Exosomal lncRNA KLF3-AS1 derived from bone marrow mesenchymal stem cells stimulates angiogenesis to promote diabetic cutaneous wound healing.

AIMS: We focused on BMSC-derived exosomal lncRNA KLF3-AS1 and its significance in diabetic cutaneous wound healing. METHODS: Potential interaction between KLF3-AS1 and miR-383, miR-383 and VEGFA were predicted using bioinformatic analysis and validated by luciferase reporter, RIP, and FISH assays. The proliferation, apoptosis, migration and tube formation of HUVECs were evaluated by CCK-8, flow cytometry, wound healing, and tube formation assays, respectively. A murine diabetic cutaneous wound model was used to investigate therapeutic effects of exosomal KLF3-AS1 in vivo. Histological alterations in skin tissues were examined using HE, Masson staining, and immunostaining of CD31. RESULTS: BMSC-derived exosomal KLF3-AS1 sufficiently promoted proliferation, migration, and tube formation, while inhibited apoptosis of HUVECs challenged by high glucose. The protective effects of exosomal KLF3-AS1 were achieved at least partially by down-regulating miR-383, and boosting the expression of its target, VEGFA. In vivo, exosomes from KLF3-AS1-expressing BMSCs demonstrated the best effects in promoting cutaneous wound healing in diabetic mice, which were associated with minimal weight loss, increased blood vessel formation, reduced inflammation, decreased miR-383 expression, and up-regulated VEGFA. CONCLUSIONS: Exosomal lncRNA KLF3-AS1 derived from BMSCs induces angiogenesis to promote diabetic cutaneous wound healing.

Quercetin protects oral mucosal keratinocytes against lipopolysaccharide-induced inflammatory toxicity by suppressing the AKT/AMPK/mTOR pathway.

BACKGROUND: Cytokines can induce a chronic inflammatory response in the periodontium, leading to periodontitis. Quercetin, a naturally occuring flavonoid, has been shown to inhibit periodontitis, but how it works is poorly understood. In this study, we assessed the impact of quercetin on lipopolysaccharide (LPS)-induced inflammatory damage in oral mucosal keratinocytes (hOMK107) and explored its underlying mechanism. METHODS: The viability and apoptosis of hOMK107 cells were measured after exposure to LPS, followed or not by quercetin. The production of IL-1ß, IL-6, IL-8, TNF-ɑ, iNOS, and COX-2 was quantified by enzyme-linked immunosorbent assay (ELISA), while levels of Akt, AMPK, and mTOR and their phosphorylation were detected semi-quantitatively by western blotting. RESULTS: Quercetin significantly improved cell viability and apoptosis by reversing LPS-induced upregulation of Bax and downregulation of Bcl-2 in hOMK107 cells. Quercetin decreased the production of IL-1ß, IL-6, IL-8, TNF-ɑ, iNOS, and COX-2, as well as signal transduction via the Akt/AMPK/mTOR pathway. Inhibitors of Akt, AMPK, and mTOR strengthened the anti-apoptotic effects of quercetin, while agonists of Akt, AMPK, or mTOR or Akt overexpression weakened the anti-apoptotic effects. CONCLUSION: These results indicate that quercetin may have a potential protective effect against the chronic inflammation-related periodontitis via suppressing Akt/AMPK/mTOR pathway.

[Construction of lentiviral vector for mouse CXC chemokine receptor 4 gene and its expression in eukaryotic cells].

This study was aimed to clone the gene coding mouse CXC chemokine receptor 4 (CXCR4), to construct the recombinant lentiviral vector carrying enhanced green fluorescence protein (EGFP) and to explore its expression in eukaryotic cells (293FT cells). The full length CXCR4 gene was cloned by RT-PCR using bone marrow cells from C57BL/6 mouse as template and inserted into PCR-Blunt vector. CXCR4 fragment was generated by digestion with restriction endonuclease and subcloned into a lentiviral vector to generate recombinant lentiviral vector LV-IRES-EGFP-CXCR4, which was co-transfected into 293FT cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. Viruses were gathered and concentrated using ultracentrifuge, and then transfected into 293FT cells. Expression of EGFP was detected by fluorescent microscopy and flow cytometry (FCM). And the expression of CXCR4 protein was detected by Western blot. The results demonstrated that mouse CXCR4 gene was cloned and the lentiviral vector was successfully constructed. The lentiviral particles were correctly packaged, and the virus titers were above 10(8) TU/ml in the supernatant after concentration. Expression of EGFP was detected by fluorescent microscopy in the transfected 293FT cells, and the transfection efficacy > 95% was determined by FCM. Expression of CXCR4 protein detected by FCM and Western blot was significantly higher than that in control group. It is concluded that the CXCR4 gene along with the gene coding EGFP is successfully inserted into a lentiviral vector to construct a recombinant lentiviral vector, which can be expressed in eukaryotic cells.