Delayed cutaneous wound closure in HO‐2 deficient mice despite normal HO‐1 expression

Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO‐1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO‐2 deficient mice is impaired with exorbitant inflammation and absence of HO‐1 expression. This study addresses the role of HO‐2 in cutaneous excisional wound healing using HO‐2 knockout (KO) mice. Here, we show that HO‐2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO‐2 KO mice compared to WT controls. Surprisingly, wound closure in HO‐2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO‐1 induction in HO‐2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C‐X‐C) ligand‐11 (CXCL‐11) in wounds of HO‐2 KO mice. Abnormal regulation of CXCL‐11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL‐11 expression in HO‐2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.     

microRNA-106b derived from endothelial cell–secreted extracellular vesicles prevents skin wound healing by inhibiting JMJD3 and RIPK3

Intriguingly, microRNAs (miRs) transferred as cargo in extracellular vesicles (EVs) can modulate wound healing through their regulation of fibroblast functions. In this study, we investigated the effects of miR-106b transfer via EVs derived from human umbilical vein endothelial cells (HUVECs) on skin wound healing. Dual-luciferase reporter gene assay identified that miR-106b could target and inhibit JMJD3. RT-qPCR analysis showed EVs isolated from HUVECs had enriched expression of miR-106b. LL29 fibroblast cells and HaCaT keratinocytes were co-cultured with HUVEC-derived EVs, in which miR-106b had been up-regulated or down-regulated by its mimic or inhibitor. The co-culture with HUVEC-derived EVs increased miR-106b expression, and reduced the viability and adhesion of LL29 and HaCaT cells, whereas the inhibition of miR-106b in HUVEC-derived EVs enhanced the viability and adhesion of LL29 and HaCaT cells through up-regulation of JMJD3. Next, we showed that JMJD3 overexpression enhanced LL29 and HaCaT cell viability and adhesion through elevating RIPK3, which induced the phosphorylation of AKT during the wound-healing process. We next developed a mouse skin wound model to investigate the actions of miR-106b in vivo after 14 days. The delivery of miR-106b via HUVEC-derived EVs delayed wound healing through suppression of collagen I content and angiogenesis, but had no effects on pro-inflammatory cytokines. In conclusion, miR-106b from HUVEC-derived EVs inhibits JMJD3 and RIPK3, leading to the inhibition of skin wound healing, thus constituting a new therapeutic target.     

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non‐diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.     

High-Glucose Inhibits Human Fibroblast Cell Migration in Wound Healing via Repression of bFGF-Regulating JNK Phosphorylation

One of the major symptoms of diabetes mellitus (DM) is delayed wound healing, which affects large populations of patients worldwide. However, the underlying mechanism behind this illness remains elusive. Skin wound healing requires a series of coordinated processes, including fibroblast cell proliferation and migration. Here, we simulate DM by application of high glucose (HG) in human foreskin primary fibroblast cells to analyze the molecular mechanism of DM effects on wound healing. The results indicate that HG, at a concentration of 30 mM, delay cell migration, but not cell proliferation. bFGF is known to promote cell migration that partially rescues HG effects on cell migration. Molecular and cell biology studies demonstrated that HG enhanced ROS production and repressed JNK phosphorylation, but did not affect Rac1 activity. JNK and Rac1 activation were known to be important for bFGF regulated cell migration. To further confirm DM effects on skin repair, a type 1 diabetic rat model was established, and we observed the efficacy of bFGF on both normal and diabetic rat skin repair. Furthermore, proteomic studies identified an increase of Annexin A2 protein nitration in HG-stressed fibroblasts and the nitration was protected by activation of bFGF signaling. Treatment with FGFR1 and JNK inhibitors delayed cell migration and increased Annexin A2 nitration levels, indicating that Annexin A2 nitration is modulated by bFGF signaling via activation of JNK. Together with these results, our data suggests that the HG-mediated delay of cell migration is linked to the inhibition of bFGF signaling, specifically through JNK suppression.     

Procyanidin B2 improves endothelial progenitor cell function and promotes wound healing in diabetic mice via activating Nrf2

One of the major reasons for the delayed wound healing in diabetes is the dysfunction of endothelial progenitor cells (EPCs) induced by hyperglycaemia. Improvement of EPC function may be a potential strategy for accelerating wound healing in diabetes. Procyanidin B2 (PCB2) is one of the major components of procyanidins, which exhibits a variety of potent pharmacological activities. However, the effects of PCB2 on EPC function and diabetic wound repair remain elusive. We evaluated the protective effects of PCB2 in EPCs with high glucose (HG) treatment and in a diabetic wound healing model. EPCs derived from human umbilical cord blood were treated with HG. The results showed that PCB2 significantly preserved the angiogenic function, survival and migration abilities of EPCs with HG treatment, and attenuated HG-induced oxidative stress of EPCs by scavenging excessive reactive oxygen species (ROS). A mechanistic study found the protective role of PCB2 is dependent on activating nuclear factor erythroid 2-related factor 2 (Nrf2). PCB2 increased the expression of Nrf2 and its downstream antioxidant genes to attenuate the oxidative stress induced by HG in EPCs, which were abolished by knockdown of Nrf2 expression. An in vivo study showed that intraperitoneal administration of PCB2 promoted wound healing and angiogenesis in diabetic mice, which was accompanied by a significant reduction in ROS level and an increase in circulating EPC number. Taken together, our results indicate that PCB2 treatment accelerates wound healing and increases angiogenesis in diabetic mice, which may be mediated by improving the mobilization and function of EPCs.     

Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing

Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3 ^-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3 ^-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3 ^-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3 ^-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3 ^-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3 ^-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3 ^-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3 ^-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3 ^-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression of wound closure.     

Necroptosis increases with age and is reduced by dietary restriction

Necroptosis is a newly identified programmed cell death pathway that is highly proinflammatory due to the release of cellular components that promote inflammation. To determine whether necroptosis might play a role in inflammaging, we studied the effect of age and dietary restriction (DR) on necroptosis in the epididymal white adipose tissue (eWAT), a major source of proinflammatory cytokines. Phosphorylated MLKL and RIPK3, markers of necroptosis, were increased 2.7‐ and 1.9‐fold, respectively, in eWAT of old mice compared to adult mice, and DR reduced P‐MLKL and P‐RIPK3 to levels similar to adult mice. An increase in the expression of RIPK1 (1.6‐fold) and MLKL (2.7‐fold), not RIPK3, was also observed in eWAT of old mice, which was reduced by DR in old mice. The increase in necroptosis was paralleled by an increase in 14 inflammatory cytokines, including the pro‐inflammatory cytokines IL‐6 (3.9‐fold), TNF‐α (4.7‐fold), and IL‐1β (5.1‐fold)], and 11 chemokines in old mice. DR attenuated the expression of IL‐6, TNF‐α, and IL‐1β as well as 85% of the other cytokines/chemokines induced with age. In contrast, inguinal WAT (iWAT), which is less inflammatory, did not show any significant increase with age in the levels of P‐MLKL and MLKL or inflammatory cytokines/chemokines. Because the changes in biomarkers of necroptosis in eWAT with age and DR paralleled the changes in the expression of pro‐inflammatory cytokines, our data support the possibility that necroptosis might play a role in increased chronic inflammation observed with age.     

High glucose-mediated oxidative stress impairs cell migration

Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we evaluate the hypothesis that high glucose concentrations inhibit cell migration. Using CHO.K1 cells, NIH-3T3 fibroblasts, mouse embryonic fibroblasts and primary skin fibroblasts from control and diabetic rats cultured in 5 mM D-glucose (low glucose, LG), 25 mM D-glucose (high glucose, HG) or 25 mM L-glucose medium (osmotic control--OC), we analyzed the migration speed, protrusion stability, cell polarity, adhesion maturation and the activity of the small Rho GTPase Rac1. We also analyzed the effects of reactive oxygen species by incubating cells with the antioxidant N-Acetyl-Cysteine (NAC). We observed that HG conditions inhibited cell migration when compared to LG or OC. This inhibition resulted from impaired cell polarity, protrusion destabilization and inhibition of adhesion maturation. Conversely, Rac1 activity, which promotes protrusion and blocks adhesion maturation, was increased in HG conditions, thus providing a mechanistic basis for the HG phenotype. Most of the HG effects were partially or completely rescued by treatment with NAC. These findings demonstrate that HG impairs cell migration due to an increase in oxidative stress that causes polarity loss, deficient adhesion and protrusion. These alterations arise, in large part, from increased Rac1 activity and may contribute to the poor wound healing observed in diabetic patients.     

IDH2 deficiency impairs cutaneous wound healing via ROS-dependent apoptosis

Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue that play a pivotal role in cutaneous wound healing by synthesizing fibronectin (a component of the extracellular matrix), secreting angiogenesis factors, and generating strong contractile forces. In wound healing, low concentrations of reactive oxygen species (ROS) are essential in combating invading microorganisms and in cell-survival signaling. However, excessive ROS production impairs fibroblasts. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is a key enzyme that regulates the mitochondrial redox balance and reduces oxidative stress-induced cell injury through the generation of NADPH. In the present study, the downregulation of IDH2 expression resulted in an increase in cell apoptosis in mouse skin through ROS-dependent ATM-mediated p53 signaling. IDH2 deficiency also delayed cutaneous wound healing in mice and impaired dermal fibroblast function. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO alleviated the apoptosis induced by IDH2 deficiency both in vitro and in vivo. Together, our findings highlight the role of IDH2 in cutaneous wound healing in association with mitochondrial ROS.     

RIPK1-RIPK3 mediates myocardial fibrosis in type 2 diabetes mellitus by impairing autophagic flux of cardiac fibroblasts

Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are critical regulators of programmed necrosis or necroptosis. However, the role of the RIPK1/RIPK3 signaling pathway in myocardial fibrosis and related diabetic cardiomyopathy is still unclear. We hypothesized that RIPK1/RIPK3 activation mediated myocardial fibrosis by impairing the autophagic flux. To this end, we established in vitro and in vivo models of type 2 diabetes mellitus with high glucose fat (HGF) medium and diet respectively. HGF induced myocardial fibrosis, and impaired cardiac diastolic and systolic function by activating the RIPK1/RIPK3 pathway, which increased the expression of autophagic related proteins such as LC3-II, P62 and active-cathepsin D. Inhibition of RIPK1 or RIPK3 alleviated HGF-induced death and fibrosis of cardiac fibroblasts by restoring the impaired autophagic flux. The autophagy blocker neutralized the effects of the RIPK1 inhibitor necrostatin-1 (Nec-1) and RIPK3 inhibitor GSK872 (GSK). RIPK1/RIPK3 inhibition respectively decreased the levels of RIPK3/p-RIPK3 and RIPK1/p-RIPK1. P62 forms a complex with RIPK1-RIPK3 and promotes the binding of RIPK1 and RIPK3, silencing of RIPK1 decreased the association of RIPK1 with P62 and the binding of P62 to LC3. Furthermore, inhibition of both kinases in combination with a low dose of Nec-1 and GSK in the HGF-treated fibroblasts significantly decreased cell death and fibrosis, and restored the autophagic flux. In the diabetic rat model, Nec-1 (1.65 mg/kg) treatment for 4 months markedly alleviated myocardial fibrosis, downregulated autophagic related proteins, and improved cardiac systolic and diastolic function. In conclusion, HGF induces myocardial fibrosis and cardiac dysfunction by activating the RIPK1-RIPK3 pathway and by impairing the autophagic flux, which is obviated by the pharmacological and genetic inhibition of RIPK1/RIPK3.Subject terms: Necroptosis, Diabetes complications     

Regulation of RIPK3- and RHIM-dependent Necroptosis by the Proteasome

Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.     

Gender specific effects on the actin-remodelling protein Flightless I and TGF-beta1 contribute to impaired wound healing in aged skin

Impaired wound healing in the elderly presents a major clinical challenge. Understanding the cellular mechanisms behind age-related impaired healing is vital for developing new wound therapies. Here we show that the actin-remodelling protein, Flightless I (FliI) is a contributing factor to the poor healing observed in elderly skin and that gender plays a major role in this process. Using young and aged, wild-type and FliI overexpressing mice we found that aging significantly elevated FliI expression in the epidermis and wound matrix. Aging exacerbated the negative effect of FliI on wound repair and wounds in aged FliI transgenic mice were larger with delayed reepithelialisation. When the effect of gender was further analysed, despite increased FliI expression in young and aged male and female mice, female FliI transgenic mice had the most severe wound healing phenotype suggesting that male mice were refractory to FliI gene expression. Of potential importance, males, but not females, up-regulated transforming growth factor-β1 and this was most pronounced in aged male FliI overexpressing wounds. As FliI also functions as a co-activator of the estrogen nuclear receptor, increasing concentrations of β-estradiol were added to skin fibroblasts and keratinocytes and significantly enhanced FliI expression and translocation of FliI from the cytoplasm to the nucleus was observed. FliI further inhibited estrogen-mediated collagen I secretion suggesting a mechanism via which FliI may directly affect provisional matrix synthesis. In summary, FliI is a contributing factor to impaired healing and strategies aimed at decreasing FliI levels in elderly skin may improve wound repair.     

RIPK4 suppresses the TGF‐β1 signaling pathway in HaCaT cells

Receptor‐interacting serine/threonine kinase 4 (RIPK4) and transforming growth factor‐β 1 (TGF‐β1) play critical roles in the development and maintenance of the epidermis. A negative correlation between the expression patterns of RIPK4 and TGF‐β signaling during epidermal homeostasis‐related events and suppression of RIPK4 expression by TGF‐β1 in keratinocyte cell lines suggest the presence of a negative regulatory loop between the two factors. So far, RIPK4 has been shown to regulate nuclear factor‐κB (NF‐κB), protein kinase C (PKC), wingless‐type MMTV integration site family (Wnt), and (mitogen‐activated protein kinase) MAPK signaling pathways. In this study, we examined the effect of RIPK4 on the canonical Smad‐mediated TGF‐β1 signaling pathway by using the immortalized human keratinocyte HaCaT cell line. According to our results, RIPK4 inhibits intracellular Smad‐mediated TGF‐β1 signaling events through suppression of TGF‐β1‐induced Smad2/3 phosphorylation, which is reflected in the upcoming intracellular events including Smad2/3‐Smad4 interaction, nuclear localization, and TGF‐β1‐induced gene expression. Moreover, the kinase activity of RIPK4 is required for this process. The in vitro wound‐scratch assay demonstrated that RIPK4 suppressed TGF‐β1‐mediated wound healing through blocking TGF‐β1‐induced cell migration. In conclusion, our results showed the antagonistic effect of RIPK4 on TGF‐β1 signaling in keratinocytes for the first time and have the potential to contribute to the understanding and treatment of skin diseases associated with aberrant TGF‐β1 signaling.     

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2^nd and 3^rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.     

Sirtuin3 deficiency exacerbates carbon tetrachloride−induced hepatic injury in mice

Sirtuin3 (SIRT3) plays an important role in maintaining normal mitochondrial function and alleviating oxidative stress. After carbon tetrachloride (CCl₄) administration, the expression of SIRT3 decreased in the liver of mice, which indicated that the SIRT3 might play a crucial role during chemical‐induced acute hepatic injury. To verify the hypothesis, CCl ₄ was given to induce acute hepatic injury in SIRT3 knockout (KO) mice and wild‐type (WT) mice. CCl ₄‐induced liver injury was more severe in SIRT3 KO mice compared with the WT mice. In addition, the oxidative stress induced by CCl ₄ was enhanced in the SIRT3 KO mice. Furthermore, the increased expression of dynamin‐related protein 1 was also aggravated in SIRT3 KO mice after CCl ₄ administration. In conclusion, our study demonstrated that SIRT3 deficiency exacerbated CCl ₄‐induced impairment of the liver in mice, and the mechanism might be related to enhanced oxidative stress.     

Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing

In addition to their well-known antinociceptive action, opioids can modulate non-neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing.     

The effect of finger millet feeding on the early responses during the process of wound healing in diabetic rats

In the present study, the role of finger millet feeding on skin antioxidant status, nerve growth factor (NGF) production and wound healing parameters in healing impaired early diabetic rats is reported. Hyperglycemic rats received food containing 50 g/100 g finger millet (FM). Non-diabetic controls and diabetic controls received balanced nutritive diet. Full-thickness excision skin wounds were made after 2 weeks prior feeding of finger millet diet. The rate of wound contraction, and the levels of collagen, hexosamine and uronic acid in the granulation tissue were determined. The skin antioxidant status and lipid peroxide concentration were also monitored during the study. In hyperglycemic rats fed with finger millet diet, the healing process was hastened with an increased rate of wound contraction. Skin levels of glutathione (GSH), ascorbic acid and alpha-tocopherol in alloxan-induced diabetic rat were lower as compared to non-diabetics. Altered activities of superoxide dismutase (SOD) and catalase (CAT) were also recorded in diabetics. Interestingly, thiobarbituric acid reactive substances (TBARS) were elevated in the wound tissues of all the groups, when compared to normal (unwounded) skin tissues. However, in diabetic rats the TBARS levels of both normal and wounded skin tissues were significantly elevated (P < 0.001) when compared with control (non-diabetic) and diabetics fed with FM. Impaired production of NGF, determined by ELISA, in diabetic rats was improved upon FM feeding and further confirmed by immunocytochemical observations reflects the increased expression of NGF in hyperglycemic rats supplemented with FM-enriched diet. Histological and electron microscopical evaluations revealed the epithelialization, increased synthesis of collagen, activation of fibroblasts and mast cells in FM-fed animals. Thus, increased levels of oxidative stress markers accompanied by decreased levels of antioxidants play a vital role in delaying wound healing in diabetic rats. However, FM feeding to the diabetic animals, for 4 weeks, controlled the glucose levels and improved the antioxidant status, which hastened the dermal wound healing process.     

Autologous blood transfusion augments impaired wound healing in diabetic mice by enhancing lncRNA H19 expression via the HIF-1α signaling pathway

Background

Impaired wound healing frequently occurs in diabetes mellitus (DM) and is implicated in impaired angiogenesis. Long non-coding RNA (lncRNA) H19 has been reported as being reduced in DM and played a critical role in inducing angiogenesis. Thus, we hypothesized that H19 may affect impaired wound healing in streptozotocin (STZ)-induced diabetic mice transfused with autologous blood preserved in standard preservative fluid or modified preservative fluid.

Methods

Fibroblasts in injured skin were isolated and cultured in vitro. After location of H19 in fibroblasts using fluorescence in situ hybridization (FISH), RNA-pull down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), Co immunoprecipitation (COIP) and dual luciferase reporter gene assay were used to verify the binding of H19 to HIF-1α.

Results

The modified preservative fluid preserved autologous blood increased the H19 expression in fibroblasts, and maintained better oxygen-carrying and oxygen release capacities as well as coagulation function. Furthermore, H19 promoted HIF-1α histone H3K4me3 methylation and increased HIF-1α expression by recruiting EZH2. H19 promoted fibroblast activation by activating HIF-1α signaling pathway in fibroblasts and enhanced wound healing in diabetic mice.

Conclusions

Taken together, H19 accelerated fibroblast activation by recruiting EZH2-mediated histone methylation and modulating the HIF-1α signaling pathway, whereby augmenting the process of modified preservative fluid preserved autologous blood enhancing the postoperative wound healing in diabetic mice.