HSFAS mediates fibroblast proliferation, migration, trans-differentiation and apoptosis in hypertrophic scars via interacting with ADAMTS8.

Hypertrophic scar (HS) is one of the most common sequelae of patients, especially after burns and trauma. The roles of regulatory long noncoding RNAs (lncRNAs) in mediating HS remain underexplored. Human hypertrophic scar-derived fibroblasts (HSFBs) have been shown to exert more potent promoting effects on extracellular matrix (ECM) accumulation than normal skin-derived fibroblasts (NSFBs) and are associated with enhanced HS formation. The purpose of this study is to search for lncRNAs enriched in HSFBs and investigate their roles and mechanisms. LncRNA MSTRG.59347.16 is one of the most highly expressed lncRNAs in HS detected by lncRNA-seq and qRT-PCR and named as hypertrophic scar fibroblast-associated lncRNA (HSFAS). HSFAS overexpression significantly induces fibroblast proliferation, migration, and myofibroblast trans-differentiation and inhibits apoptosis in HSFBs, while knockdown of HSFAS results in augmented apoptosis and attenuated proliferation, migration, and myofibroblast trans-differentiation of HSFBs. Mechanistically, HSFAS suppresses the expression of A disintegrin and metalloproteinase with thrombospondin motifs 8 (ADAMTS8). ADAMTS8 knockdown rescues downregulated HSFAS-mediated fibroblast proliferation, migration, myofibroblast trans-differentiation and apoptosis. Thus, our findings uncover a previously unknown lncRNA-dependent regulatory pathway for fibroblast function. Targeted intervention in the HSFAS-ADAMTS8 pathway is a potential therapy for HS.

LncRNA FPASL suppresses fibroblast proliferation through its DNA methylation via DNMT3b in hypertrophic scar.

Long noncoding RNAs (lncRNAs) are increasingly being implicated as key regulators of cell proliferation, apoptosis, and differentiation. However, the molecular mechanisms of specific lncRNAs in the context of hypertrophic scar remain largely unclear. Here, we find that the lncRNA FPASL (fibroblast proliferation-associated LncRNA) is downregulated in HS, and FPASL reduces fibroblast proliferation and colony formation and blocks cell cycle progression. Using GO annotation enrichment analysis along with AZC (a specific inhibitor of DNA methylation), we identify that DNA methylation is responsible for downregulating FPASL in hypertrophic scar. Subsequent studies demonstrate that high expression of DNMT3b inhibits FPASL expression in HS. Mechanistic study reveals a significant increase in fibroblast proliferation after transfection with LNA-FPASL, which is further inhibited by knockdown of DNMT3b. Thus, our study reveals that DNMT3b mediates hypermethylation of the lncRNA FPASL promoter and the downregulation of lncRNA FPASL promotes fibroblast proliferation in hypertrophic scar.

[Homocysteine promotes human hepatocyte autophagy through activating the activating molecule in beclin-1-regulated autophage (AMBRA1)].

Objective To investigate the role of activating molecule in beclin-1-regulated autophage (AMBRA1) in homocysteine (Hcy)-induced hepatocytes autophagy. Methods Hepatocytes were cultured in vitro and divided into control group (0 µmol/L Hcy) and Hcy treatment group (100 µmol/L Hcy). Western blotting was used to detect the expression of microtubule-associated protein 1 light chain 3B (LC3BII, LC3BI); hepatocytes were treated with 0, 25, 50, 100 µmol/L chloroquine (CQ), CCK-8 assay was used to detect the inhibitory effect of CQ on hepatocyte proliferation and Western blotting was performed to detect the expression of LC3B and AMBRA1; After hepatocytes were transfected with AMBRA1 small interfering RNA, real-time fluorescent quantitative PCR and Western blotting were used to detect the interference efficiency of AMBRA1 expression; After the transfected hepatocytes were treated with Hcy, the expression of LC3B was detected by Western blot analysis. mRFP-GFP-LC3 adenovirus was transfected with hepatocytes and the autophagy flow was observed by laser scanning confocal microscopy. Results Compared with the control group, the ratio of LC3BII/LC3BIincreased in the Hcy treatment group; the inhibition rate of 50 µmol/L CQ on hepatocyte proliferation was close to 50%; compared with the control group, the ratio of LC3BII/LC3BI and the expression of AMBRA1 increased significantly in the Hcy group , and the ratio of LC3BII/LC3BI and the expression of AMBRA1 in the Hcy combined with CQ group were significantly lower than those in the Hcy group; the ratio of LC3BII/LC3BI decreased after knocking down AMBRA1; compared with the control group, the autophagosomes and autophagolysosomes increased in Hcy group and decreased after knocking down AMBRA1. Conclusion Hcy can promote hepatocyte autophagy by activating AMBRA1.

A novel lncRNA FPASL regulates fibroblast proliferation via the PI3K/AKT and MAPK signaling pathways in hypertrophic scar.

Hypertrophic scar is a problem for numerous patients, especially after burns, and is characterized by increased fibroblast proliferation and collagen deposition. Increasing evidence demonstrates that lncRNAs contribute to the development and progression of various diseases. However, the function of lncRNAs in hypertrophic scar formation remains poorly characterized. In this study, a novel fibroblast proliferation-associated lncRNA, named lncRNA FPASL (MSTRG.389905.1), which is mainly localized in the cytoplasm, is found to be downregulated in hypertrophic scar, as detected by lncRNA microarray and qRT-PCR. The full-length FPASL is characterized and further investigation confirms that it has no protein-coding potential. FPASL knockdown in fibroblasts triggers fibroblast proliferation, whereas overexpression of FPASL directly attenuates the proliferation of fibroblasts. Furthermore, target genes of the differentially expressed lncRNAs in hypertrophic scars and the matched adjacent normal tissues are enriched in fibroblast proliferation signaling pathways, including the PI3K/AKT and MAPK signaling pathways, as determined by GO annotation and KEGG enrichment analysis. We also demonstrate that knockdown of FPASL activates the PI3K/AKT and MAPK signaling pathways, and specific inhibitors of the PI3K/AKT and MAPK signaling pathways can reverse the proliferation of fibroblasts promoted by FPASL knockdown. Our findings contribute to a better understanding of the role of lncRNAs in hypertrophic scar and suggest that FPASL may act as a potential novel therapeutic target for hypertrophic scar.

Homocysteine facilitates endoplasmic reticulum stress and apoptosis of hepatocytes by suppressing ERO1α expression via cooperation between DNMT1 and G9a.

Endoplasmic reticulum (ER) stress and apoptosis play a critical role in liver injury. Endoplasmic reticulum oxidoreductase 1α (ERO1α) is an oxidase that exists in the luminal side of the ER membrane, participating in protein folding and secretion and inhibiting apoptosis, but the underlying mechanism on liver injury induced by homocysteine (Hcy) remains obscure. In this study, hyperhomocysteinemia (HHcy) mice model was established in cbs+/- mice by feeding a high-methionine diet for 12 weeks; and cbs+/- mice fed with high-methionine diet exhibited more severe liver injury compared to cbs+/+ mice. Mechanistically, we found that Hcy promoted ER stress and apoptosis of hepatocytes and thereby aggravated liver injury through inhibiting ERO1α expression; accordingly, overexpression of ERO1α remarkably alleviated ER stress and apoptosis of hepatocytes induced by Hcy. Epigenetic modification analysis revealed that Hcy significantly increased levels of DNA methylation and H3 lysine 9 dimethylation (H3K9me2) on ERO1α promoter, which attributed to upregulated DNA methyltransferase 1 (DNMT1) and G9a, respectively. Further study showed that DNMT1 and G9a cooperatively regulated ERO1α expression in hepatocytes exposed to Hcy. Taken together, our work demonstrates that Hcy activates ER stress and apoptosis of hepatocytes by downregulating ERO1α expression via cooperation between DNMT1 and G9a, which provides new insight into the mechanism of Hcy-induced ER stress and apoptosis of hepatocytes in liver injury.

Walnut oil promotes healing of wounds and skin defects in rats via regulating the NF-kB pathway.

The effects of walnut oil on wound healing and skin injury repair was observed in Sprague-Dawley (SD) rats, and mechanism of action was investigated. Normal SD rats were divided into an experimental group and a control group. Each group was observed at4 time points (day [D]3, D7, D14, and D21). In both groups, a skin wound was created on the back of the rats, with the spine as the central axis. In the experimental group, the wound was covered with walnut oil, and then bandaged and fixed with sterile gauze. In the control group, the wound was bandaged with vaseline gauze. At each corresponding time point, the wound area and wound healing time of each rat were examined. Epithelial cells of the wound tissues were observed using haematoxylin and eosin staining and immunohistochemical analysis,and the numbers of inflammatory cells and capillaries were counted. A western blot method was used to detect the expression of nuclear factor (NF)-κB and epidermal growth factor (EGF) in the wound tissues of both groups. Meanwhile, enzyme-linked immunosorbent analysis (ELISA) was used to detect the expression of transforming growth factor (TGF)-ß1 and matrix metalloproteinase (MMP)-1 in rat sera. A total of 48 SD rats completed the experiment. Healing time of residual wounds in the experimental group was 10.0±3.5 days, which was significantly shorter than that in the control group (18.0±6.0 days) (p<0.05). The wound healing rates in the experimental group were 54.14 % (D3) and 91.2 3% (D7), whereas those in the control group were 22.12% (D3) and 54.84% (D7 (p<0.05).Histological examinations revealed no epithelial cells on D3, D7, D14, and D21 in both the experimental and control groups. However, the number of inflammatory cells decreased significantly and the number of capillaries increased significantly in the experimental group compared to control (p<0.05). NF-κB expression was significantly lower, EGF expression significantly higher in the in the experimental group. Conversely, ELISA showed a significant increase in the expression of TGF-ß1 and MMP-1 in rat sera in the experimental group. So we conclude that walnut oil has significant effects in promoting the healing of skin defect wounds in SD rats.

[Experimental study of the effect of liposome-mediated vascular endothelial growth factor gene on the flap survival in rats].

OBJECTIVE: To investigate the effect of VEGF gene on the random flap survival after pedicle division at different time in rats. METHODS: The random-pattern flaps were formed on the back of the 120 SD rats. PcDNAVEGF165 (gene) wrapped with liposome was injected into the flaps in experimental group (n = 40). The flaps in the two control groups were injected with PcDNA (n = 40) or saline (n = 40). 1, 3, 5, 7 days after injection, 10 rats in each group were randomly selected to performed pedicle division. 7 days after pedicle division, the rats were killed to measure the flap survival rate. The microvessels was studied by histologic examination. The expression of VEGF was assessed by immunohistochemical staining. The flaps were also examined under the electron ultrastructure microscopy. RESULTS: 1) Flap survival rate after pedicle division in experimental group at 1 day, 3 days, 5 days, 7 days after injection, were (45.45 +/- 12.24)%, (82.95 +/- 3.81)%, (85.00 +/- 3.38)%, (85.96 +/- 3.25)%, respectively. The flap survival rates were significant different between experimental group and the control groups at 3, 5, 7 days after injection (P < 0.05), but not at 1 days after injection (P > 0.05). 2) The average microvascular diameter and number in experimental group were significantly higher than those in control groups (P < 0.05). 3) The expression of VEGF in experimental group was significantly higher than that in the two control groups (P < 0.05). 4) Ultrastructure study showed more angiogenesis in experimental group. CONCLUSIONS: Subcutaneous injection of liposome-mediated VEGF gene can increase the survival rate of flap with early pedicle division. It is a simple, efficient, economic, and the relatively safe gene therapy.