GDNF facilitates the differentiation of ADSCs to Schwann cells and enhances nerve regeneration through GDNF/MTA1/Hes1 axis.

Adipose tissue-derived stem cells (ADSCs) are a kind of stem cells with multi-directional differentiation potential, which mainly restore tissue repair function and promote cell regeneration. It can be directionally differentiated into Schwann-like cells to promote the repair of peripheral nerve injury. Glial cell line-derived neurotrophic factor (GDNF) plays an important role in the repair of nerve injury, but the underlying mechanism remains unclear, which seriously limits its further application.The study aimed to identify the molecular mechanism by which overexpression of glial cell line-derived neurotrophic factor (GDNF) facilitates the differentiation of ADSCs into Schwann cells, enhancing nerve regeneration after injury. In vitro, ADSCs overexpressing GDNF for 48 h exhibited changes in their morphology, with 80% of the cells having two or more prominences. Compared with that of ADSCs, GDNF-ADSCs exhibited increased expression of the Schwann cell marker S100, nerve damage repair-related factors.ADSC cells in normal culture and ADSC cells were overexpressing GDNF(GDNF-ADSCs) were analysed using TMT-Based Proteomic Analysis and revealed a significantly higher expression of MTA1 in GDNF-ADSCs than in control ADSCs. Hes1 expression was significantly higher in GDNF-ADSCs than in ADSCs and decreased by MTA1 silencing, along with a simultaneous decrease in the expression of S100 and nerve damage repair factors. These findings indicate that GDNF promotes the differentiation of ADSCs into Schwann cells and induces factors that accelerate peripheral nerve damage repair.

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.

Exosome/metformin-loaded self-healing conductive hydrogel rescues microvascular dysfunction and promotes chronic diabetic wound healing by inhibiting mitochondrial fission.

Chronic diabetic wounds remain a globally recognized clinical challenge. They occur due to high concentrations of reactive oxygen species and vascular function disorders. A promising strategy for diabetic wound healing is the delivery of exosomes, comprising bioactive dressings. Metformin activates the vascular endothelial growth factor pathway, thereby improving angiogenesis in hyperglycemic states. However, multifunctional hydrogels loaded with drugs and bioactive substances synergistically promote wound repair has been rarely reported, and the mechanism of their combinatorial effect of exosome and metformin in wound healing remains unclear. Here, we engineered dual-loaded hydrogels possessing tissue adhesive, antioxidant, self-healing and electrical conductivity properties, wherein 4-armed SH-PEG cross-links with Ag+, which minimizes damage to the loaded goods and investigated their mechanism of promotion effect for wound repair. Multiwalled carbon nanotubes exhibiting good conductivity were also incorporated into the hydrogels to generate hydrogen bonds with the thiol group, creating a stable three-dimensional structure for exosome and metformin loading. The diabetic wound model of the present study suggests that the PEG/Ag/CNT-M + E hydrogel promotes wound healing by triggering cell proliferation and angiogenesis and relieving peritraumatic inflammation and vascular injury. The mechanism of the dual-loaded hydrogel involves reducing the level of reactive oxygen species by interfering with mitochondrial fission, thereby protecting F-actin homeostasis and alleviating microvascular dysfunction. Hence, we propose a drug-bioactive substance combination therapy and provide a potential mechanism for developing vascular function-associated strategies for treating chronic diabetic wounds.

miR-125b-5p in adipose derived stem cells exosome alleviates pulmonary microvascular endothelial cells ferroptosis via Keap1/Nrf2/GPX4 in sepsis lung injury.

BACKGROUND: Sepsis is a fatal disease with a high rate of morbidity and mortality, during which acute lung injury is the earliest and most serious complication. Injury of pulmonary microvascular endothelial cells (PMVECs) induced by excessive inflammation plays an important role in sepsis acute lung injury. This study is meant to explore the protective effect and mechanism of ADSCs exosomes on excessive inflammation PMVECs injury. RESULTS: We successfully isolated ADSCs exosomes, the characteristic of which were confirmed. ADSCs exosomes reduced excessive inflammatory response induced ROS accumulation and cell injury in PMVECs. Besides, ADSCs exosomes inhibited excessive inflammatory response induced ferroptosis while upregulated expression of GPX4 in PMVECs. And further GPX4 inhibition experiments revealed that ADSCs exosomes alleviated inflammatory response induced ferroptosis via upregulating GPX4. Meanwhile, ADSCs exosomes could increase the expression and nucleus translocation of Nrf2, while decrease the expression of Keap1. miRNA analysis and further inhibition experiments verified that specific delivery of miR-125b-5p by ADSCs exosomes inhibited Keap1 and alleviated ferroptosis. In CLP induced sepsis model, ADSCs exosomes could relieve the lung tissue injury and reduced the death rate. Besides, ADSCs exosomes alleviated oxidative stress injury and ferroptosis of lung tissue, while remarkably increase expression of Nrf2 and GPX4. CONCLUSION: Collectively, we illustrated a novel potentially therapeutic mechanism that miR-125b-5p in ADSCs exosomes could alleviate the inflammation induced PMVECs ferroptosis in sepsis induced acute lung injury via regulating Keap1/Nrf2/GPX4 expression, hence improve the acute lung injury in sepsis.

Deacetylation of IRF8 inhibits iNOS expression and inflammation via SIRT1 in macrophages.

AIMS: Dysregulated interferon regulatory factor 8 (IRF8) mediated inducible nitric oxide synthase (iNOS) transcription is crucial to the pathogenesis of several inflammatory disorders. However, the molecular mechanism that control the transcription activity of IRF8 in the regulation of iNOS is not fully elucidated. This study is undertaken to determine whether SIRT1 impacts IRF8 acetylation level in the macrophages. MAIN METHODS: The silver stain, mass spectrum, bone marrow-derived monocytes differentiation, lentiviral transduction, immunoprecipitation and chromatin immunoprecipitation assay were used to investigate the relationship between IRF8 and SIRT1. KEY FINDINGS: We demonstrate that deacetylation of IRF8 is induced by lipopolysaccharide (LPS) and suppresses iNOS expression. Macrophages expressing acetylation-defective iNOS are highly septic upon transfer to macrophages cleaned up mice. Mechanistically, deacetylation IRF8 facilitates the binding of silent information regulator 1 (SIRT1) to the iNOS promoter and restricts iNOS transcription. The expression of iNOS was enhanced in the macrophages from SIRT1 conditional knockout mice and the progression of sepsis is more serious. SIGNIFICANCE: The discovery of the IRF8-SIRT1 interaction that governs iNOS expression may exploit new therapeutic strategies for inflammatory disorders.

The Notch pathway attenuates burn-induced acute lung injury in rats by repressing reactive oxygen species.

Background: Acute lung injury (ALI) is a common complication following severe burns. The underlying mechanisms of ALI are incompletely understood; thus, available treatments are not sufficient to repair the lung tissue after ALI. Methods: To investigate the relationship between the Notch pathway and burn-induced lung injury, we established a rat burn injury model by scalding and verified lung injury via lung injury evaluations, including hematoxylin and eosin (H&E) staining, lung injury scoring, bronchoalveolar lavage fluid and wet/dry ratio analyses, myeloperoxidase immunohistochemical staining and reactive oxygen species (ROS) accumulation analysis. To explore whether burn injury affects Notch1 expression, we detected the expression of Notch1 and Hes1 after burn injury. Then, we extracted pulmonary microvascular endothelial cells (PMVECs) and conducted Notch pathway inhibition and activation experiments, via a γ-secretase inhibitor (GSI) and OP9-DLL1 coculture, respectively, to verify the regulatory effect of the Notch pathway on ROS accumulation and apoptosis in burn-serum-stimulated PMVECs. To investigate the regulatory effect of the Notch pathway on ROS accumulation, we detected the expression of oxidative-stress-related molecules such as superoxide dismutase, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 2, NOX4 and cleaved caspase-3. NOX4-specific small interfering RNA (siRNA) and the inhibitor GKT137831 were used to verify the regulatory effect of the Notch pathway on ROS via NOX4. Results: We successfully established a burn model and revealed that lung injury, excessive ROS accumulation and an inflammatory response occurred. Notch1 detection showed that the expression of Notch1 was significantly increased after burn injury. In PMVECs challenged with burn serum, ROS and cell death were elevated. Moreover, when the Notch pathway was suppressed by GSI, ROS and cell apoptosis levels were significantly increased. Conversely, these parameters were reduced when the Notch pathway was activated by OP9-DLL1. Mechanistically, the inhibition of NOX4 by siRNA and GKT137831 showed that the Notch pathway reduced ROS production and cell apoptosis by downregulating the expression of NOX4 in PMVECs. Conclusions: The Notch pathway reduced ROS production and apoptosis by downregulating the expression of NOX4 in burn-stimulated PMVECs. The Notch-NOX4 pathway may be a novel therapeutic target to treat burn-induced ALI.

A Novel 3D Culture Model of Human ASCs Reduces Cell Death in Spheroid Cores and Maintains Inner Cell Proliferation Compared With a Nonadherent 3D Culture.

3D cell culture technologies have recently shown very valuable promise for applications in regenerative medicine, but the most common 3D culture methods for mesenchymal stem cells still have limitations for clinical application, mainly due to the slowdown of inner cell proliferation and increase in cell death rate. We previously developed a new 3D culture of adipose-derived mesenchymal stem cells (ASCs) based on its self-feeder layer, which solves the two issues of ASC 3D cell culture on ultra-low attachment (ULA) surface. In this study, we compared the 3D spheroids formed on the self-feeder layer (SLF-3D ASCs) with the spheroids formed by using ULA plates (ULA-3D ASCs). We discovered that the cells of SLF-3D spheroids still have a greater proliferation ability than ULA-3D ASCs, and the volume of these spheroids increases rather than shrinks, with more viable cells in 3D spheroids compared with the ULA-3D ASCs. Furthermore, it was discovered that the SLF-3D ASCs are likely to exhibit the abovementioned unique properties due to change in the expression level of ECM-related genes, like COL3A1, MMP3, HAS1, and FN1. These results indicate that the SLF-3D spheroid is a promising way forward for clinical application.

Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts.

BACKGROUND: MicroRNA-101 (miR-101) is a tumor suppressor microRNA (miRNA) and its loss is associated with the occurrence and progression of various diseases. However, the biological function and target of miR-101 in the pathogenesis of hypertrophic scars (HS) remains unknown. METHODS: We harvested HS and paired normal skin (NS) tissue samples from patients and cultured their fibroblasts (HSF and NSF, respectively). We used quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), enzyme-linked immunosorbent assays (ELISA) and Western blot analyses to measure mRNA levels and protein expression of miR-101, enhancer of zeste homolog 2 (EZH2), collagen 1 and 3 (Col1 and Col3) and α-smooth muscle actin (α-SMA) in different in vitro conditions. We also used RNA sequencing to evaluate the relevant signaling pathways and bioinformatics analysis and dual-luciferase reporter assays to predict miR-101 targets. We utilized a bleomycin-induced fibrosis mouse model in which we injected miR-101 mimics to evaluate collagen deposition in vivo. RESULTS: We found low expression of miR-101 in HS and HSF compared to NS and NSF. Overexpressing miR-101 decreased Col1, Col3 and α-SMA expression in HSF. We detected high expression of EZH2 in HS and HSF. Knockdown of EZH2 decreased Col1, Col3 and α-SMA in HSF. Mechanistically, miR-101 targeted the 3'-untranslated region (3'UTR) of EZH2, as indicated by the decreased expression of EZH2. Overexpressing EZH2 rescued miR-101-induced collagen repression. MiR-101 mimics effectively suppressed collagen deposition in the bleomycin-induced fibrosis mouse model. CONCLUSIONS: Our data reveal that miR-101 targets EZH2 in HS collagen production, providing new insight into the pathological mechanisms underlying HS formation.

Advances on Graphene-Based Nanomaterials and Mesenchymal Stem Cell-Derived Exosomes Applied in Cutaneous Wound Healing.

Graphene is a new type of carbon nanomaterial discovered after fullerene and carbon nanotube. Due to the excellent biological properties such as biocompatibility, cell proliferation stimulating, and antibacterial properties, graphene and its derivatives have become emerging candidates for the development of novel cutaneous wound dressings and composite scaffolds. On the other hand, pre-clinical research on exosomes derived from mesenchymal stem cells (MSC-Exos) has been intensified for cell-free treatment in wound healing and cutaneous regeneration, via ameliorating the damaged microenvironment of the wound site. Here, we provide a comprehensive understanding of the latest studies and observations on the various effects of graphene-based nanomaterials (GBNs) and MSC-Exos during the cutaneous wound repair process, as well as the putative mechanisms thereof. In addition, we propose the possible forward directions of GBNs and MSC-Exos applications, expecting to promote the clinical transformation.

Exosomes from adipose-derived stem cells alleviate the inflammation and oxidative stress via regulating Nrf2/HO-1 axis in macrophages.

ADSCs exosomes, an important means of intercellular communication, can regulate an array of biological processes, including promoting tissue repairs and regeneration, and attenuating inflammation. In this study, we found that ADSCs exosomes could polarize macrophage to an anti-inflammatory phenotype via regulating the expression of Nrf2 and HO-1, and improve inflammatory reaction and injury of multi-organ in sepsis. We revealed that ADSCs exosomes could alleviate LPS induced accumulation of ROS and the expression of inflammatory cytokines IL-1ß, TNF-α, and IL-6 in macrophages. Western blot and Flow cytometry results indicated that expression of M1 markers (iNOS and CD86) in LPS stimulated macrophages were significantly declined, while M2 (Arg1 and CD206) were enhanced when pretreated with ADSCs exosomes. Besides, the stress-related molecule HO-1 was upregulated when pretreated with ADSCs exosomes. Further H0-1 interference experiment indicated that anti-inflammatory effect of ADSCs exosomes was dependent on HO-1. Moreover, ADSCs exosomes enhanced expression and nucleus translocation of Nrf2, while downregulated its negative mediator Keap1. In in vivo sepsis models, intravenous injection of ADSCs exosomes relieved inflammatory cytokines storm and organ injury, while promoted expression of HO-1. In conclusion, we proved that ADSCs exosomes alleviated LPS induced inflammation and exerted protective effect in sepsis via regulating Nrf2/HO-1 expression.

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.

Streptococcus thermophilus Attenuates Inflammation in Septic Mice Mediated by Gut Microbiota.

Sepsis is a life-threatening organ dysfunction condition caused by a dysregulated host response to infection and lack of effective treatment method. Supplementation of probiotics has emerged as a potential biotherapy for inflammatory diseases in recent years, but its role in protecting viscera against the damage caused by sepsis and the underlying mechanism is poorly understood. Streptococcus thermophilus 19 is one of the most well-studied probiotics, which is selected in this study among seven strains isolated from homemade yogurt due to its optimal ability of suppressing the inflammation response in vitro. It showed significant decrease in the expression of TNF-α, IL-1ß, and IL-6 in the co-culture of S. thermophilus 19 and LPS-treated mouse macrophage. The effect of S. thermophilus 19 in mice and the response of mice gut microbiota were subsequently investigated. In LPS-induced septic mouse model, S. thermophilus 19 was highly resistant to LPS and exhibited significantly decreased expressions of inflammatory factors compared to LPS-treated mice. A MiSeq-based 16S rDNA sequence analysis revealed that the decrease of gut microbial diversity in mice intraperitoneally injected with 1 mg/ml LPS were mitigated by the administration of S. thermophilus 19. Fusobacterium significantly decreased during the development of sepsis and rose again after supplement strain 19, while Flavonifractor showed the opposite trend, which demonstrated these two genera were the key bacteria that may function in the mice gut microbiota for alleviation of LPS-induced inflammation reaction. To conclude, S. thermophilus 19 may be a potential candidate for novel biotherapeutic interventions against inflammation caused by sepsis.

Smad interacting protein 1 influences transforming growth factor-ß1/Smad signaling in extracellular matrix protein production and hypertrophic scar formation.

The transforming growth factor (TGF)-ß/Smad signal transduction pathway is closely associated with hypertrophic scar (HS) formation. Smad interacting protein 1 (SIP1) is a cytoplasmic protein that efficiently regulates Smad2-/3-dependent signaling within the TGF-ß1 pathway. SIP1 influences collagen synthesis in the HS through a heretofore unknown mechanism. This study investigated the role of the SIP1-mediated TGF-ß1/Smad signaling pathway in extracellular matrix (ECM) protein production and hypertrophic scarring. SIP1 expression was markedly lower in HS vs. normal skin (NS) tissue, and α-smooth muscle actin (α-SMA) content and collagen I/III (Col I/III) synthesis were inversely correlated with SIP1 expression. Furthermore, SIP1 inhibited Smad2/3 phosphorylation in vitro, and improved the collagen-based architecture of the scar while reducing collagen expression and overall scar formation in a rabbit ear model of HS. Based on these findings, we propose that SIP1 acts as a molecular modulator capable of altering Smad2-/3-facilitated signaling through the control of Smad phosphorylation, thus inhibiting α-SMA and collagen upregulation in fibroblasts and, ultimately, HS formation. The low SIP1 content in scar tissue also suggests that SIP1 (and positive regulation thereof) is a prospective target for selective HS drug therapy.

MicroRNA-130a has pro-fibroproliferative potential in hypertrophic scar by targeting CYLD.

Hypertrophic scars are dermal fibrosis diseases that protrude from the surface of the skin and irregularly extend to the periphery, seriously affecting the appearance and limb function of the patient. In this study, we found that microRNA-130a (miR-130a) was increased in hypertrophic scar tissues and derived primary fibroblasts, accompanied by up-regulation of collagen1/3 and α-SMA. Inhibition of miR-130a in hypertrophic scars fibroblasts suppressed the expression of collagen1/3 and α-SMA as well as the cell proliferation. Bioinformatics analysis combined with luciferase reporter gene assay results indicated that CYLD was a target gene of miR-130a, and the miR-130a mimic could reduce the level of CYLD. In contrast to miR-130a, the expression of CYLD was downregulated in hypertrophic scars and their derived fibroblasts. Overexpressing CYLD inhibited the expression of collagen 1/3 and α-SMA, slowed cell proliferation, and inhibited Akt activity. As expected, further study showed that the overexpression of CYLD could prevent the pro-fibroproliferative effects of miR-130a. Consistent with the in vitro results, the inhibitor of miR-130a effectively ameliorated excessive collagen deposition in bleomycin-induced skin fibrosis mouse model. Taken together, our results indicate that miR-130a promotes collagen secretion, myofibroblast transformation and cell proliferation by targeting CYLD and enhancing Akt activity. Therefore, the miR-130a/CYLD/Akt pathway may serve as a novel entry point for future skin fibrosis research.

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.

ROR alpha protects against LPS-induced inflammation by down-regulating SIRT1/NF-kappa B pathway.

Systemic inflammatory response syndrome (SIRS) is associated with excessive inflammatory response, however, the pathophysiology of inflammation is poorly understood. The retinoid-related orphan receptor α (RORα) is a key inflammatory regulator, but the mechanisms underlying its role remain unclear. The aim of this study was to investigate how RORα was involved in the regulation of inflammatory response. Here we put forward a hypothesis that RORα might negatively regulate inflammatory response by controlling silent information regulator Sirtuin 1 (SIRT1) expression. Stimulation of macrophages in vitro with LPS and LPS administration in vivo were used to explore the function of RORα and the relationship between RORα and SIRT1. We found that the level of RORα was suppressed in macrophages stimulated with LPS and overexpression or knockdown of RORα by transfection with lentivirus or siRNAs significantly decreased or increased, respectively, the pro-inflammatory cytokines IL-1ß, TNF, IL-6 and MCP-1. Importantly, overexpression of RORα suppressed inflammation and alleviated LPS-induced organ injury in vivo. Further study showed that RORα could regulate SIRT1 expression and, consequently, affect deacetyation and nuclear translocation of nuclear factor-kappa B (NF-κB) subunit p65. Moreover, the activation of SIRT1 by its specific agonist, SR1720, could reduce the expression of proinflammatory cytokines in RORα knockdown macrophages stimulated with LPS. In conclusion, we demonstrated that RORα could alleviate LPS-induced inflammation and organ injury both in vivo and in vitro by blocking NF-κB p65 nuclear translocation and restricting acetylation of NF-κB p65 at lysine 310 via the regulation of SIRT1 expression. Targeting RORα might be a promising therapeutic strategy to regulate inflammatory disorders.

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.

Acetylation-Dependent Regulation of Notch Signaling in Macrophages by SIRT1 Affects Sepsis Development.

SIRT1 is reported to participate in macrophage differentiation and affect sepsis, and Notch signaling is widely reported to influence inflammation and macrophage activation. However, the specific mechanisms through which SIRT1 regulates sepsis and the relationship between SIRT1 and Notch signaling remain poorly elucidated. In this study, we found that SIRT1 levels were decreased in sepsis both in vitro and in vivo and that SIRT1 regulation of Notch signaling affected inflammation. In lipopolysaccharide (LPS)-induced sepsis, the levels of Notch signaling molecules, including Notch1, Notch2, Hes1, and intracellular domain of Notch (NICD), were increased. However, NICD could be deacetylated by SIRT1, and this led to the suppression of Notch signaling. Notably, in macrophages from myeloid-specific RBP-J-/- mice, in which Notch signaling is inhibited, pro-inflammatory cytokines were expressed at lower levels than in macrophages from wild-type littermates and in RBP-J-/- macrophages, and the NF-κB pathway was also inhibited. Accordingly, in the case of RBP-J-/- mice, LPS-induced inflammation and mortality were lower than in wild-type mice. Our results indicate that SIRT1 inhibits Notch signaling through NICD deacetylation and thus ultimately alleviates sepsis.

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.