Activation of Sestrin2 accelerates deep second-degree burn wound healing through PI3K/AKT pathway.

Deep second-degree burns heal slowly, and promoting the healing process is a focus of clinical research. Sestrin2 is a stress-inducible protein with antioxidant and metabolic regulatory effects. However, its role during acute dermal and epidermal re-epithelialization in deep second-degree burns is unknown. In this study, we aimed to explore the role and molecular mechanism of sestrin2 in deep second-degree burns as a potential treatment target for burn wounds. To explore the effects of sestrin2 on burn wound healing, we established a deep second-degree burn mouse model. Then we detected the expression of sestrin2 by western blot and immunohistochemistry after obtaining the wound margin of full-thickness burned skin. The effects of sestrin2 on burn wound healing were explored in vivo and in vitro through interfering sestrin2 expression using siRNAs or the small molecule agonist of sestrin2, eupatilin. We also investigated the molecular mechanism of sestrin2 in promoting burn wound healing by western blot and CCK-8 assay. Our in vivo and in vitro deep second-degree burn wound healing model demonstrated that sestrin2 was promptly induced at murine skin wound edges. The small molecule agonist of sestrin2 accelerated the proliferation and migration of keratinocytes, as well as burn wound healing. Conversely, the healing of burn wounds was delayed in sestrin2-deficient mice and was accompanied by the secretion of inflammatory cytokines as well as the suppression of keratinocyte proliferation and migration. Mechanistically, sestrin2 promoted the phosphorylation of the PI3K/AKT pathway, and inhibition of PI3K/AKT pathway abrogated the promoting role of sestrin2 in keratinocyte proliferation and migration. Therefore, sestrin2 plays a critical role in activation of the PI3K/AKT pathway to promote keratinocyte proliferation and migration, as well as re-epithelialization in the process of deep second-degree burn wound repair.

A Composite of Hepatocyte Growth Factor- and 5α-Dihydrotestosterone-Gelatin Microspheres with Adipose-Derived Stem Cells Enhances Wound Healing.

INTRODUCTION: Reconstructing sebaceous glands is one goal of functionally healing patients who have suffered severe burns, instead of the simple pursuit of wound closure. Effective regeneration of skin appendages remains a challenge in skin wound management and research. OBJECTIVE: The aim of this study was to evaluate the differentiation of adipose-derived stem cells (ADSCs) into sebaceous glands and clarified the involvement of hepatocyte growth factor (HGF) and 5α-dihydrotestosterone (5α-DHT) in this process. METHODS: This study used HGF- and 5α-DHT-gelatin microspheres to treat human ADSCs and investigated the reconstruction of sebaceous glands. HGF- and 5α-DHT-gelatin microspheres were constructed using microcapsule slow-release technology. A mice full-thickness skin-wound model was established to evaluate wound healing, and hematoxylin-eosin staining was utilized to determine the skin structure. RESULTS: In vitro analyses found that HGF- and 5α-DHT-gelatin microspheres promoted migration of and tube formation by ADSCs. Furthermore, AKT/ERK signaling, which is related to sebocyte and sweat gland epithelial-cell growth, was activated after HGF and 5α-DHT treatment. An in vivo wound healing model demonstrated that ADSCs primed with amnion-loaded HGF- and 5α-DHT-gelatin microspheres promoted wound healing and increased sebaceous gland formation compared to the control group. CONCLUSIONS: This study confirms the efficacy of ADSCs treated with amnion and HGF- and 5α-DHT-gelatin microspheres in accelerating wound healing and effectively restoring sebaceous glands. This engineered tissue provides insight into and a novel therapeutic material for burns and full-thickness skin wounds.

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts.

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL-10 is a potent anti-inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL-10 alleviates lipopolysaccharide (LPS)-induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS-derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS-derived fibroblasts (NSFs). LPS could up-regulate the expression of TLR4, pp65, Col I, Col III and α-SMA in NSFs, but IL-10 could down-regulate their expression in both HSFs and LPS-induced NSFs. Blocking IL-10 receptor (IL-10R) or the phosphorylation of STAT3, their expression was up-regulated. In addition, in vitro and in vivo models results showed that IL-10 could alleviate LPS-induced fibroblast-populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL-10 alleviates LPS-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL-10 can alleviate the LPS-induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down-regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.

Wnt4 negatively regulates the TGF-ß1-induced human dermal fibroblast-to-myofibroblast transition via targeting Smad3 and ERK.

Abnormal activation of Wnt signaling has been demonstrated in the wound healing process and the pathogenesis of fibrotic disorders, with Wnt4 specifically identified as having a key role in the pathogenesis of renal, pulmonary and liver fibrosis. Wnt4 also was found to be upregulated by transforming growth factor-ß1 (TGF-ß1) in fetal and postnatal murine fibroblasts and bone marrow mesenchymal cells, suggesting an underlying cooperation between Wnt4 and TGF-ß1 in fibrosis. However, the specific roles of Wnt4 in TGF-ß1-induced skin myofibroblast transition and hypertrophic scar formation remain unclear. In the present study, we first observed reduced Wnt4 expression in hypertrophic scar tissue compared with that in normal skin tissue. Following upregulation by TGF-ß1, Wnt4 inhibited the TGF-ß1-induced transdifferentiation of fibroblasts into myofibroblasts. Using fibroblast-populated collagen lattice contraction assays, we showed that the increased contractility induced by TGF-ß1 was significantly blocked by exogenous Wnt4 and the α-smooth muscle actin (α-SMA) expression was decreased in fibroblasts in the collagen lattices. In addition, knockdown of Wnt4 resulted in further increases in α-SMA and collagen I expressions. Further investigation showed that Wnt4 could inhibit the autocrine effect of TGF-ß1 as well as block the phosphorylation of Smad3 and ERK but not of AKT or JNK. Lastly, using hypertrophic scar-derived fibroblasts, we showed that the elevated α-SMA and collagen I levels were markedly reduced after treatment with Wnt4. Taken together, our results suggest that Wnt4 negatively regulates TGF-ß1-induced fibroblast activation, which may represent a novel therapeutic strategy for the treatment and prevention of hypertrophic scars.

Highly-expressed micoRNA-21 in adipose derived stem cell exosomes can enhance the migration and proliferation of the HaCaT cells by increasing the MMP-9 expression through the PI3K/AKT pathway.

OBJECTIVE: Wound healing remains a challenge in burns and trauma fields. Adipose derived stem cells exosomes (AD-exos) had been confirmed to have a positive effect on the wound healing and the migration and proliferation of keratinocyte. However, the mechanism of the AD-exos is still unclear. The objective of this article is to observe the function of the miR-21 expressed in the adipose AD-exos and the effect on migration and proliferation of the HaCaT cells. MATERIALS AND METHODS: The full layer dermal wound of BALb/c mouse was used to observe the vitro effect of the AD-exos and detect the expression of miR-21.The co-culture systems were established by transwell plates for observing the migration, proliferation, apoptosis rate, detecting the RNA, and protein expression in different treated groups. MiR-21 plasmid was used to over-express miR-21 by transfection of HaCaT cells. GW4869 was used to inhibit the secreting of exosomes from ADSCs. RESULTS: The results showed that both ADSCs and the AD-exos could improve the wound healing process of BALb/c mouse full layer skin wound at a similar level, especially at the 7th day post surgery when compared to the control group (p < 0.01) and the highly expressed miR-21 was detected (p < 0.01 compared with control group and p < 0.001 compared to other microRNAs) in the treated groups at the same time point. AD-exos could obviously enhance the migration and proliferation of the HaCaT cells (p < 0.01), and fell back to the same level when the exosomes inhibitor--GW4869 was added compared with control group (p > 0.05). Over-expressed miR-21 could also significantly improve the migration and proliferation of HaCaT cells. But both AD-exos and miR-21 had no significantly effect on the apoptosis rate of HaCaT cells (p > 0.05 compared with each other). Over-expression of miR-21 plasmid could decrease the TGF-ßI protein level (p < 0.001 vs. control group) in HaCaT cells while TGF-ßI protein level increased again when antagomiR-21 was added in (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 expression of HaCaT cells could be increased by the transfect ion of miR-21 plasmid (p < 0.001 vs. empty plasmid group) and decreased by antagomiR-21 (p < 0.01 vs. empty plasmid group, p < 0.001 vs. miR-21 plasmid group). MiR-21 appeared to have influence on MMP-9 and TIMP-2 (p < 0.001 compared to control group and p < 0.001 compared to TGF-ßI group) but not MMP-2 and TIMP-1 (p > 0.05 compared to control group and TGF-ßI group). These processes might act through PI3K/AKT pathway. CONCLUSION: This research provide the experimental evidence that the miR-21 is highly expressed in AD-exos and can significantly accelerate the wound healing process and enhance the migration and proliferation of the HaCaT cells. Over-expressed miR-21 can inhibit the TGF-ßI expression and excess TGF-ßI can also have negative feedback influence on miR-21. We have found a reliable evidence that these two factors can act on HaCaT cells by influencing MMP-2 and TIMP-1 protein expression through the PI3K/AKT signal pathway. These results may provide a potential perspectives on improving the wound healing.

Notch signal deficiency alleviates hypertrophic scar formation after wound healing through the inhibition of inflammation.

Pathological scar is a common complication after wound healing. One of the most important factors that affects scar formation is inflammation. During this process, macrophages play a critical role in the wound healing process, as well as in scar formation. Notch signaling is reported to participate in inflammation and fibrosis; however, whether it affects scar formation is still unclear. In this study, RBP-J knockout mice, in which Notch signaling was down-regulated, and control mice were used, and a skin incision model was established. Sirius red staining and Masson staining suggested that RBP-J knockout could significantly reduce collagen sedimentation after wound healing. Western blot analysis and RT-PCR also confirmed the results. During wound healing, the expression of inflammatory cytokines and macrophage infiltration were decreased in RBP-J knockout mice. In vitro, it was also verified that RBP-J deficiency in macrophages effectively suppressed the expression of inflammatory cytokines and chemotaxis of macrophages after LPS stimulation. In conclusion, blocking Notch signaling in macrophages effectively alleviated scar formation by suppressing the inflammatory response and collagen sedimentation.

Encapsulation of troglitazone and AVE0991 by gelation microspheres promotes epithelial transformation of adipose-derived stem cells.

Deformities in human soft tissue caused by trauma or burn present a difficult problem in plastic surgery. In this study, we encapsulated troglitazone and angiotensin 1-7 mimetic AVE0991 in gelation microspheres with the goal of inducing epithelial transformation for potential applications in tissue reconstruction. After troglitazone or AVE0991 were encapsulated to gelation microspheres, their release kinetics and bioactivity were examined. Surface morphology and diameter of the gelation microspheres were evaluated using light microscopy. The release of the drugs was assessed in the presence of human adipose-derived stem cells (ADSCs). Treatment with troglitazone microspheres increased cell viability and activated the ß-catenin in ADSCs. Moreover, the AVE0991 microspheres also increased cell viability and C-myc expression of ADSCs. These results showed that troglitazone and AVE0991 microspheres promoted the activity of ADSCs. Furthermore, ADSCs were co-treated with troglitazone and AVE0991 microspheres. Western blot and immunofluorescent staining showed that co-treatment with troglitazone and AVE0991 microspheres elevated the expression of epithelialization associated protein CK14 in ADSCs. In conclusion, our findings indicate that microspheres with troglitazone and AVE0991 can significantly improve the viability and epithelialization of ADSCs, which provides a new approach for the construction of tissue-engineered skin.

MCPIP1 alleviated lipopolysaccharide-induced liver injury by regulating SIRT1 via modulation of microRNA-9.

The severity of sepsis is associated with excessive inflammatory responses. MCP-1 induced protein (MCPIP1) could negatively regulate inflammatory responses by deubiquitinating K48 or K63 polyubiquitins of TNF receptor-associated factors. The function of MCPIP1 in negative regulation of inflammation is known, however, only the exact molecular pathway remains unknown. The aim of this study was to investigate whether and how MCPIP1 is involved in the regulation of lipopolysaccharides (LPS)-induced liver injury. Macrophages and a mouse model were induced by LPS treatment. Several in vitro assays, such as quantitative real-time PCR, immunoblotting, cell transfection, dual luciferase reporter assay, Enzyme-linked immunosorbent assay, and Hematoxylin-Eosin staining assay were used to explore the role of MCPIP1 and the interaction between MCPIP1, Sirtuin 1 (SIRT1), and microRNA-9 (miR-9). We found that the level of MCPIP1 increased and the level of SIRT1 decreased in LPS induced Kupffer cells or RAW 264.7 macrophages. Overexpression of MCPIP1 alleviated cytokine secretion and p65 nuclear translocation. Further study showed that MCPIP1 regulated p65 nuclear translocation by controlling p65 acetylation via promoting SIRT1 expression. Meanwhile, we found that miR-9 could directly regulate SIRT1 transcription by binding to the 3'-Untranslated Region of SIRT1 messenger RNA and that miR-9 was negatively regulated by MCPIP1. Importantly, overexpression of MCPIP1 in vivo could alleviate LPS-induced inflammation responses and liver injury in septic mice. These results demonstrated that MCPIP1 could alleviate inflammation responses and sepsis associated liver injury by promoting the expression of SIRT1, and miR-9 was involved in the MCPIP1-mediated regulation of SIRT1. Collectively, our results provide a possible novel signaling axis involving MCPIP1/miR-9/SIRT1 in LPS-induced septic mice.

IL-17 Promotes Scar Formation by Inducing Macrophage Infiltration.

Trauma or burn injuries that affect the deep dermis often produce a hypertrophic scar, which limits patients' joint movement and generates an aesthetic problem. Inflammation is believed to be one of the main pathogenic mechanisms. We found that IL-17 was increased in scar tissues from patients with hypertrophic scar compared with normal skin. Recombinant mouse IL-17 was subcutaneously injected into mice that underwent full-thickness excision surgery to investigate the role of IL-17 in scar formation. Mice stimulated with IL-17 showed aggravated fibrogenesis, delayed wound healing, and increased inflammation. In addition, macrophage infiltration was also increased. According to the results of the Transwell assay, IL-17 promoted macrophage infiltration through an indirect mechanism. After depleting macrophages with clodronate liposomes, the effect of IL-17 disappeared. Levels of monocyte chemotactic protein (MCP) 1, MCP2, and MCP3 (together referred to as MCPs) were increased by IL-17 stimulation. Bindarit (an inhibitor of MCPs) was used to verify the role of MCPs. In addition, the Ly6C-low macrophages were responsible for wound fibrogenesis in mice. In this study, we detected the increased levels of IL-17 for the first time and revealed that IL-17 induced the infiltration of a specific subtype of macrophages to aggravate fibrosis through an MCP-dependent mechanism. Thus, our results provide a better understanding of scar formation and new strategies for scar prevention.

Inhibition of SIRT1 by microRNA-9, the key point in process of LPS-induced severe inflammation.

Severe inflammation may lead to multiple organs dysfunction syndrome, which has a high mortality. MicroRNA is found participated in this process. In this study we developed a lipopolysaccharide-induced inflammation cell model on macrophages and a lipopolysaccharide-induced inflammation mouse model. It was found that during inflammation, microRNA-9 was increased, accompanied with the up-regulation of pro-inflammatory cytokines and anti-inflammatory cytokines. Down-regulation of microRNA-9 inhibited the up-regulation of inflammatory cytokines, promoted the up-regulation of anti-inflammatory cytokines and induced the remission of organ damage, showing a protective effect in inflammation. Bioinformatics analysis combined with luciferase reporter assay showed that SIRT1 was the target gene of microRNA-9. Transfection of microRNA-9 inhibitor could increase the level of SIRT1 and decrease the activation of NF-κB pathway in macrophages. Myeloid specific sirt1 knockout mice were included and we found that lack of SIRT1 in mice macrophages led to aggravated inflammation, cell apoptosis and organ injury, and eliminated the protective property of microRNA-9 inhibitor. In conclusion, we demonstrated that inhibition of microRNA-9 could alleviate inflammation through the up-regulation of SIRT1 and then suppressed the activation of NF-κB pathway. This is a meaningful explore about the specific mechanism of microRNA-9 in inflammation.

SIRT1 activation promotes angiogenesis in diabetic wounds by protecting endothelial cells against oxidative stress.

OBJECTIVE: Chronic wounds are a devastating complication of diabetes and can lead to amputations or even death. Current medical therapies are insufficient to accelerate its repair. The objective of this study was to explore the role of Sirtuin1 (SIRT1) in diabetic wounds. METHODS AND MATERIALS: Perilesional skin tissue samples from diabetic ulcers and normoglycemic trauma wounds were used to detect SIRT1 expression and oxidative stress levels. In a diabetic mouse model, SIRT1 was pharmacologically activated to attenuate angiogenesis and accelerate wound closure. Finally, in vitro experiments were performed to elucidate some of the mechanisms by which SIRT1 activation promotes angiogenesis in diabetic wound healing. RESULTS: We found that skin tissue from diabetes patients showed lower expression of SIRT1 and severe oxidative stress. Decreased SIRT1 expression was observed in skin tissue from streptozocin (STZ)-induced diabetic mice and was associated with impaired wound healing. In addition, the wounds of STZ-induced diabetic mice treated with SRT1720 (a specific SIRT1 activator) demonstrated locally improved wound healing and angiogenesis. In the in vitro experiment, similar results were observed. Under hyperglycemia conditions, human umbilical vein endothelial cells (HUVECs) showed lower expression of SIRT1 and higher levels of reactive oxygen species (ROS) production. Furthermore, the migration, proliferation and in vitro tube formation ability of HUVECs were impaired under hyperglycemia conditions, and SRT1720 treatment rescued these impairments and decreased ROS production in HUVECs. CONCLUSIONS: This study provides experimental evidence that SIRT1 activation could improve angiogenesis in wounds in vitro and in vivo and that sirtuin1 activation accelerates wound healing in diabetic mice by promoting angiogenesis. These positive therapeutic effects may be mediated by protecting vascular endothelial cells from oxidative stress injury. This study suggested that SIRT1 may serve as a potentially important and potent therapeutic target for treating diabetic ulcers.

Cell-free therapy based on adipose tissue stem cell-derived exosomes promotes wound healing via the PI3K/Akt signaling pathway.

INTRODUCTION: Adipose tissue-derived stem cells (ADSCs) have been shown to enhance wound healing via their paracrine function. Exosomes, as one of the most important paracrine factors, play an essential role in this process. However, the concrete mechanisms that underlie this effect are poorly understood. In this study, we aim to explore the potential roles and molecular mechanisms of exosomes derived from ADSCs in cutaneous wound healing. METHODS: Normal human skin fibroblasts and ADSCs were isolated from patient skin and adipose tissues. ADSCs were characterized by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Exosomes were purified from human ADSCs by differential ultracentrifugation and identified by electron microscopy, nanoparticle tracking, fluorescence confocal microscopy and western blotting. Fibroblasts were treated with different concentrations of exosomes, and the synthesis of collagen was analyzed by western blotting; the levels of growth factors were analyzed by real-time quantitative PCR (RT-PCR) and ELISA; and the proliferation and migration abilities of fibroblasts were analyzed by real-time cell analysis, CCK-8 assays and scratch assays. A mouse model with a full-thickness incision wound was used to evaluate the effect of ADSC-derived exosomes on wound healing. The level of p-Akt/Akt was analyzed by western blotting. Ly294002, a phosphatidylinositol 3-kinases (PI3K) inhibitor, was used to identify the underlying mechanisms by which ADSC-derived exosomes promote wound healing. RESULTS: ADSC-derived exosomes were taken up by the fibroblasts, which showed significant, dose-dependent increases in cell proliferation and migration compared to the behavior of cells without exosome treatment. More importantly, both the mRNA and protein levels of type I collagen (Col 1), type III collagen (Col 3), MMP1, bFGF, and TGF-ß1 were increased in fibroblasts after stimulation with exosomes. Furthermore, exosomes significantly accelerated wound healing in vivo and increased the level of p-Akt/Akt in vitro. However, Ly294002 alleviated these exosome-induced changes, suggesting that exosomes from ADSCs could promote and optimize collagen deposition in vitro and in vivo and further promote wound healing via the PI3K/Akt signaling pathway. CONCLUSIONS: This study demonstrates that ADSC-derived exosomes can promote fibroblast proliferation and migration and optimize collagen deposition via the PI3K/Akt signaling pathway to further accelerate wound healing. Our results suggest that ADSCs likely facilitate wound healing via the release of exosomes, and the PI3K/Akt pathway may play a role in this process. Our data also suggest that the clinical application of ADSC-derived exosomes may shed new light on the use of cell-free therapy to accelerate full-thickness skin wound healing and attenuate scar formation.

Wild-type p53-modulated autophagy and autophagic fibroblast apoptosis inhibit hypertrophic scar formation.

Hypertrophic scarring is a serious fibrotic skin disease, and the abnormal activation of hypertrophic scar fibroblasts (HSFs) intensifies its pathogenesis. Our previous studies have demonstrated that the dysregulation of autophagy in HSFs is associated with fibrosis. However, knowledge regarding the regulation of HS fibrosis by p53-modulated autophagy is limited. Here, we investigated the effect of p53-modulated autophagy on HS fibrosis. The overexpression of wtp53 (Adp53) promoted autophagic capacity and inhibited collagen and α-SMA expression in HSFs. In contrast, LC3 (AdLC3) overexpression did not suppress Col 1, Col 3, or α-SMA expression, but LC3 (shLC3) knockdown downregulated collagen expression. Adp53-modulated autophagy altered Bcl-2 and Bcl-xL expression, but AdLC3 affected only Bcl-xL expression. Silencing Bcl-xL suppressed collagen expression, but autophagy was also inhibited. Flow cytometry showed that the silencing of Bcl-2 (sibcl-2), Bcl-xL (sibcl-xL), and Adp53 significantly increased apoptosis in the HSFs. Therefore, wtp53 inhibited fibrosis in the HSFs by modulating autophagic HSF apoptosis; moreover, the inhibition of autophagy by sibcl-xL had antifibrotic effects. In addition, treatment with Adp53, AdLC3, shLC3, sibcl-2, and sibcl-xL reduced scar formation in a rabbit ear scar model. These data confirm that wtp53-modulated autophagy and autophagic HSF apoptosis can serve as potential molecular targets for HS therapy.

MicroRNA-138 Aggravates Inflammatory Responses of Macrophages by Targeting SIRT1 and Regulating the NF-κB and AKT Pathways.

BACKGROUND/AIMS: With increased understanding of sepsis, mortality is decreasing. However, there is still a lack of effective therapeutic strategy. The inflammatory response of macrophages is critical during sepsis. METHODS: Macrophages were stimulated with LPS. Western blotting and qRT-PCR were used to detect inflammatory responses. Then, the inhibitor of microRNA-138 was transfected and Western blotting, qRT-PCR, H&E staining and ELISA were used to verify the role of microRNA-138 in inflammation. Then target gene prediction databases were used to predict the potential target of microRNA-138. Both animal and cell models under LPS challenges were established to verify the regulation of SIRT1 and microRNA-138 during inflammation. RESULTS: The present study showed that microRNA-138 was increased in macrophages stimulated with LPS. Additionally, the NF-κB and AKT pathways were both activated. The pre-treatment of microRNA-138 inhibitor decreased inflammatory factors, downregulated the NF-κB pathway, activated the AKT pathway and protected against organ damage in mice challenged with LPS. SIRT1 was demonstrated as a potential target of microRNA-138In macrophages stimulated with LPS, the inhibition effect of microRNA-138 inhibitor on inflammation was lost by SIRT1 siRNA pre-treatment. In the animal model, the protective effect of microRNA-138 antagomir disappeared in SIRT1 knockout mice. CONCLUSION: We demonstrated that miR-138 participated in the inflammatory process by inhibiting SIRT1 and activating the NF-κB pathway.

Prolonged skin grafts survival time by IFN-γ in allogeneic skin transplantation model during acute rejection through IFN-γ/STAT3/IDO pathway in epidermal layer.

Allogeneic skin transplantation is the life-saving therapy for multiple diseases, including extensive burn, large-scale trauma and certain post-surgical complications. However, acute rejection impedes clinical application of allogeneic skin transplantation. Although a lot of novel immunosuppressant drugs have been developed, there is still great need for ideal therapy with less complication and more therapeutic effects. Here, we found interferon gamma (IFN-γ) as an immunomodulatory cytokine prolonged the survival time of allografts from (8.50 ±â€¯1.517) days to (14.83 ±â€¯2.714) days at best. Indoleamine-2, 3-dioxygenase (IDO) has been proposed to play key roles in induction of immune tolerance. Using in vitro tissue culture and primary keratinocytes and fibroblasts, we investigated the regulatory effects of IFN-γ on the IDO expression. IFN-γ upregulated IDO expression through STAT3 phosphorylation and this upregulation was reduced by abolition of STAT3 phosphorylation through a STAT3 phosphorylation inhibitor. Interestingly, IFN-γ induced IDO expression predominately in epidermis rather than dermis. In consistent with these results, IFN-γ significantly triggered IDO expression in keratinocytes but not fibroblasts. Taken together, this suggests that IFN-γ might be a potential immunomodulatory drug in acute rejection and keratinocytes in epidermis may play a main role in immune tolerance after allogeneic skin transplantation.

SIRT1 regulates inflammation response of macrophages in sepsis mediated by long noncoding RNA.

Molecular mechanisms for macrophage immune responses modulated by SIRT1 during sepsis remain unclear. Here, we show that SIRT1 expression is down-regulated in macrophages from mouse sepsis model or LPS stimulation. SIRT1 expression in macrophages correlates with low levels of a long noncoding RNA (lncRNA)-NONMMUT003701 [named as lncRNA-CCL2]. SIRT1 inhibits lncRNA-CCL2 expression via sustaining a repressive chromatin state in the lncRNA-CCL2 locus. The inflammation cytokines expression is downregulated by knockdown of lncRNA-CCL2. Such inhibition can be reversed partly by decreased SIRT1 activity. Thus, this work uncovers previously unidentified mechanisms in which SIRT1 associates with lncRNA and lncRNA regulates macrophage inflammatory response.

Allogeneic adipose-derived stem cells promote survival of fat grafts in immunocompetent diabetic rats.

Autologous adipose-derived stem cells (ADSCs) can protect fat grafts in cell-assisted lipotransfer (CAL). However, diabetes alters the intrinsic properties of ADSCs and impairs their function so that they lack these protective effects. We investigate whether allogeneic ADSCs from healthy donors could protect fat grafts in immunocompetent diabetic rats. Syngeniec adipose tissues and ADSCs were derived from diabetic Lewis (LEW) rats, whereas allogeneic ADSCs were from healthy brown-Norway rats. A grafted mixture containing 0.7 ml granule fat and 0.3 ml 6 × 10(6) allogeneic/syngeneic ADSCs was injected subcutaneously on the skulls of diabetic LEW rats. Fat samples were harvested to evaluate the levels of injury and vascularization as shown by perilipin A, CD34 and VEGF at 14 days. The immune response was evaluated with a lymphocytotoxicity test and the CD4/CD8 ratio in peripheral blood at 14 days. The volume retention of fat grafts was measured at 3 months. Healthy allogeneic ADSCs increased the expression levels of perilipin A, CD34 and VEGF at 14 days. The volume retention of fat grafts was improved by allogeneic ADSCs at 3 months. ADSCs were demonstrated to have low immunogenicity by the lymphocyte proliferation test and immunophenotype including MHC and co-stimulatory markers. The lymphocytotoxicity test and CD4/CD8 ratio indicated no obvious immune response elicited by allogeneic ADSCs. Thus, healthy allogeneic ADSCs can promote the survival of fat grafts in this immunocompetent diabetic rat model, with little or no obvious immune rejection.

Loureirin B Inhibits Hypertrophic Scar Formation via Inhibition of the TGF-ß1-ERK/JNK Pathway.

BACKGROUND/AIMS: Our previous study confirmed that Loureirin B (LB) can inhibit hypertrophic scar formation. However, the mechanism of LB-mediated inhibition of scar formation is still unknown. METHODS: Immunohistochemistry was used to detect expression of Col1, FN and TGF-ß1 in skin and scar tissue. Fibroblasts were stimulated with TGF-ß1 to mimic scar formation. LB or MAPK inhibitors were used to study the pathways involved in the process. Western blotting was used to evaluate the expression of p-JNK, p-ERK, p-p38, Col1 and FN. The contractile capacity of fibroblasts was evaluated using a gel contraction assay. Tissues were cultured ex vivo with LB to further investigate the participation of ERK and JNK in the LB-mediated inhibition of scar formation. RESULTS: FN and Col1 were up regulated in hypertrophic scars. LB down regulated p-ERK and p-JNK in TGF-ß1-stimulated fibroblasts, while levels of phosphorylated p38 did not change. The down regulation of p-ERK and p-JNK was associated with a reduction of Col1 and FN. Similarly, inhibition of ERK and JNK down regulated the expression of Col1 and FN in TGF-ß1-stimulated fibroblasts. LB down regulated protein levels of p-ERK and p-JNK in cultured hypertrophic scar tissue ex vivo. CONCLUSIONS: This study suggests that LB can inhibit scar formation through the ERK/JNK pathway.

Loureirin B inhibits fibroblast proliferation and extracellular matrix deposition in hypertrophic scar via TGF-ß/Smad pathway.

The ethanolic extract of Resina Draconis (RDEE) has been reported beneficial to normal wound healing yielding more regularly arranged collagen fibres. Loureirin B, a major component in RDEE, has been supposed to be effective on the prevention and treatment of pathological scars. To investigate the therapeutic effects of loureirin B on hypertrophic scar (HS), fibroblasts from human HS and normal skin (NS) were isolated. Results showed that loureirin B dose-dependently downregulated both mRNA and protein levels of type I collagen (ColI), type III collagen (ColIII) and α-smooth muscle actin (α-SMA) in HS fibroblasts. Loureirin B also suppressed fibroblast proliferative activity and redistributed cell cycle, but did not affect cell apoptosis. In vivo rabbit ear scar model, loureirin B significantly improved the arrangement and deposition of collagen fibres, decreased protein levels of ColI, ColIII and α-SMA and suppressed myofibroblast differentiation and scar proliferative activity. In NS fibroblasts, loureirin B effectively inhibited TGF-ß1-induced upregulation of ColI, ColIII and α-SMA levels, myofibroblast differentiation and the activation of Smad2 and Smad3. Loureirin B also affected mRNA levels of major MMPs and TIMPs in TGF-ß1-stimulated fibroblasts. Taken together, this study demonstrates that loureirin B could downregulate the expression of fibrosis-related molecules by regulating MMPs and TIMPs levels, inhibit scar fibroblast proliferation and suppress TGF-ß1-induced fibrosis, during which TGF-ß1/Smad2/3 pathway is likely involved. These findings suggest that loureirin B is a potential therapeutic compound for HS treatment.

Allogeneic Adipose-Derived Stem Cells Protect Fat Grafts at the Early Stage and Improve Long-Term Retention in Immunocompetent Rats.

BACKGROUND: Syngeneic adipose-derived stem cells (ASCs) promote the survival of fat grafts. But it is unclear whether allogeneic ASCs have a similar protective effect. In this study, we investigated the protective effect of allogeneic ASCs in a fat graft model of immunocompetent rats. METHODS: Syngeneic and allogeneic ASCs were derived from Lewis (LEW) and Norway-Brown rats, respectively. Fifty-four LEW rats were divided into three groups. Each LEW rat was injected subcutaneously at two paravertebral spots with adipose granules premixed with DMEM (AFT group), syngeneic ASCs (SYNG group), or allogeneic ASCs (ALLG group). Fat grafts were harvested at 7 and 14 days to examine apoptosis rates and immunochemistry staining was performed for Perilipin A and CD34. At 3 months, fat graft volume retentions were measured. The proportion of regulatory T (Treg) cells and the ratio of CD4/CD8 cells in blood were analyzed at 7 days. RESULTS: Expression of Perilipin A and CD34 was higher in the ALLG group than the AFT group at 14 days (P < 0.05). The apoptosis rate in the ALLG group decreased in comparison with the AFT group at 7 and 14 days (P < 0.05). At 3 months, allogeneic ASCs increased fat graft volume retentions (P < 0.05). No difference was found in the proportion of Treg cells and CD4/CD8 cells ratio between groups. There were no statistically significant difference between ALLG and SYNG groups at all time points (P > 0.05). CONCLUSIONS: Allogeneic ASCs protected fat grafts at the early stage and improved long-term volume retention in the fat graft model of immunocompetent rats with no or little obvious immune rejection.