Toll-like receptor expression and signaling in human diabetic wounds

AIM: To examine the contribution of toll-like receptors(TLRs) expression and activation to the prolonged inflammation often seen in human diabetic wounds.METHODS: Debridement wound tissue was collected from diabetic patients with informed consent. Total RNA and protein were isolated and subjected to real-time polymerase chain reaction and Western blot analyses. RESULTS: TLR1, 2, 4, and 6 mRNA expressions were increased significantly in wounds of diabetic patients compared with non-diabetic wounds(P 0.05). MyD88 protein expression was significantly increased in diabetic wounds compared to non-diabetic wounds. Interleukin-1beta, tumor necrosis factor-alpha concentration nuclear factor-kappa B activation, and thiobarbituric acid reactive substances were increased in diabetic wounds compared to non-diabetic wounds(P 0.01). CONCLUSION: Collectively, our novel findings show that increased TLR expression, signaling, and activation may contribute to the hyper inflammation in the human diabetic wounds.     

Deficient cytokine expression and neutrophil oxidative burst contribute to impaired cutaneous wound healing in diabetic,biofilm‐containing chronic wounds

Diabetic patients exhibit dysregulated inflammatory and immune responses that predispose them to chronic wound infections and the threat of limb loss. The molecular underpinnings responsible for this have not been well elucidated, particularly in the setting of wound biofilms. This study evaluates host responses in biofilm‐impaired wounds using the TallyHo mouse, a clinically relevant polygenic model of type 2 diabetes. No differences in cytokine or Toll‐like receptor (TLR) expression were noted in unwounded skin or noninoculated wounds of diabetic and wild‐type mice. However, diabetic biofilm‐containing wounds had significantly less TLR 2, TLR 4, interleukin‐1β, and tumor necrosis factor‐α expression than wild‐type wounds with biofilm (all p < 0.001). Both groups had similar bacterial burden and neutrophil infiltration after development of biofilms at 3 days postwounding, but diabetic wounds had significantly less neutrophil oxidative burst activity. This translated into a log‐fold greater bacterial burden and significant delay of wound epithelization for biofilm‐impaired diabetic wounds at 10 days postwounding. These results suggest that impaired recognition of bacterial infection via the TLR pathway leading to inadequate cytokine stimulation of antimicrobial host responses may represent a potential mechanism underlying diabetic susceptibility to wound infection and ulceration.     

Activation of TLR signalling regulates microwave radiation‐mediated impairment of spermatogenesis in rat testis

Microwave radiation could increase the expression of pro‐inflammatory cytokines in rat Sertoli cells, which may impair spermatogenesis. However, the mechanisms that microwave radiation induces the cytokine expression in Sertoli cells remain to be clarified. The activation of TLRs by their ligands can trigger a common signalling pathway to upregulate inflammatory cytokines such as IL‐1, IL‐6, IL‐12 and TNF‐α. Microwave radiation can increase the expression of TLRs in lymphocytes. The purpose of this study was to determine the effect of microwave radiation on the TLRs in rat testis. We focus on the effect of TLR2‐5 (which is expressed relatively highly) by microwave radiation. The results showed that the expression of TLR2‐5 and the pro‐inflammatory cytokines (IL‐1β, IL‐6 and TNF‐α) was increased both in mRNA and in protein. Furthermore, p‐p38, p‐ERK1/2, p‐JNK and p‐NF‐κB p65, the key factors of TLR signalling, were also elevated by microwave exposure. And the NF‐κB can be induced more dominantly. These results suggest that TLRs signalling can be activated by microwave radiation in testis, which may provide the molecular basis for the in‐depth study.     

Diabetic wound healing in a MMP9‐/‐ mouse model

Reduced mobilization of endothelial progenitor cells (EPCs) from the bone marrow (BM) and impaired EPC recruitment into the wound represent a fundamental deficiency in the chronic ulcers. However, mechanistic understanding of the role of BM‐derived EPCs in cutaneous wound neovascularization and healing remains incomplete, which impedes development of EPC‐based wound healing therapies. The objective of this study was to determine the role of EPCs in wound neovascularization and healing both under normal conditions and using single deficiency (EPC) or double‐deficiency (EPC + diabetes) models of wound healing. MMP9 knockout (MMP9 KO) mouse model was utilized, where impaired EPC mobilization can be rescued by stem cell factor (SCF). The hypotheses were: (1) MMP9 KO mice exhibit impaired wound neovascularization and healing, which are further exacerbated with diabetes; (2) these impairments can be rescued by SCF administration. Full‐thickness excisional wounds with silicone splints to minimize contraction were created on MMP9 KO mice with/without streptozotocin‐induced diabetes in the presence or absence of tail‐vein injected SCF. Wound morphology, vascularization, inflammation, and EPC mobilization and recruitment were quantified at day 7 postwounding. Results demonstrate no difference in wound closure and granulation tissue area between any groups. MMP9 deficiency significantly impairs wound neovascularization, increases inflammation, decreases collagen deposition, and decreases peripheral blood EPC (pb‐EPC) counts when compared with wild‐type (WT). Diabetes further increases inflammation, but does not cause further impairment in vascularization, as compared with MMP9 KO group. SCF improves neovascularization and increases EPCs to WT levels (both nondiabetic and diabetic MMP9 KO groups), while exacerbating inflammation in all groups. SCF rescues EPC‐deficiency and impaired wound neovascularization in both diabetic and nondiabetic MMP9 KO mice. Overall, the results demonstrate that BM‐derived EPCs play a significant role during wound neovascularization and that the SCF‐based therapy with controlled inflammation could be a viable approach to enhance healing in chronic diabetic wounds.     

The analgesic hybrid of dermorphin/substance P and analog of enkephalin improve wound healing in streptozotocin‐induced diabetic rats

The purpose of this study was to investigate the effect of the peptide analgesic hybrid compounds: AWL3106 analog of dermorphin and substance P (7‐11), and biphalin enkephalin analog on wound healing in streptozotocin‐induced diabetic rats. The diabetes was induced in 6–7 week‐old male Wistar rats by intraperitoneal injection of streptozotocin. After 70 days, the wounds were created on the back of the rats and then, once a day for 21 days, the dressing containing lanolin ointment, 10% of keratin scaffolds, and 1 mM of AWL3106 or biphalin was applied. The wounds histology were analyzed by hematoxylin and eosin staining. The orientation and organization of collagen was analyzed by Masson's trichome staining. The number of macrophages, blood vessels, and fibroblasts were visualized by CD68, CD34, and vimentin immunoreactivity, respectively. Our results demonstrated that the wound area of AWL3106‐ and biphalin‐treated groups was greatly reduced (up to 47% on the 7 day) in comparison with untreated diabetic groups. The immunohistochemical staining of macrophages demonstrated that AWL3106 and biphalin accelerated inflammatory progression and subsequently decreased persistent inflammation. The histological analysis showed that the structure of tissue in the groups under the study was very similar to the one of wound tissue in N‐DM group. The H&E and Masson's trichome staining demonstrated that the orientation and organization of collagen as well as the number and shape of blood vessels were better in 3106‐ and BIF‐treated group than in DM group. In conclusion, the obtained data suggested that our hybrid peptides enhanced wound healing, particularly by accelerating the inflammatory phase and promoted the wound closure.     

Time course study of delayed wound healing in a biofilm‐challenged diabetic mouse model

Bacterial biofilm has been shown to play a role in delaying wound healing of chronic wounds, a major medical problem that results in significant health care burden. A reproducible animal model could be very valuable for studying the mechanism and management of chronic wounds. Our previous work showed that Pseudomonas aeruginosa (PAO1) biofilm challenge on wounds in diabetic (db/db) mice significantly delayed wound healing. In this wound time course study, we further characterize the bacterial burden, delayed wound healing, and certain aspects of the host inflammatory response in the PAO1 biofilm‐challenged db/db mouse model. PAO1 biofilms were transferred onto 2‐day‐old wounds created on the dorsal surface of db/db mice. Control wounds without biofilm challenge healed by 4 weeks, consistent with previous studies; none of the biofilm‐challenged wounds healed by 4 weeks. Of the biofilm‐challenged wounds, 64% healed by 6 weeks, and all of the biofilm‐challenged wounds healed by 8 weeks. During the wound‐healing process, P. aeruginosa was gradually cleared from the wounds while the presence of Staphylococcus aureus (part of the normal mouse skin flora) increased. Scabs from all unhealed wounds contained 10⁷ P. aeruginosa, which was 100‐fold higher than the counts isolated from wound beds (i.e., 99% of the P. aeruginosa was in the scab). Histology and genetic analysis showed proliferative epidermis, deficient vascularization, and increased inflammatory cytokines. Hypoxia inducible factor expression increased threefold in 4‐week wounds. In summary, our study shows that biofilm‐challenged wounds typically heal in approximately 6 weeks, at least 2 weeks longer than nonbiofilm‐challenged normal wounds. These data suggest that this delayed wound healing model enables the in vivo study of bacterial biofilm responses to host defenses and the effects of biofilms on host wound healing pathways. It may also be used to test antibiofilm strategies for treating chronic wounds.     

Anti‐inflammatory effects of haptoglobin on LPS‐stimulated macrophages: Role of HMGB1 signaling and implications in chronic wound healing

Nonhealing wounds possess elevated numbers of pro‐inflammatory M1 macrophages, which fail to transition to anti‐inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb‐Hp), can elicit M2‐like macrophages through the heme oxygenase‐1 (HO‐1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non‐inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)‐stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb‐Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. Hp treatment decreased a majority of inflammatory factors, Hb increased many, and Hb‐Hp had intermediate trends, indicating that Hp attenuated overall inflammation to the greatest extent. From this data, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway—strongly down‐regulated from Hp, strongly up‐regulated from Hb, and slightly activated from Hb‐Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp—and not Hb‐Hp, which is known to be superior in noninflammatory conditions—reduces inflammation in LPS‐stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound‐healing models.     

High‐dose folic acid and its effect on early stage diabetic foot ulcer wound healing

High‐dose folic acid (HDFA; vitamin B9)—5 mg, given daily, has not been evaluated as a treatment to improve early stage‐diabetic foot ulcer (ES‐DFU) wound healing. However, HDFA has been demonstrated to correct: (a) endothelial dysfunction and decreased nitric oxide (NO) bioavailability, associated with type‐2 diabetes mellitus (T2DM); and (b) hyperhomocysteinemia (HHcy) that may promote impaired DFU‐wound healing. Measures of wound area (cm²) reduction (wound closure; WC), over a 4‐week period (4 W‐WC), greater than 50% of the wound area, have been reported as a robust indicator of the potential for DFU‐wound healing. By using this model, we examined the effectiveness of a wound treatment in promoting progressive healing and complete wound closure for the chronic, nonhealing DFU‐wound. To investigate this possible relationship between HDFA and ES‐DFU wound healing, a retrospective cohort study of medical records, between November 2018 and April 2019, was performed for Veterans with T2DM and ES‐DFUs following treatment with HDFA. During the study period 29 (n = 29) Veterans with ES‐DFU wounds who received HDFA treatment were identified. Medical record reviews of this retrospective cohort of ES‐DFU Veterans receiving HDFA report 90% (26/29) experiencing complete DFU‐wound closure during the study period. Of the 29 Veterans with ES‐DFUs receiving HDFA, the medical records of nine (30%), with healed wounds, provided documentation suitable for 4 W‐WC, pre‐ and post‐HDFA treatment study comparisons. This study documents significant (P < .05) improvements comparing 4 W‐WC values for standard treatment for Veterans with poorly progressing, worsening or stagnating ES‐DFU‐wounds to those for the same subjects following HDFA treatment. These observations suggest that chronic ES‐DFUs treated with HDFA may experience significantly improved wound closure and complete healing (re‐epithelialization) when compared with standard treatments without HDFA. With validation from RCTs, HDFA may be established as an effective treatment to promote wound healing and closure for nonhealing ES‐DFUs.     

Blocking Interleukin-1β Induces a Healing-Associated Wound Macrophage Phenotype and Improves Healing in Type 2 Diabetes

Diabetes is associated with persistent inflammation and defective tissue repair responses. The hypothesis of this study was that interleukin (IL)-1β is part of a proinflammatory positive feedback loop that sustains a persistent proinflammatory wound macrophage phenotype that contributes to impaired healing in diabetes. Macrophages isolated from wounds in diabetic humans and mice exhibited a proinflammatory phenotype, including expression and secretion of IL-1β. The diabetic wound environment appears to be sufficient to induce these inflammatory phenomena because in vitro studies demonstrated that conditioned medium of both mouse and human wounds upregulates expression of proinflammatory genes and downregulates expression of prohealing factors in cultured macrophages. Furthermore, inhibiting the IL-1β pathway using a neutralizing antibody and macrophages from IL-1 receptor knockout mice blocked the conditioned medium–induced upregulation of proinflammatory genes and downregulation of prohealing factors. Importantly, inhibiting the IL-1β pathway in wounds of diabetic mice using a neutralizing antibody induced a switch from proinflammatory to healing-associated macrophage phenotypes, increased levels of wound growth factors, and improved healing of these wounds. Our findings indicate that targeting the IL-1β pathway represents a new therapeutic approach for improving the healing of diabetic wounds.Chronic wounds associated with diabetes, venous insufficiency, or pressure represent a major health problem, with millions of patients afflicted and the associated treatment costing billions of dollars per year (1). Despite the socioeconomic impact of chronic wounds, the underlying causes of impaired healing are not well-understood and effective treatments remain elusive. A common characteristic of these poorly healing wounds is a persistent inflammatory response, with prolonged accumulation of macrophages and elevated levels of proinflammatory cytokines (2–5). Translational research of the dysregulation of inflammation associated with impaired healing in diabetes should provide insight into the development of new therapeutic approaches.During normal wound healing in mice, inflammatory cells such as macrophages promote healing indirectly by killing pathogens and clearing the wound of damaged tissue, but also promote healing directly by producing growth factors that induce angiogenesis, collagen deposition, and wound closure (6–9). In contrast, during impaired healing of diabetic mice, wounds exhibit prolonged accumulation of macrophage associated with elevated levels of proinflammatory cytokines and proteases and reduced levels of various growth factors, all of which mimic chronic wounds in humans (10–12). We recently demonstrated that in wounds of diabetic mice, macrophages exhibit a sustained proinflammatory phenotype with an impaired upregulation of healing-associated factors that is observed in nondiabetic mice as healing progresses (13). However, the underlying causes of the dysregulation of macrophage in diabetic wounds remain to be elucidated.Multiple factors can influence macrophage phenotype and the actual phenotypes expressed in chronic wounds are likely determined by the balance of the proinflammatory and anti-inflammatory stimuli present in the wound environment. The proinflammatory environment observed in diabetic wounds has the potential to sustain a proinflammatory macrophage phenotype, which, in turn, would contribute to sustaining the proinflammatory environment. In fact, hyperglycemia is known to induce expression of interleukin (IL)-1β in a number of different cell types, including macrophages (14–16), and IL-1β, in turn, is known to induce a proinflammatory macrophage phenotype in part by inducing itself (17). Thus, the IL-1β pathway may be part of a positive feedback loop that sustains inflammation in chronic wounds and contributes to impaired healing. However, little is known about the actual role of IL-1β in diabetic wounds.The central hypothesis of this study is that sustained activity of the IL-1β pathway in diabetic wounds contributes to impaired healing of these wounds. The results of this study demonstrate that sustained IL-1β expression in wounds of diabetic humans and mice is associated with a proinflammatory macrophage phenotype, and that inhibiting the IL-1β pathway in wounds of diabetic mice induces the switch from proinflammatory to healing-associated macrophage phenotypes and improves healing of these wounds.     

Autophagy ameliorates Pseudomonas aeruginosa-infected diabetic wounds by regulating the toll-like receptor 4/myeloid differentiation factor 88 pathway

Diabetic foot ulcers (DFUs) are among the most common complications in patients with diabetes and a leading cause of lower extremity amputation. DFUs are exacerbated by prolonged bacterial infection; therefore, there is an urgent need for effective treatments to alleviate the burden associated with this condition. Although autophagy plays a unique role in pathogen phagocytosis and inflammation, its role in diabetic foot infections (DFIs) remains unclear. Pseudomonas aeruginosa (PA) is the most frequently isolated gram-negative bacterium from DFUs. Here, we evaluated the role of autophagy in ameliorating PA infection in wounds in a diabetic rat model and a bone marrow-derived macrophage (BMDM) hyperglycemia model. Both models were pretreated with or without rapamycin (RAPA) and then infected with or without PA. Pretreatment of rats with RAPA significantly enhanced PA phagocytosis, suppressed wound inflammation, reduced the M1:M2 macrophage ratio, and improved wound healing. In vitro investigation of the underlying mechanisms revealed that enhanced autophagy resulted in decreased macrophage secretion of inflammatory factors such as TNF-α, IL-6, and IL-1β but increased that of IL-10 in response to PA infection. Additionally, RAPA treatment significantly enhanced autophagy in macrophages by increasing LC3 and beclin-1 levels, which led to altered macrophage function. Furthermore, RAPA blocked the PA-induced TLR4/MyD88 pathway to regulate macrophage polarisation and inflammatory cytokine production, which was validated by RNA interference and use of the autophagy inhibitor 3-methyladenine (3-MA). These findings suggest enhancing autophagy as a novel therapeutic strategy against PA infection to ultimately improve diabetic wound healing.     

Tissue-engineered provisional matrix as a novel approach to enhance diabetic wound healing

Inherent pathologies associated with diabetic wound microenvironment including increased proteolysis, inflammatory dysregulation, and impaired neovascularization prevent timely resolution of chronic diabetic ulcers. It is hypothesized that augmentation of local wound microenvironment with a stable provisional matrix formed by proteolysis-resistant angiogenic peptide nanofibers (NFs) will create permissive environment for attenuated inflammation, enhanced neovascularization, and improved diabetic wound healing. Using murine excisional wound healing models, full-thickness dorsal skin wounds were treated with either NFs or control solutions (phosphate buffered saline; hyaluronic acid) and analyzed for morphology, inflammatory response, neovascularization, and biomechanical properties. NF treatment of diabetic wounds stimulated formation of a robust pro-angiogenic in situ tissue-engineered provisional matrix leading to a significant decrease in wound inflammatory cell infiltration and proinflammatory interleukin-6 levels, a significant increase in endothelial and endothelial progenitor cell infiltration, vascular endothelial growth factor levels, and neovascularization (day 7), as well as improved wound morphology, accelerated wound closure, and significantly stronger repair tissue (day 28). These results suggest that appropriate design of provisional matrix may compensate for some of the complex disruptions in diabetic wound microenvironment and provide missing cues to cells and direct in situ responses toward improved healing, which is promising for future development of new therapies for diabetic ulcers.     

Comparative analysis of global gene expression profiles between diabetic rat wounds treated with vacuum‐assisted closure therapy,moist wound healing or gauze under suction

How differential gene expression affects wound healing is not well understood. In this study, Zucker diabetic fatty (fa/fa) male inbred rats were used to investigate gene expression during wound healing in an impaired wound‐healing model. Whole genome microarray surveys were used to gain insight into the biological pathways and healing processes in acute excisional wounds treated with vacuum‐assisted closure (V.A.C.^®) Therapy, moist wound healing (MWH) or gauze under suction (GUS). Global gene expression analyses after 2 days of healing indicated major differences with respect to both number of genes showing fold changes and pathway regulation between the three different wound treatments. Statistical analysis of expression profiles indicated that 5072 genes showed a >1·6‐fold change with V.A.C. Therapy compared with 3601 genes with MWH and 3952 genes with GUS. Pathways and related genes associated with the early phases of wound healing diverged between treatment groups. For example, pathways involving angiogenesis, cytoskeletal regulation and inflammation were associated with elevated gene expression following V.A.C. Therapy. This study is the first to assess wound healing by whole genome interrogation in a diabetic rat model treated with different healing modalities.     

Chemokines and diabetic wound healing

Chemokines are critical for white blood cell recruitment to injured tissues and play an important role in normal wound healing processes. In contrast, impaired wound healing in diabetic patients is accompanied by decreased early inflammatory cell infiltration but persistence of neutrophils and macrophages in the chronic, nonhealing wounds. These changes in inflammatory cell recruitment occur in conjunction with alterations in chemokine and growth factor expression. In addition to leukocyte trafficking, many different cell types, including endothelial cells, fibroblasts, and keratinocytes, produce and respond to chemokines, and these interactions are altered in diabetic wounds. Thus, the chemokine system may have both direct and inflammatory-mediated effects on many different aspects of diabetic wound healing. The potential roles of chemokines and inflammatory or immune cells in nonhealing diabetic wounds, including impairments in growth factor expression, angiogenesis, extracellular matrix formation, and reepithelialization, are examined.     

Delayed wound healing in diabetic (db/db) mice with Pseudomonas aeruginosa biofilm challenge: a model for the study of chronic wounds

Chronic wounds are a major clinical problem that lead to considerable morbidity and mortality. We hypothesized that an important factor in the failure of chronic wounds to heal was the presence of microbial biofilm resistant to antibiotics and protected from host defenses. A major difficulty in studying chronic wounds is the absence of suitable animal models. The goal of this study was to create a reproducible chronic wound model in diabetic mice by the application of bacterial biofilm. Six‐millimeter punch biopsy wounds were created on the dorsal surface of diabetic (db/db) mice, subsequently challenged with Pseudomonas aeruginosa (PAO1) biofilms 2 days postwounding, and covered with semiocclusive dressings for 2 weeks. Most of the control wounds were epithelialized by 28 days postwounding. In contrast, none of biofilm‐challenged wounds were closed. Histological analysis showed extensive inflammatory cell infiltration, tissue necrosis, and epidermal hyperplasia adjacent to challenged wounds—all indicators of an inflammatory nonhealing wound. Quantitative cultures and transmission electron microscopy demonstrated that the majority of bacteria were in the scab above the wound bed rather than in the wound tissue. The model was reproducible, allowed localized cutaneous wound infections without high mortality, and demonstrated delayed wound healing following a biofilm challenge. This model may provide an approach to study the role of microbial biofilms in chronic wounds as well as the effect of specific biofilm therapy on wound healing.     

Low flow oxygenation of full‐excisional skin wounds on diabetic mice improves wound healing by accelerating wound closure and reepithelialization

Oxygen‐based therapies have proven effective in treating chronic and difficult‐to‐heal skin wounds, but the current therapeutic approaches suffer from major limitations and they do not allow for continuous wound treatment. Here we examined whether the continuous treatment of wounds with pure oxygen at low flow rates accelerates wound closure and improves wound healing in a murine model of diabetic skin wounds. Two full‐excisional dorsal skin wounds were generated on 15‐week‐old diabetic db/db mice and treated for 10 weeks continuously with pure oxygen (>99·9%) at low flow rates (3 ml/h). After 6 days, oxygen treatment resulted in a mean reduction of the original wound size by 60·2% as compared with only 45·2% in wounds on control mice that did not receive pure oxygen.(P = 0·022). After 10 days, oxygen‐treated wounds were 83·1% closed compared with 71·2% in wounds on control mice. While reepithelialisation was complete after 10 days in over 57% of wounds receiving low flow oxygen treatment, significant epithelial gaps remained in 75% wounds from mice that did not receive oxygen. Continuous low flow oxygenation significantly improves healing of diabetic skin wounds in mice and may therefore be an effective treatment for chronic cutaneous and possibly other slow‐healing wounds in diabetic patients.     

Curcumin accelerates cutaneous wound healing via multiple biological actions: The involvement of TNF‐α, MMP‐9, α‐SMA,and collagen

Curcumin, a constituent of the turmeric plant, has antitumor, anti‐inflammatory, and antioxidative effects, but its effects on wound healing are unclear. We created back wounds in 72 mice and treated them with or without topical curcumin (0.2 mg/mL) in Pluronic F127 gel (20%) daily for 3, 5, 7, 9, and 12 days. Healing in wounds was evaluated from gross appearance, microscopically by haematoxylin and eosin staining, by immunohistochemistry for tumour necrosis factor alpha and alpha smooth muscle actin, and by polymerase chain reaction amplification of mRNA expression levels. Treatment caused fast wound closure with well‐formed granulation tissue dominated by collagen deposition and regenerating epithelium. Curcumin increased the levels of tumour necrosis factor alpha mRNA and protein in the early phase of healing, which then decreased significantly. However, these levels remained high in controls. Levels of collagen were significantly higher in curcumin‐treated wounds. Immunohistochemical staining for alpha smooth muscle actin was increased in curcumin‐treated mice on days 7 and 12. Curcumin treatment significantly suppressed matrix metallopeptidase‐9 and stimulated alpha smooth muscle levels in tumour necrosis factor alpha‐treated fibroblasts via nuclear factor kappa B signalling. Thus, topical curcumin accelerated wound healing in mice by regulating the levels of various cytokines.     

Differential expression of TLRs and AKAP3 in cigarette smoked mice testis

Toll‐like receptors (TLRs) are expressed in Sertoli cells and Leydig cells and can initiate testicular innate immune responses. The A‐kinase anchor protein 3 (AKAP3), a family of scaffolding protein, was reported to be expressed only in testis and plays important regulatory roles during spermatogenesis. Our present study aimed to investigate the differential expression of TLRs family and AKAP3 in cigarette smoked Kunming mice testis. To check the effect of cigarette smoke, mice were randomly divided (n = 5 each) and exposed to cigarette smoke (2 hr/day with 10 cigarettes) for six consecutive days followed by one exposure‐free day. The exposure lasted for zero (control), 1, 3, 5 and 7 months respectively. The IHC results showed that expression of AKAP3 protein is mainly located in sperm cells and the mean density of which was significantly lower than that of control mice. Real‐time PCR results showed that expression of AKAP3 was significantly increased at early CS exposure (1 month) and then returned to normal in subsequent months. TLR2‐7, TLR13, Myd88 and Traf6 mRNA expression are much lower compared to control, especially after 3‐month cigarette smoke exposure, the time of which is almost consistent with sperm cycle. The present study suggests that TLR signal pathway and AKAP3 may play roles in spermatogenesis.     

Modulation of the inflammatory response by increasing fetal wound size or interleukin‐10 overexpression determines wound phenotype and scar formation

Wound size impacts the threshold between scarless regeneration and reparative healing in the fetus with increased inflammation showed in fetal scar formation. We hypothesized that increased fetal wound size increases pro‐inflammatory and fibrotic genes with resultant inflammation and fibroplasia and that transition to scar formation could be reversed by overexpression of interleukin‐10 (IL‐10). To test this hypothesis, 2‐mm and 8‐mm dermal wounds were created in mid‐gestation fetal sheep. A subset of 8‐mm wounds were injected with a lentiviral vector containing the IL‐10 transgene (n = 4) or vehicle (n = 4). Wounds were harvested at 3 or 30 days for histology, immunohistochemistry, analysis of gene expression by microarray, and validation with real‐time polymerase chain reaction. In contrast to the scarless 2‐mm wounds, 8‐mm wounds showed scar formation with a differential gene expression profile, increased inflammatory cytokines, decreased CD45+ cells, and subsequent inflammation. Lentiviral‐mediated overexpression of the IL‐10 gene resulted in conversion to a regenerative phenotype with decreased inflammatory cytokines and regeneration of dermal architecture. In conclusion, increased fetal wounds size leads to a unique gene expression profile that promotes inflammation and leads to scar formation and furthermore, these results show the significance of attenuated inflammation and IL‐10 in the transition from fibroplasia to fetal regenerative healing.