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Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-β1 were treated with IL-10 to analyze the mRNA and some of proteins' expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring. [ABSTRACT FROM AUTHOR]

Autophagy is a tightly regulated physiological process essential for cellular maintenance, differentiation, development, and homeostasis. Aberration of this process associates with the pathogeneses of several diseases in mammals. Hypertrophic scar (HS) is characterized by an abundance of collagenous tissue with hypercellularity. However, the molecular mechanism in HS formation is poorly understood. We compared the autophagic capacity in HS and its normal skin (NS) counterparts and explored the molecular mechanism of autophagy during the formation of HS. Microtubule-associated protein 1 light chain 3 (LC3) proteins in HS and NS were detected by immunohistochemistry, Western blot and quantitative real-time PCR (qPCR). The data showed that LC3 positive staining in HS was less intensive relative to NS group ( p < 0.05). Three forms of LC3, with molecular weights of about 19 kDa (proLC3), 18 kDa (LC3-I) and 16 kDa (LC3-II), respectively, expressed in NS by Western blot. In contrast, only proLC3 expressed while both LC3-I and LC3-II were significantly downregulated in HS. The protein level of beclin 1 in HS was significantly lower compared with NS ( p < 0.05). LC3 and beclin 1 mRNA levels in HS were significantly lower than that in NS ( p < 0.05). These results suggest that the generation of LC3-I and LC3-II are interrupted in HS, and that the resultant decrease of autophagic capacity may associate with the pathogenesis of HS. [ABSTRACT FROM AUTHOR]

The shale gas adsorption and flow characteristics play essential roles in improving shale gas recovery. Motivated by the desire to clarify these characteristics carefully and precisely, a series of shale models with different water contents from 0.6 to 2.4 wt% were established. Presumably, these characteristics were sought to pin down answers by using the grand canonical Monte Carlo (GCMC) and molecular dynamics (MD) methods respectively. Importantly, the analysis of the pore structure of these models is firstly taken into account considering its microstructure to meet the demand for the explanation of the adsorption characteristics of methane. The results showed that the enterable volumes decrease significantly with the increase of water content due to the diffusion and aggregation of water molecules in the middle of enterable pores. Intuitively, it could lead to a marked linear decrease in the adsorption amount of methane from 1.2 mmol/g to 0.6 mmol/g. A curiosity of this study is that the diffusion coefficients of methane increase as the increase of temperature and ensuring the low pressure outside the channel could boost the flux of methane intriguingly. Suffice to say, the optimum development shale gas conditions in this work are at the temperature of 358 K and in the presence of water content of 2.4 wt%. Hence, there is an expectation that this study would provide a guidance for the exploitation of shale gas in the presence of water. [ABSTRACT FROM AUTHOR]

[Display omitted] • We constructed a model of AKI induced by cisplatin and ischaemia–reperfusion. • RG108, a DNA methyltransferase inhibitor, reduces inflammation and kidney damage. • RG108 reduces inflammation through the P38 MAPK/FOS and JNK/JUN pathways. • RG108 may be a novel clinical candidate for the treatment of acute kidney injury. Acute kidney injury (AKI) is an important clinical syndrome characterised by a sudden decline in renal function, often accompanied by renal inflammation and tubular epithelial cell damage. It has been reported that inhibiting DNA methylation significantly suppress the progression of AKI. In the current study, we investigate the effect of the DNA methyltransferase (DNMT) inhibitor RG108 in cisplatin- and hypoxia-reoxygenation-induced AKI. The expression of kidney injury molecules and inflammatory factors was examined by immunofluorescence, Western blotting and Real-time PCR. The results demonstrated that RG108 treatment significantly reduced kidney inflammation and injury. Furthermore, RNA-seq analysis was performed to reveal the regulatory mechanism of RG108 in AKI. The expression of the FOS and JUN genes, which are downstream of the MAPK pathway, were significant increased in AKI. Meanwhile, the expression of FOS and JUN were both inhibited by RG108, which is similar to what we found treatment with a specific JNK inhibitor and a specific p38 MAPK inhibitor, and thus attenuated renal inflammation and injury. In conclusion, we suggest that RG108 inhibits P38 MAPK/FOS and JNK/JUN pathways and attenuates renal injury and inflammatory responses. In these results, RG108 may become a novel MAPK pathway inhibitor and a clinical candidate for the treatment of AKI. [ABSTRACT FROM AUTHOR]

Background and Purpose: Sirtuin1 (SIRT1), the founding member of mammalian class III histone deacetylases, is reported to be a drug target involved in fibrotic diseases. However, whether it is an effective drug target in hypertrophic scar treatment is still not known.Experimental Approach: In the present study, we observed that SIRT1 localized to both the epidermis and the dermis of skin tissues by immunohistochemistry. After knock-down of SIRT1 by shRNA or up-regulating SIRT1 by resveratrol, the expression of α-SMA, Col1 and Col3 in fibroblasts were detected by western blots. A mouse excision wound healing model was used to observe the changes in collagen fibre associated with the different expression levels of SIRT1.Key Results: SIRT1 expression was inhibited in hypertrophic scar tissue. The down-regulation of SIRT1 resulted in an increased expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts. In contrast, the up-regulation of SIRT1 not only inhibited the expression of α-SMA, Col1 and Col3 in hypertrophic scar-derived fibroblasts but also blocked the activation of TGFβ1-induced normal skin-derived fibroblasts. In the mouse model of wound healing, the deletion of SIRT1 resulted in denser collagen fibres and a more disordered structure, whereas resveratrol treatment led to a more organized and thinner collagen fibre, which was similar to that observed during normal wound healing.Conclusions and Implications: The results revealed that SIRT1 negatively regulates TGFβ1-induced fibroblast activation and inhibits excessive scar formation and is, therefore, a promising drug target for hypertrophic scar formation. [ABSTRACT FROM AUTHOR]

Perchlorate, as an oxidizer, has many applications such as explosives and pyrotechnics, especially in rocket propellants and missile motors. Because it was found in water including wells and drinking water in the US, its effect on human health was being noted. However, the reproductive toxic effect on perchlorate is still unclear. In present study, the effects of repeated exposure to perchlorate on reproductive toxicity were evaluated in Wistar rats. The rats were treated orally with perchlorate at doses of 0.05, 1.00 or 10.00 mg/kg body weight (b.w.) daily for 8 weeks. The levels of T 3 and T 4 hormones in the rat serum were detected by radioimmunoassay kit. The indexes of reproduction, percentage of organ in body weight (%) and frequency of abnormal sperm cells were also analyzed in this study. DNA damage in testicular cells was evaluated by Comet assay. The levels of MDA, GSH and SOD were examined in testicle tissues of rats by ELISA. The expression of c-fos and fas protein was examined in testicle tissues by immunohistochemistry. The results showed that perchlorate did not affect the body weight of rats. Perchlorate also significantly decreased indexes of live birth and weaning in the groups of 1.00 and 10.00 mg/kg, and viability index only in the 10.00 mg/kg group (P < 0.05). Perchlorate also significantly decreased the serum level of T 3 in male rats of 1.00 and 10.00 mg/kg groups, increased the rate of sperm abnormality (10.00 mg/kg), potentially caused DNA damage in testicular cells and altered the status of oxidative stress in male rats. In addition, because of the increase in the expression of fas and c-fos protein in testicle tissues, perchlorate could induce apoptosis in spermatogenesis. Thus, these findings indicate that perchlorate could cause DNA damage in testicular tissues and reduce testicular spermatogenic ability, resulting in reproductive toxicity. • Perchlorate disturbs the level of T3 hormone in male rats. • Perchlorate induces ROS to DNA damage in testicular tissues and reduces the testicular spermatogenic ability. • Perchlorate pollution results in the reproductive toxicity in rats. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fibrosis, tightly associated with wound healing, is a significant symptomatic clinical problem. Inflammatory response was reported to be one of the reasons. MiR-155 is relatively related with the development and requirement of inflammatory cells, so we thought reduce the expression of miR-155 in wound sites could improve the quality of healing through reduce inflammatory response. To test this hypothesis, locally antagonizing miR-155 by directly injecting antagomir to wound edge was used to reduce the expression of miR-155. We found wounds treated with miR-155 antagomir had an obvious defect in immune cells requirements, pro-inflammatory factors IL-1β and TNF-α reduced while anti-inflammatory factor IL-10 increased. With treatment of miR-155 antagomir, the expression of α-smooth muscle actin (α-SMA), Col1 and Col3 at wound sites all reduced both from mRNA levels and protein expressions. Wounds injected with antagomir resulted in the structure improvement of collagen, the collagen fibers were more regularly arranged. Meanwhile the rate of healing did not change significantly. These results provide direct evidences that miR-155 play an important role in the pathogenesis of fibrosis and show that miR-155 antagomir has the potential therapy in prevention and reduction of skin fibrosis. [ABSTRACT FROM AUTHOR]

Background: As an important oncogenic miRNA, microRNA-21 (miR-21) is associated with various malignant diseases. However, the precise biological function of miR-21 and its molecular mechanism in hypertrophic scar fibroblast cells has not been fully elucidated. Methodology/Principal Findings: Quantitative Real-Time PCR (qRT-PCR) analysis revealed significant upregulation of miR-21 in hypertrophic scar fibroblast cells compared with that in normal skin fibroblast cells. The effects of miR-21 were then assessed in MTT and apoptosis assays through in vitro transfection with a miR-21 mimic or inhibitor. Next, PTEN (phosphatase and tensin homologue deleted on chromosome ten) was identified as a target gene of miR-21 in hypertrophic scar fibroblast cells. Furthermore, Western-blot and qRT-PCR analyses revealed that miR-21 increased the expression of human telomerase reverse transcriptase (hTERT) via the PTEN/PI3K/AKT pathway. Introduction of PTEN cDNA led to a remarkable depletion of hTERT and PI3K/AKT at the protein level as well as inhibition of miR-21-induced proliferation. In addition, Western-blot and qRT-PCR analyses confirmed that hTERT was the downstream target of PTEN. Finally, miR-21 and PTEN RNA expression levels in hypertrophic scar tissue samples were examined. Immunohistochemistry assays revealed an inverse correlation between PTEN and hTERT levels in high miR-21 RNA expressing-hypertrophic scar tissues. Conclusions/Significance: These data indicate that miR-21 regulates hTERT expression via the PTEN/PI3K/AKT signaling pathway by directly targeting PTEN, therefore controlling hypertrophic scar fibroblast cell growth. MiR-21 may be a potential novel molecular target for the treatment of hypertrophic scarring. [ABSTRACT FROM AUTHOR]

Abstract: This study aimed to investigate the feasibility of using an immortal keratinocyte cell line, HaCaT cells, to effectively deliver epidermal growth factor (EGF) in a skin substitute to treat burn wounds. The skin equivalent was constructed with human EGF (hEGF) gene modified HaCaT cells obtained through stable gene transfection; these were applied to full thickness burn wounds in a rat model. The results showed that the hEGF gene modified HaCaT cells produced more than 390ng/l of bioactive hEGF in the culture supernatant. K19 and integrin-β1 as keratinocyte differentiation markers were elevated in the hEGF gene modified HaCaT cells which were shown to be non-tumorigenic. The skin equivalent constructed with hEGF gene modified HaCaT cells demonstrated improved epidermal morphogenesis with a thick and compact epidermis. Wound healing was accelerated noticeably when applied with this skin substitute seeded with hEGF gene modified HaCaT cells in vivo. The results suggest that HaCaT cells modified with hEGF gene might be promising seed cells for construction of genetically modified skin substitute which can effectively secrete hEGF to accelerate wound repair and regeneration. [Copyright &y& Elsevier]

Keloids and hypertrophic scars are significant symptomatic clinical problems characterized by the excessive and abnormal deposition of collagen-based extracellular matrix (ECM) components. However, the molecular basis of keloid and hypertrophic scar formation has not been fully elucidated. Here, we demonstrated that down-regulation of the transcription factor Smad interacting protein 1 (SIP1) could be relevant to keloid and hypertrophic scar formation. The results of the present study show that the level of SIP1 mRNA is significantly decreased in pathological scar tissues and in normal skin and pathological scar fibroblasts treated with transforming growth factor β1 (TGF-β1). In contrast, the expression of SIP1 mRNA is not decreased in normotrophic scar samples. The SIP1 mRNA level inversely correlates with the mRNA level of type I collagen (COL1A2) and directly correlates with the mRNA level of matrix metalloproteinase-1 (MMP1). Overexpression of SIP1 in keloid and hypertrophic scar fibroblasts represses TGF-β1-stimulated COL1A2 expression and induces MMP1 expression. Alternatively, knockdown of SIP1 in normal skin fibroblasts enhance TGF-β1-induced COL1A2 levels. These findings suggest that SIP1 could be a regulator of skin fibrosis, and depletion of SIP1 in pathological scar tissues could result in an up-regulation of collagen and down-regulation of matrix metalloproteinase, leading to an abnormal accumulation of ECM along with fibrosis and pathological scar formation. [Copyright &y& Elsevier]