enters the bloodstream and is absorbed by the fibroblasts or vascular endothelial cells of the ulcer wound. Then, miR‐191 by its action on its target gene for zonula occludens‐1 (ZO‐1, also known as tight junction protein‐1), inhibits angiogenesis and cell migration, ultimately leading to a delay in chronic wound healing (Dangwal et al., 2015).

4.2. miRNAs and epithelialization of wound epidermis

Many studies have reported the involvement of miRNAs in wound epithelialization during the process of wound healing. Ghatak et al. (2015) investigated a wound healing model in mice and found that the expression of enzymes involved in wound healing was regulated by miRNAs, which accelerated wound healing. For example, in epithelial keratinocytes, the expression of mir‐146a is increased through the activation of TLRs (TLR2, TLR3, TLR4, and TLR5 (Meisgen et al., 2014). Li, Wang, et al. (2015) revealed a significant change in the expression level of miR‐132 during the transition from the inflammatory phase of wound healing to the proliferative phase. In vascular endothelial cells, miR‐132 was involved in the activation, proliferation, migration, and capillary formation of static endothelial cells through the regulation of the GTPase‐activating protein p120RasGAP (Anand et al., 2010). Recent studies have highlighted protective roles of miR‐21 in the proliferative phase of epidermal cells, as miR‐21 promoted the migration of keratinocytes and fibroblasts in vitro. It is believed that TIMP3 and TIAM1 are the direct target genes of miR‐21 and participate in the migration of keratinocytes. When miR‐21 is inhibited, the epithelialization process of the epidermis is delayed, and wound healing is slowed down (Madhyastha, Madhyastha, Nakajima, Omura, & Maruyama, 2012). In endothelial cells, the overexpression of miR‐21 inhibits the death domain‐related protein (DAXX), a known direct miR‐21 target that is also a key tumour suppressor gene (Liu et al., 2018), while the silenced target mRNA of DAXX reverses the inhibition of miR‐21 on the hyperglycaemia‐induced apoptosis of endothelial cells. These findings suggest that miR‐21 prevents apoptosis of endothelial cells by inhibiting expression of the DAXX protein.

In DCU, inhibiting the expression of miR‐26a causes granulation tissue hyperplasia and angiogenesis and promotes wound healing through the ID1/SMAD1 signalling pathway in endothelial cells. The inhibition of miR‐26a also enhances the aggregation of muscular fibroblasts, which is conducive to wound contraction (Icli et al., 2013). In a model of skin ulcers in a mouse model of Type 2 diabetes mellitus (T2DM), inhibition of the expression of miR‐26a promoted the formation of blood capillaries, increased wound perfusion, increased proliferation of granulation tissue, and promoted wound healing. Further, as PTEN is the direct target gene of miR‐26a, this miRNA promotes angiogenesis by inhibiting PTEN expression and then negatively regulating the PI3K/Akt signalling pathway (Liu et al., 2012).

Overexpressed miR‐27b directly interacts with the target genes TSP1, TSP2, p66shc, and semaphorin 6A(SEAM6A), promoting cell proliferation and migration, lumen formation, and adhesion enhancement and thereby accelerating wound healing (Wang et al., 2014). Also, miR‐27b is very sensitive to oxygen concentration and stimulates HIF to activate VEGF and glucose transporter type 1 (GLUT1) in a hypoxic environment, hence promoting wound surface angiogenesis (Brancato & Albina, 2011). Another study showed that the expression of the miR‐99 family is reduced in DCU and that the miRNAs family mediates PI3K/Akt signalling and keratinocyte migration and proliferation by regulating IGF1R, MTOR, and Akt1 target genes. These findings reveal the role of this miRNA family in epidermis epithelialization and wound healing (Jin et al., 2013). Furthermore, there is substantial evidence to show that miR‐31 can make keratinocytes continuously up‐regulated by stimulation of TGF‐β2 in wounds, inhibiting epithelial membrane protein as a consequence. This promotes keratinocyte proliferation and migration and accelerates epidermal epithelialization of wounds (Li et al., 2015).

A wide range of miRNAs has been associated with the proliferation and migration of keratinocytes and epithelial cells. In damaged skin, miR‐203 is down‐regulated in highly proliferating keratinocytes, and the target gene p63 has fundamental roles in cell proliferation. The new target genes p63, RAN, and RAPH1 are also essential in the epidermal epithelialization of wounds (Viticchiè et al., 2012). High expression of miR‐204 inhibits the proliferation and migration of cells, while down‐regulated expression increases the wound healing rate, and the expression of target genes SIRT1 and SMAD4 inhibits the proliferation and migration of epithelial cells (An et al., 2015). During the epithelialization of wound epidermis, the down‐regulation of miR‐205 expression affects the migration of keratinocytes from the wound edge to the wound site. In human epithelium, the expression of the target gene (SHIP2) of miR‐205 is increased, which reduces the healing ability of the wound. Local inhibition of miR‐205 promotes the differentiation and migration of keratinocytes in the wound site (Wang et al., 2016).

On the other hand, overexpression of miR‐210 is a feature of the hypoxia response, and the expression of miR‐210 is related to the oxygen concentration in cells. Recent findings have emphasized that in ischaemic wounds, HIF‐1α levels are dependent on miR‐210 expression. HIF‐1α is a transcription factor that regulates the hypoxia cycle and inhibits the proliferation of keratinocytes. In a local ischaemic chronic wound, the expression of the target gene of E2F3 of miR‐210 was found to be decreased, due to its importance in the proliferation of epithelial cells; thus, miR‐210 may inhibit the proliferation of epithelial cells in chronic wounds (Huang & Zuo, 2014).

In DCU, the expression of miR‐145 was proposed to be negatively correlated with TGF‐β1 regulation. TGF‐β1 can stimulate the secretion of ECM and has several important regulatory roles in morphological changes as well as proliferation and differentiation of monocytes, brain cells, and osteoblasts. in vivo studies have been clarified that when the expression of miR‐145 is down‐regulated, TGF‐β1 expression is up‐regulated, thus promoting wound healing and typical granulation tissue formation (Okizaki et al., 2015). Thus, miR‐145 may be a potential target gene or marker for the diagnosis or treatment of DCU. Otherwise, its specific mechanism remains to be elucidated.

4.3. miRNAs and tissue remodelling

When the wound is closed, the remodelling phase begins, which is the last stage of wound healing. A large number of miRNAs are reported to be involved in tissue remodelling.

The remodelling stage includes the recalibration of ECM, the transformation of type III collagen into type I collagen, and the cessation of scar formation in granulation tissue. In ECM, type I collagen rapidly replaces type III collagen (Xue & Jackson, 2015). Subsequently, granulation tissue stops growing, angiogenesis is decreased, and then wound healing and scar formation are completed (Fahs, Bi, Yu, Zhou, & Mi, 2015).

The miR‐29 family (miR‐29a, miR‐29b, and miR‐29c) plays an important regulatory role in the biological processes of cell proliferation and apoptosis. miR‐29a and their target genes of TABL1 can directly affect the expression of collagen in fibroblasts (Ciechomska, O'Reilly, Suwara, Bogunia‐Kubik, & van Laar, 2014). miR‐29b is related to the proliferation of fibroblasts induced by TGF‐β1, and several target genes of miR‐29b are COL3A1, COL4A1, COL4A2, and COL5A1, which are all involved in the regulation of ECM remodelling. Moreover, the up‐regulated expression of miR‐29b on the surface of a wound reduces wound contraction and increases the proportion of collagen III/I (Monaghan, Browne, Schenke‐Layland, & Pandit, 2014). Besides, miR‐29b inhibits the TGF‐β1/Smad/CTGF signalling pathway to reduce the formation of wound scars (Guo, Lin, Shao, Rong, & Zhang, 2017). In skin fibroblasts, a reduction in miR‐98 expression significantly increased the expression of the collagen type I α1 (Col1α1) gene and decreased apoptosis. Thus, miR‐98 has important roles in wound healing by enhancing the proliferation of fibroblasts and controlling scar formation (Bi, Chai, Yuan, Cao, & Li, 2017).

miR‐192/215 enhanced the expression of the target gene e‐calcium mucin (E‐cadherin) in wounds by inhibiting the translation of the ZEB2 gene, thus playing an important role in the reconstruction of the skin barrier in the wound (Wang et al., 2010). In addition to ZEB2, ZEB1 also plays important role in epithelial cell dynamics during development and in diabetic wound healing impairment (Singh et al., 2019; Vannier, Mock, Brabletz, & Driever, 2013). Singh et al. (2019) reported that Zeb1 ^+/− mice exhibited compromised wound closure complicated by defective EMT and poor wound angiogenesis, indicating that ZEB1 expression may be associated with progression of DCU healing. Also, ZEB1 is target of several miRNAs including the miR‐200 family, miR‐203, miR‐655, and miR‐1199 (Diepenbruck et al., 2017; Harazono et al., 2013; Singh, Prasad, & Katiyar, 2016).

A recent discovery suggested that miR‐185 can directly interact with target genes TGF‐β1 and Col‐1 and inhibit the growth of fibroblasts, which in turn has a key role in the formation of proliferative scars (Zhao, Wang, Zhou, Zheng, & Zhao, 2017). In summary, fewer miRNAs have been studied as regulators of tissue remodelling. Therefore, further work is needed at all stages of wound healing to identify more of the various miRNAs involved in regulation of wound healing.

5.1. Epigenetic regulation of miRNA expression in DCU

Clinical studies have found that people with diabetes are more likely to develop chronic complications of diabetes even though their blood glucose has returned to normal or close to normal after treatment. This is due to poor blood glucose control before or at the beginning of diagnosis. At these early stages of the disease, the body forms high glucose metabolic memory, a phenomenon known as “vascular glycaemic memory” or “metabolic memory” (Diabetes Control and Complications Trial Research Group, et al., 1993).

The mechanism(s) underlying “metabolic memory” is not clear. Epigenetic regulation may be involved in the pathogenesis of diabetes and its complications by affecting the occurrence of metabolic memory effect (Reddy, Zhang, & Natarajan, 2015). Recent studies have shown that epigenetic regulation of DNA methylation, histone modification, and miRNAs are involved in the four stages of inflammation, angiogenesis, re‐epithelialization, and remodelling of diabetic wound healing (Ling & Rönn, 2019).

Recently, the epigenetic regulation of miRNA has been studied and found to be associated with diabetic complications (Morales, Monzo, & Navarro, 2017). Singh et al. (2017) have demonstrated that hyperglycaemia may drive vasculopathy by epigenetic modification of a miR promoter, and they have provided the first evidence of the miR‐200b promoter methylation as a critical determinant of angiogenesis in diabetic wounds.

5.2. Wnt/β‐catenin signalling pathway

The Wnt signalling pathway is divided into two subcategories: the classical signalling pathway (Wnt/β‐catenin pathway) and the non‐classical signalling pathway. Classical Wnt signalling is transduced by frizzled receptor proteins (Bhanot et al., 1996) and LRP5/LRP6 (Pinson, Brennan, Monkley, Avery, & Skarnes, 2000), while non‐classical Wnt signalling pathways, such as the planar cell polar signalling pathway and the Wnt‐Ca²⁺ pathway, are replaced by an alternative effect of molecular transduction and that is independent of the β‐catenin protein (Gao & Chen, 2010). In 1976, the wingless gene wg1, necessary for the development of the wings of fruit flies, was found (Sharma & Chopra, 1976). A few years later, the mammalian oncogene Int1 was the first identified Wnt gene (Wnt1) (Nusse & Varmus, 1982). With the discovery of these two homologous genes, the Wnt signalling pathway was recognized (Nusse & Varmus, 2012). At present, the Wnt pathway is composed of 19 secretory protein families, including the frizzled protein family of transmembrane receptors (Nusse & Clevers, 2017), the LDL receptor‐related protein (LRP) family, and several downstream pathways involving the composition protein and effect protein (Green, Nusse, & van Amerongen, 2014). Thus, Wnt proteins play an important role in developing and maintaining balanced cells in the body (Archbold, Yang, Chen, & Cadigan, 2012). β‐Catenin is a transcription‐assisted activation factor that binds to T cells or lymphocytes and serves essential roles in the proliferation phase of wound healing. It is also a key regulator of the Wnt pathway (Bielefeld et al., 2011). It has been demonstrated that several Wnt family glycoproteins in the Wnt/β‐catenin signalling pathway can directly or indirectly enhance chronic wound healing, and the activation of the Wnt/β‐catenin signalling pathway has important functional roles in cell proliferation and angiogenesis, as well as epithelial remodelling in wound repair (Amini‐Nik et al., 2014). A large number of experimental studies have reported that proteins and gene molecules in the Wnt/β‐catenin signalling pathway are involved in the regulation of a range of biological processes, including cell proliferation, differentiation, and apoptosis, as well as in maintaining the multifunctional properties of stem cells. In the process of chronic wound repair, Zhao et al. (2019) reported that miR‐218‐5p regulates skin and hair follicle development through Wnt/β‐catenin signalling pathway by targeting SFRP2, but how miR‐218‐5p and its target genes regulate the signalling pathway and affect the healing of skin ulcer wounds with the formation of scars require further studies.

5.3. NF‐κB signalling pathway

NF‐κB is a transcriptional factor that forms dimeric complexes with the Rel protein family (Zhang, Lenardo, & Baltimore, 2017). The five protein factors of the Rel protein families include p65 (RelA), Relb, C‐rel, p50/p105 (NFκB 1), and p52/p100 (NFκB 2). The classical NF‐κB signalling pathway primarily uses p65:p50 heterodimer transcription factors to activate downstream target genes for IL‐6, IL‐8, IL‐1β, and TNF‐α. Moreover, two key regulatory molecules, IRAK1 and TRAF6, also increase the activity of the NF‐κB signalling pathway (Ben et al., 2019; Perkins, 2012). In unstimulated cells, the RelA:p50 heterogeneous dimer binds to an inhibitor of the NF‐κB‐IκB protein, preventing it from entering the nucleus and blocking gene transcription as a result (Oeckinghaus, Hayden, & Ghosh, 2011). In summary, the NF‐κB signalling pathway is involved in a range of inflammatory and immune response processes of cell growth, proliferation, and development. The NF‐κB signalling pathway also responds to a variety of signalling pathways, which include cytokines, pathogens, trauma, and stress environments of other signalling (Lopez‐Dee & Ling, 2015). In the process of chronic wound repair, the NF‐κB signalling pathway not only regulates the scar TGF‐α signal in the wound remodelling stage but also binds with the specific molecular iNOS that regulates the promoter region of the inflammatory response target gene, thereby regulating the proliferation and apoptosis process of cells (Huang, Akaishi, & Ogawa, 2012).Non‐coding gene miRNA was also involved in the regulation of the NF‐κB signalling pathway and the healing of chronic skin ulcer wounds. For example, after the activation of TLR4 in monocytes and neutrophils during the inflammatory response to wound healing, miR‐9 directly combines with the target p50 mRNA to inhibit the NF‐κB signalling pathway and control cell activation. This indicates that the gene is a key transcription regulator of the inflammatory response (Bazzoni et al., 2009). Similarly, Xu et al. (2012) highlighted that the expression of miR‐146a was down‐regulated in diabetic skin ulcer wounds, and the expressions of pro‐inflammatory target genes IRAK1 and TRAF6 were increased, which resulted in activation of the NF‐κB signalling pathway, leading to up‐regulation of the IL‐6 and IMP‐2 genes. This caused a delay in wound repair and healing. Subsequently, Gantier et al. (2012) identified that in fibroblasts, miR‐19 inhibits the expression of the 3′UTR reporter gene of the target genes Kdm2a and Zbtb16 through the NF‐κB signalling pathway and high expression of the 3′UTR reporter gene of the target gene Rnf11. These findings indicate that the Rnf11 target gene is a key element in the miR‐19‐regulated NF‐κB signalling pathway. In addition, miR‐19 also regulates the expression of the suppressor of cytokine signalling 1 (SOCS1) by blocking the TLR2/4 signalling pathway, thereby reducing the activity of NF‐κB in the inflammatory response (Strebovsky, Walker, Lang, & Dalpke, 2011) (Figure 2). In summary, miRNAs can regulate the inflammatory response by regulating the NF‐κB signalling pathway, and thus contributing to the repair and healing of wounds.

5.4. PI3K/Akt/mTOR signalling pathway

The phosphoinositide‐3‐kinase/Akt/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway plays a pivotal role in regulating cell proliferation, growth, metabolism, and angiogenesis, and this signalling pathway also interacts with other pathways such as the Ras/MAPK/NF‐κB/Wnt/β‐catenin signalling pathway. Excessive activation of the PI3K/Akt/mTOR pathway causes significant dysfunction of normal cellular function, which in turn leads to competitive growth metabolism and angiogenesis (Yang, Tsai, Korivi, Chang, & Hseu, 2017). In this signalling pathway, PI3K activates phosphorylation and leads to the activation of Akt. Akt regulates cell proliferation, protein translation, and autophagy through indirect activation of mTOR, which exists in the form of two multi‐protein complexes of mTORC1 and mTORC2 and acts as a downstream effector of the PI3K/Akt signalling pathway (Keppler‐Noreuil, Parker, Darling, & Martinez‐Agosto, 2016).

The PI3K/Akt/mTOR pathway not only participates in cell proliferation and migration but also regulates protein synthesis within cells to control metabolism. Abnormal activation of the PI3K/Akt/mTOR pathway is usually observed in malignant gastric cancer (Wang et al., 2018), prostate cancer (Xu, Ge, Zhang, & Zhou, 2017), and oral squamous cell carcinoma (Wang, Zhang, Song, & Zhang, 2018), whereas the pathway also plays an important role in the chronic wound healing process (Xiao et al., 2017).

Squarize, Castilho, Bugge, and Gutkind (2010) demonstrated that in the process of promoting chronic wound repair, PI3K/Akt/mTOR activation is achieved by activation of the mTOR protein. More importantly, the more active the mTOR protein is, the more significantly it accelerates wound healing. Jin et al. (2013) suggested that the non‐coding gene miRNA‐99 family (miRNA‐99a/b and miRNA‐100) regulates chronic wound healing by targeting multiple genes in the PI3K/Akt/mTOR signalling pathway. Regulation of the target genes IGF1R, MTOR, and ATK1 further regulates the migration and proliferation of keratinocytes at the wound surface. The IGF1R, MTOR, and ATK1 genes are also major and direct targets in the PI3K/Akt/mTOR signalling pathway. When the skin is damaged, down‐regulation of the miR‐99 family leads to the activation of the PI3K/Akt/mTOR signalling pathway and regulation target genes of the miR‐99 family for cell proliferation and migration, thus promoting wound closure (Emmerson et al., 2012; Jin et al., 2013) (Figure 3).

5.5. TGF‐β/Smad signalling pathway

An increasing number of growth factors and cytokines have been identified to be involved in the repair and healing of chronic wounds. These include TGF‐βs, PDGFs, EGFs, FGFs, VEGFs, and various pro‐inflammatory interleukins (Schultz & Wysocki, 2009). These factors are released by different types of cells. In this review, we focus on the role of one cytokine mediator, TGF‐β, in the healing of chronic skin wounds (Figure 4).

In the process of chronic wound repair, TGF‐β is secreted by various types of cells, including macrophages and fibroblasts and is also released from the storage site of ECM (Margadant & Sonnenberg, 2010). Early secretion of TGF‐β1 can promote wound fibrosis and scar formation. TGF‐β2 promotes the development of hair follicles, while TGF‐β3 has a role in anti‐scar formation (Foitzik, Paus, Doetschman, & Dotto, 1999). Zhang, Wu, Qin, Chen, and Zhang (2014) identified that during wound healing in elderly rats, TGF‐β1 could cause ECM deposition, intracellular flow and proliferation of fibroblasts, the phenotypic transformation of fibroblasts into myofibroblasts, angiogenesis, inflammatory cell infiltration, wound contraction, and epithelial closure. Wang et al. (2006) showed that compared with fibroblasts in normal skin, fibroblasts in chronic ulcer wounds can express higher levels of TGF‐β1, TGF‐β2, and collagen α1 at mRNA and protein levels.

In the TGF‐β/Smad signalling pathway, not only related cytokines, growth factors, and proteins play a role but also genes, such as non‐coding genes miRNAs, siRNA, mRNAs, whic jointly regulate this pathway. Li, Li, et al. (2015) reported that up‐regulating the expression of miR‐31 induces TGF‐β1 and TGF‐β2 expression in wound marginal keratinocytes, and high miR‐31 expressions in the TGF‐β signalling pathway are involved in chronic wound healing. This expression negatively regulates the epithelial membrane protein 1 (EMP‐1), which promotes the proliferation and migration of keratinocytes and accelerates the repair and healing of wounds. Moreover, Ramirez et al. (2014) also indicated that when the skin is damaged, the expression of miR‐31 mediates the high expression of TGF‐β1 and TGF‐β2, and the migration of keratinocytes at the wound surface is accelerated, leading to epithelialization of the wound surface.

In the process of transformation from inflammation to epidermal epithelialization in chronic wound healing, miR‐132, as a key regulatory molecule in the TGF‐β signalling pathway, up‐regulates the TGF‐β1 and TGF‐β2 expression by regulating the target gene of HB‐EGF. At the same time, miR‐132 promotes the migration of keratinocytes and later induces epidermal regeneration and accelerates wound healing (Li, Wang, et al., 2015).

In the remodelling stage of chronic wound healing, miR‐1908 regulates the TGF‐β signalling pathway through the target gene Ski, and the TGF‐β signalling pathway promotes the proliferation of fibroblasts and increases the expression levels of TGF‐β1, IL‐1α, TNF‐α, and type I collagen, thereby promoting the formation scarring (Xie, Shi, Zhang, Zhao, & Yu, 2016).

In DCU wounds, the increase in TGF‐β1 expression in the TGF‐β signalling pathway can promote the healing of the ulcer wounds and the formation of granulation tissue. Zhang, Ren, Liu, Ren, and Wang (2016) found that in DCU patients with amputation of the muscle, miR‐145 expression was increased, and TGF‐β1 expression was significantly decreased. miR‐145 regulated the expression of TGF‐β1, indicating that this may be one of the factors affecting ulcer wound healing. While it appears that TGF‐β1 may be a key regulatory molecule involved in wound healing, its specific mechanism of action requires further investigation in future studies (Okizaki et al., 2015).

5.6. VEGF signalling pathway

VEGF is a 34–42 kD homodimer heparin‐binding glycoprotein molecule produced by cells. It is also an important angiogenic factor affecting endothelial cells, inflammatory cells, and neuronal cells. Several studies have reported that, in the process of chronic wound healing in DCU, the formation of blood vessels in the wound is related to damage to the VEGF‐2 receptors (VEGFR‐2) (Zhou, Ma, & Brogan, 2015). In the skin tissue of DCU patients, the expression levels of VEGF‐A and VEGFR‐2 in the VEGF signalling pathway are decreased. In an animal DCU model, the mRNA expression levels of VEGF on the wound surface of DCU mice were decreased, compared with normal mice (Frank et al., 1995). Krishnan, Quattrini, Jeziorska, Malik, and Rayman (2007) reported that the expression of VEGFR‐2 is decreased in epidermal and dermal neovascularization of the wound surface in DCU patients, while the expression of VEGFR‐1/2 is increased. Further, VEGFR‐1‐mediated macrophages inhibited the production of inflammatory factor IL‐1, and the expression of VEGF‐A was found to be decreased in streptozotocin (STZ)‐induced animal models (Okizaki et al., 2016).

We know that the healing of chronic ulcer wounds is a complex physiological process. When the wound secretes a hypoxia‐responsive transcription factor HIF‐1α, it can stimulate the release of VEGF‐A. VEGF‐A combines with VEGFRs on vascular endothelial cells, triggers the TK pathway to promote angiogenesis, and accelerates the healing of ulcer wounds (Palmer & Clegg, 2014). Conversely, several studies have found that many non‐coding RNAs can promote the healing of chronic wounds by regulating the VEGF signalling pathway. Brouillet et al. (2010) concluded that the expression of the VEGF gene in the VEGF signalling pathway is up‐regulated by the overexpression of two small interference RNAs (siRNAs), which targeted LITAF and STAT6B. When the target genes mLITAF and mSTAT6B were missing, the expression of the VEGF protein and the mVEGF gene was found to be decreased significantly, and endothelial cell migration and angiogenesis were inhibited. Neither of these two changes is conducive to chronic wound healing (Tang et al., 2013). Cakmak, Gokmen, Bozkurt, Kocaturk, and Ergin (2018) showed that, during the generation of granulation tissue and the inflammation stage of wound healing, miR‐15b and miR‐21a regulate HIF‐1α secretion from the wound, further promoting the release of VEGF‐A. VEGF‐A combined with VEGFR1/2 promotes angiogenesis and accelerates wound healing.

5.7. Other signalling pathways

In addition to the above main signalling pathways, several other signalling pathways are involved in the regulation of healing of chronic ulcer wounds, including MAPK signalling pathway, insulin signalling pathway, and ErbB pathway. Several miRNAs involved in the regulation of the MAPK signalling pathway in chronic wound repair have been reported including miR‐31, ‐204, ‐335, ‐127, ‐34b/c, ‐182, ‐150, let‐7d, ‐149‐5p, ‐203a, ‐222‐3p, and ‐122‐5p. miRNAs involved in regulation of the insulin signalling pathway include miR‐338, ‐150, ‐346, ‐542, ‐34b, ‐203a, and ‐122‐5p. Those involved in the regulation of the ErbB pathways are miR‐222, ‐200c, ‐34b/c, ‐150, and ‐222‐3p. In contrast, the specific mechanism underlying the effect of these miRNAs on each pathway remains unclear (Liu et al., 2015; Zhao et al., 2019). Therefore, to date, few studies have reported that non‐coding genes participate in signalling pathways to regulation of the known major signal pathways involved in chronic wound repair. However, further evidence is needed to establish the specific mechanisms of non‐coding genes in other signalling pathways.

6.1. Genetic drugs and stem cells

Currently, several methods of treatment based on miRNAs are at different stages of clinical trials. In the field of diabetic complications, miR‐103/107 are currently in the Phase 1 clinical trials. There are indications that they can increase insulin sensitivity in patients with T2DM to comprehensively regulate the disease condition and further control the occurrence and development of diabetic complications (Trajkovski et al., 2011). What is more, Gallant‐Behm et al. (2018) found that miRNA‐92a‐3p is a potent anti‐angiogenic miRNA whose inhibition has led to increases in angiogenesis in multiple organ systems, resulting in an improvement in function following myocardial infarction, limb ischaemia, vascular injury, and bone fracture. These authors investigated the effect of a development stage locked nucleic acid‐modified miR‐92a inhibitor, MRG‐110, in excisional wounds in db/db mice and in normal pigs. These studies suggested that MRG‐110 has the potential to accelerate both chronic and acute wound healing, and provide support for future clinical trials of MRG‐110. Besides, Xu et al. (2012) showed that in DCU wounds, the expression of miRNA‐146a decreased, and the expression of pro‐inflammatory target genes IRAK1, TRAF6, and NF‐kB increased significantly. This results in an increase in the expression of IL‐6 and CXCL‐2 genes, which causes abnormal regulation of the inflammatory response in the wound. By comparison, after treatment with mesenchymal stem cells (MSC), miRNA‐146a expression was up‐regulated, and the expression of pro‐inflammatory‐related IL‐6 and CXCL‐2 genes was down‐regulated, thus promoting wound healing.

6.2. Natural products

Skin fibroblasts are important players in chronic wound healing and the natural product Notoginsenoside Ftl promoted cell proliferation and collagen formation through the PI3K/Akt/mTOR signalling pathway (Huang et al., 2017). At the same time, Ftl accelerated wound epithelial repair and the formation of granulation tissue and increased the expression of α‐SMA, COL1A1, COL3A1, TGFβ1, TGFβ3, and fibronectin mRNA. Further, Ftl promoted new angiogenesis and increased the mRNA expression of VEGF, PDGF, and FGF, and the expression levels of TNF‐α and IL‐6 mRNA were reduced, and the infiltration of monocytes in the inflammatory response was reduced, thus promoting wound healing (Zhang, Gao, Yang, Wu, & Wang, 2016).

Emodin (1,3,8‐trihydroxy‐6‐methyl‐9,10‐anthraquinone; C₁₅H₁₀O₅ MW 270‐23) is a natural anthraquinone derivative, found in rhubarb, Polygonum multiflorum, Semen Cassiae, the vine of multi flowers knotweed, Polygonum cuspidatum, and other plants. Emodin has anti‐inflammatory (Chen et al., 2016), antibacterial (Li et al., 2016), antioxidant (Chen et al., 2017), anti‐fibrosis, anti‐tumour, and other pharmacological activities (Ma et al., 2017). In an analysis of diabetic peripheral neuropathy, the cell line PC‐12 was exposed to a high glucose environment and treated with emodin. The results showed that emodin protected PC‐12 cells from apoptosis and autophagy induced by the high glucose environment. The expression of non‐coding gene miR‐9 was also up‐regulated and the protective effect of emodin was lost when miR‐9 was inhibited. Meanwhile, emodin induced the activation of PI3K/Akt signalling and inhibited NF‐κB signalling, effects which was also reversed when miR‐9 expression was low. It appears that emodin may be an effective means of preventing DCU (Fan et al., 2018).

Lucidone is a naturally occurring cyclopentenedione, isolated from the dried fruits of Lindera erythrocarpa Makino (Lauraceae). The dried fruits of L. erythrocarpa have been used in traditional Chinese medicine as a digestive, analgesic, diuretic, antidote, and antibacterial substances (Wang et al., 2008). The isolated monomer component lucidone has pharmacological value, with antioxidant, anti‐inflammatory, neuroprotective, and antiviral effects (Chen et al., 2013). In keratinocytes, lucidone inhibited oxidative stress and inflammatory reactions induced by free radicals (Kumar et al., 2013). Recently, Hseu et al. (2015) have demonstrated that lucidone can not only protect UV‐induced DNA damage and mitochondrial dysfunction in skin keratinocytes but also activate the PI3K/Akt, Wnt/β‐catenin, and NF‐κB pathways during chronic wound healing. When these pathways were activated, lucidone accelerated the growth and migration of keratinocytes, fibroblasts, and endothelial cells, thus promoting the repair and healing of chronic wounds (Yang et al., 2017).

Furthermore, we have recently developed a promising approach using asiaticoside plus NO to successfully develop a gel as a new form of treatment for DCU (Nie et al., 2020). One of the important functions of the gel was to provide a vehicle for NO that would adhere to the skin and to make the gel stable on the wound surface for long periods of time. The combination of asiaticoside and NO accelerated the healing rate of DCU wounds. The asiaticoside‐NO gel also inhibited the growth of bacteria in the wound surface, alleviated the inflammatory reaction of wound, and increased the expression of VEGF, iNOS, eNOS, and CD34. Our results showed that the asiaticoside‐NO gel promoted DCU wound healing by regulating the Wnt/β‐catenin signalling pathway. It is hoped that these results will provide new targets and strategies for the diagnosis and treatment of DCU (Nie et al., 2020).

6.3. Growth‐promoting factors

To date, growth‐promoting factors such as EGF, TGF‐β, and PDGF have been shown to improve wound healing through the synergetic effect on promoting angiogenesis, re‐epithelialization and collagen deposition on the wound surface of DCU.

PDGF is a polypeptide secreted by many types of cells, including platelets, keratinocytes, and fibroblasts. It can stimulate the proliferation and division of cells such as macrophages and neutrophils and also promotes the process of wound epithelium formation. At present, with the approval of the FDA, a PDGF gel preparation, becaplermin, is widely used in the clinical treatment of diabetic foot skin ulcers, with significant therapeutic effect (Waycaster, Gilligan, & Motley, 2016). Nonetheless, recent results have reported an increased risk of cancer in patients treated with becaplermin gel, especially at high doses (Park et al., 2014) The FDA has issued a warning that patients who use more than three gels have a significantly increased risk of cancer. Therefore, future studies are necessary to evaluate the therapeutic effects and side effects of becaplermin gel.

EGF and its receptors promote the proliferation and division of a variety of cells such as epithelial cells, fibroblasts, and smooth muscle cells, in vitro and in vivo. In the process of diabetic ulcer wound healing, the mRNA for EGF receptors increases rapidly. As EGF receptors regulate cell growth, promote the repair of DNA and RNA, are involved in protein synthesis, and regulate the glycolytic pathway of cells, their increase promotes re‐epithelialization of chronic ulcer wounds (Tuyet et al., 2009) and accelerates the rate of wound healing. Further, EGF can also induce fibroblast proliferation, keratinocyte thickening, and inhibition of protein hydrolysis in ulcer wounds, and it is also involved in oxidative stress at the wound site in order to balance the systemic redox response of the patients (Ojalvo et al., 2017). Sommeling et al. (2013) confirmed that EGF can promote the formation of granulation tissue at the bottom of a wound and improve the quality of wound healing. Therefore, the local application of EGF to the DCU can promote angiogenesis and epidermal re‐epithelialization, improve the microenvironmental state of the wound, reduce the healing time of ulcer wounds, and achieve therapeutic effects on chronic refractory wounds.

Recombinant human GM‐CSF (rhGM‐CSF) is a new multifunctional growth factor and mitotic factor that is involved in multiple pathological processes associated with chronic ulcer wound healing (Hu, Sun, Han, Wang, & Yu, 2011). In recent years, rhGM‐CSF gels and injections have been used clinically for the treatment of ulcer wounds. It promotes wound healing through the following four functions: (a) activating the proliferation, differentiation and phagocytosis of neutrophils, monocytes, and macrophages and promoting the migration of epithelial cells and the proliferation of keratinocytes; (b) promoting the expression of VEGF and its receptor Flt‐1 and the growth of granulation tissue; (c) accelerating the re‐epithelialization of epidermis, ensuring the normal deposition of collagen fibres, and shortening the formation time of epidermis; (d) at the same time, it also relieves the pain at the wound and jointly achieves the effect of promoting wound healing (Ansurudeen et al., 2012; Herter & Xu Landén, 2017; Huang et al., 2014) (Figure 5).

In summary, although some drugs can be used to relieve or treat DCU, clearly greater clinical efficacy has not yet been achieved (Eleftheriadou, Tentolouris, Tentolouris, & Papanas, 2019). miRNA gene and stem cell therapies demonstrate promising development prospects, but the development and utilization of such treatments are difficult and time‐consuming. Further, the specificity and safety of miRNA and stem cell therapy remain unclear. Therefore, in future effort, it is crucial to develop a clearer understanding of the relationship between drugs, the body, and diseases and to better understand the structure of the human genome and the mechanism(s) of regulating gene expression. With further development of gene technology, there is likely to be a revolutionary breakthrough in the treatment of DCU with therapies based on miRNA genes.

7.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Fabbro et al., 2019a, 2019b; Alexander, Kelly et al., 2019).