Shichao Han

ROS-Mediated NLRP3 Inflammasome Activity Is Essential for Burn-Induced Acute Lung Injury

The NLRP3 inflammasome is necessary for initiating acute sterile inflammation. However, its role in the pathogenesis of burn-induced acute lung injury (ALI) is unknown. This study aimed to determine the role of the NLRP3 inflammasome and the signaling pathways involved in burn-induced ALI. We observed that the rat lungs exhibited enhanced inflammasome activity after burn, as evidenced by increased levels of NLRP3 expression and Caspase-1 activity and augmented inflammatory cytokines. Inhibition of NLRP3 inflammasome by BAY11-7082 attenuated burn-induced ALI, as demonstrated by the concomitant remission of histopathologic changes and the reduction of myeloperoxidase (MPO) activity, inflammatory cytokines in rat lung tissue, and protein concentrations in the bronchoalveolar lavage fluid (BALF). In the in vitro experiments, we used AMs (alveolar macrophages) challenged with burn serum to mimic the postburn microenvironment and noted that the serum significantly upregulated NLRP3 inflammasome signaling and reactive oxygen species (ROS) production. The use of ROS scavenger N-acetylcysteine (NAC) partially reversed NLRP3 inflammasome activity in cells exposed to burn serum. These results indicate that the NLRP3 inflammasome plays an essential role in burn-induced ALI and that burn-induced NLRP3 inflammasome activity is a partly ROS-dependent process. Targeting this axis may represent a promising therapeutic strategy for the treatment of burn-induced ALI.

MiR-10a and miR-181c regulate collagen type I generation in hypertrophic scars by targeting PAI-1 and uPA

Urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor‐1 (PAI‐1) have been proposed to play key roles in extracellular matrix (ECM) deposition in hypertrophic scars (HS). Here, we found that in HS fibroblasts (HFs) miR‐181c and miR‐10a were differentially‐expressed and targeted uPA and PAI‐1, respectively. The production of Type 1 collagen (Col1) was inhibited by miR‐181c knockdown or miR‐10a overexpression in HFs, and this resulted in increased levels of metalloproteinase 1 (MMP1). These results suggest that the miR‐181c–uPA and miR‐10a–PAI‐1 regulatory pathways have an integral role in HS pathogenesis.

Adipose tissue-derived stem cells suppress hypertrophic scar fibrosis via the p38/MAPK signaling pathway

BackgroundHypertrophic scars (HS) generally occur after injury to the deep layers of the dermis, resulting in functional deficiency for patients. Growing evidence has been identified that the supernatant of adipose tissue-derived stem cells (ADSCs) significantly ameliorates fibrosis of different tissues, but limited attention has been paid to its efficacy on attenuating skin fibrosis. In this study, we explored the effect and possible mechanism of ADSC-conditioned medium (ADSC-CM) on HS.MethodReal-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to detect the expression of collagen I (Col1), collagen III (Col3), and α-smooth muscle actin (α-SMA) after fibroblasts and cultured HS tissues were stimulated with ADSC-CM and p38 inhibitor/activator. Immunofluorescence staining was performed to test the expression of α-SMA. Masson’s trichrome staining, hematoxylin and eosin (H&E) staining, and immunohistochemistry staining were carried out to assess the histological and pathological change of collagen in the BALB/c mouse excisional model. All data were analyzed by using SPSS17.0 software. Statistical analysis was performed by Student’s t tests.ResultsThe in vitro and ex vivo study revealed ADSC-CM decreased the expression of Col1, Col3, and α-SMA. Together, thinner and orderly arranged collagen was manifested in HS tissues cultured with ADSC-CM. Dramatically, the assessed morphology showed an accelerated healing rate, less collagen deposition, and col1- and col3-positive cells in the ADSC-CM treated group. Importantly, the protein level of p-p38 was downregulated in a concentration-dependent manner in HS-derived fibroblasts with ADSC-CM treatment, which further decreased the expression of p-p38 after the application of its inhibitor, SB203580. SB203580 led to an obvious decline in the expression of Col1, Col3, and α-SMA in fibroblasts and cultured HS tissues and presented more ordered arrangement and thinner collagen fibers in BALB/c mice. Lastly, anisomycin, an agonist of p38, upregulated the expression of fibrotic proteins and revealed more disordered structure and denser collagen fibers.ConclusionThis study demonstrated that ADSC-CM could decrease collagen deposition and scar formation in in vitro, ex vivo and in vivo experiments. The regulation of the p38/MAPK signaling pathway played an important role in the process. The application of ADSC-CM may provide a novel therapeutic strategy for HS treatment, and the anti-scarring effect can be achieved by inhibition of the p38/MAPK signaling pathway.

Melatonin prevents acute kidney injury in severely burned rats via the activation of SIRT1

Acute kidney injury (AKI) is a common complication after severe burns. Melatonin has been reported to protect against multiple organ injuries by increasing the expression of SIRT1, a silent information regulator that regulates stress responses, inflammation, cellular senescence and apoptosis. This study aimed to investigate the protective effects of melatonin on renal tissues of burned rats and the role of SIRT1 involving the effects. Rat severely burned model was established, with or without the administration of melatonin and SIRT1 inhibitor. The renal function and histological manifestations were determined to evaluate the severity of kidney injury. The levels of acetylated-p53 (Ac-p53), acetylated-p65 (Ac-p65), NF-κB, acetylated-forkhead box O1 (Ac-FoxO1), Bcl-2 and Bax were analyzed to study the underlying mechanisms. Our results suggested that severe burns could induce acute kidney injury, which could be partially reversed by melatonin. Melatonin attenuated oxidative stress, inflammation and apoptosis accompanied by the increased expression of SIRT1. The protective effects of melatonin were abrogated by the inhibition of SIRT1. In conclusion, we demonstrate that melatonin improves severe burn-induced AKI via the activation of SIRT1 signaling.

Loureirin B Inhibits Hypertrophic Scar Formation via Inhibition of the TGF-β1-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-β1 in skin and scar tissue. Fibroblasts were stimulated with TGF-β1 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-β1-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-β1-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.

Knockdown of lncRNA-ATB suppresses autocrine secretion of TGF-β2 by targeting ZNF217 via miR-200c in keloid fibroblasts

Abnormally high activation of transforming growth factor-β (TGF-β) signaling has been demonstrated to be involved in the initiation and progression of keloids. However, the functional role of long non-coding RNA (lncRNA)-activated by TGF-β (lncRNA-ATB) in keloids has not been documented. Here we investigated the role of lncRNA-ATB in the autocrine secretion of TGF-β in keloid fibroblasts (KFs) and explored the underlying molecular mechanism. Using immunohistochemistry and quantitative RT-PCR analysis, we showed that lncRNA-ATB and ZNF217, a transcriptional activator of TGF-β, were overexpressed and miR-200c, which targets ZNF217, was under-expressed in keloid tissue and keloid fibroblasts. Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-β2 and ZNF217 expression but upregulated expression of miR-200c in KFs. Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-β2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs. Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-β2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-β2. These findings may provide potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.

Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways

Wound healing is a highly orchestrated physiological process consisting in a complex interaction of cellular and biochemical events. Human amniotic epithelial stem cells (HAESCs) have been shown to be an attractive resource for wound healing because they are primitive stem cells. However, the exact effects of amnion-derived stem cells on the migration or proliferation of keratinocytes and their potential mechanism are not fully understood. We have found that HAESCs accelerate the migration of keratinocytes and induce a remarkable increase in the activity of phospho-ERK, phospho-JNK, and phospho-AKT, the blockade of which by their specific inhibitors significantly inhibits migration induced by HAESC-conditioned medium (CM). Furthermore, the co-culture of keratinocytes with HAESCs up-regulates the expression levels of cell proliferation proteins Cyclin D1, Cyclin D3 and Mdm2. In vivo animal experiments have shown that HAESC-CM improves wound healing, whereas blockade with ERK, JNK and AKT inhibitors significantly impairs wound healing. Taken together, these results reveal, for the first time, that HAESCs promote wound healing by facilitating the migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways and might be a potential therapy in skin wound healing.

Notch1 Pathway Protects against Burn-Induced Myocardial Injury by Repressing Reactive Oxygen Species Production through JAK2/STAT3 Signaling

Oxidative stress plays an important role in burn-induced myocardial injury, but the cellular mechanisms that control reactive oxygen species (ROS) production and scavenging are not fully understood. This study demonstrated that blockade of Notch signaling via knockout of the transcription factor RBP-J or a pharmacological inhibitor aggravated postburn myocardial injury, which manifested as deteriorated serum CK, CK-MB, and LDH levels and increased apoptosis in vitro and in vivo. Interruption of Notch signaling increased intracellular ROS production, and a ROS scavenger reversed the exacerbated myocardial injury after Notch signaling blockade. These results suggest that Notch signaling deficiency aggravated postburn myocardial injury through increased ROS levels. Notch signaling blockade also decreased MnSOD expression in vitro and in vivo. Notably, Notch signaling blockade downregulated p-JAK2 and p-STAT3 expression. Inhibition of JAK2/STAT3 signaling with AG490 markedly decreased MnSOD expression, increased ROS production, and aggravated myocardial injury. AG490 plus GSI exerted no additional effects. These results demonstrate that Notch signaling protects against burn-induced myocardial injury through JAK2/STAT3 signaling, which activates the expression of MnSOD and leads to decreased ROS levels.

Src promotes cutaneous wound healing by regulating MMP-2 through the ERK pathway.

Wound healing is a highly orchestrated, multistep process, and delayed wound healing is a significant symptomatic clinical problem. Keratinocyte migration and re-epithelialization play the most important roles in wound healing, as they determine the rate of wound healing. In our previous study, we found that Src, one of the oldest proto-oncogenes encoding a membrane-associated, non-receptor protein tyrosine kinase, promotes keratinocyte migration. We therefore hypothesized that Src promotes wound healing through enhanced keratinocyte migration. In order to test this hypothesis, vectors for overexpressing Src and small interfering RNAs (siRNAs) for silencing of Src were used in the present study. We found that the overexpression of Src accelerated keratinocyte migration in vitro and promoted wound healing in vivo without exerting a marked effect on cell proliferation. The extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways play important roles in Src-accelerated keratinocyte migration. Further experiments demonstrated that Src induced the protein expression of matrix metallopro-teinase-2 (MMP-2) and decreased the protein expression of E-cadherin. We suggest that ERK signaling is involved in the Src-mediated regulation of MMP-2 expression. The present study provided evidence that Src promotes keratinocyte migration and cutaneous wound healing, in which the regulation of MMP-2 through the ERK pathway plays an important role, and thus we also demonstrated a potential therapeutic role for Src in cutaneous wound healing.

Rho kinase inhibitor Y-27632 promotes the differentiation of human bone marrow mesenchymal stem cells into keratinocyte-like cells in xeno-free conditioned medium

IntroductionBone marrow mesenchymal stem cells (BMSCs), which have the ability to self-renew and to differentiate into multiple cell types, have recently become a novel strategy for cell-based therapies. The differentiation of BMSCs into keratinocytes may be beneficial for patients with burns, disease, or trauma. However, the currently available cells are exposed to animal materials during their cultivation and induction. These xeno-contaminations severely limit their clinical outcomes. Previous studies have shown that the Rho kinase (ROCK) inhibitor Y-27632 can promote induction efficiency and regulate the self-renewal and differentiation of stem cells. In the present study, we attempted to establish a xeno-free system for the differentiation of BMSCs into keratinocytes and to investigate whether Y-27632 can facilitate this differentiation.MethodsBMSCs isolated from patients were cultured by using a xeno-free system and characterised by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Human primary keratinocytes were also isolated from patients. Then, the morphology, population doubling time, and β-galactosidase staining level of these cells were evaluated in the presence or absence of Y-27632 to determine the effects of Y-27632 on the state of the keratinocytes. Keratinocyte-like cells (KLCs) were detected at different time points by immunocytofluorescence analysis. Moreover, the efficiency of BMSC differentiation under different conditions was measured by quantitative real-time-polymerase chain reaction (RT-PCR) and Western blot analyses.ResultsThe ROCK inhibitor Y-27632 promoted the proliferation and lifespan of human primary keratinocytes. In addition, we showed that keratinocyte-specific markers could be detected in BMSCs cultured in a xeno-free system using keratinocyte-conditioned medium (KCM) independent of the presence of Y-27632. However, the efficiency of the differentiation of BMSCs into KLCs was significantly higher in the presence of Y-27632 using immunofluorescence, quantitative RT-PCR, and Western blot analyses.ConclusionsThis study demonstrated that Y-27632 could promote the proliferation and survival of human primary keratinocytes in a xeno-free culture system. In addition, we found that BMSCs have the ability to differentiate into KLCs in KCM and that Y-27632 can facilitate this differentiation. Our results suggest that BMSCs are capable of differentiating into KLCs in vitro and that the ROCK pathway may play a critical role in this process.