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also demonstrated that wound healing in diabetic patients is associated with an increase in the synthesis of HIF-1α protein. In this study, we investigated the role of HIF-1α in AgNP-mediated cell proliferation and IL-8 upregulation in hMSCs.
The present study measured cell proliferation and the expression of HIF-1α and IL-8 in hMSCs treated with AgNPs. In addition, this study investigated the effect of small interfering RNA specific for HIF-1α (HIF-1α siRNA) on cell proliferation and IL-8 expression in hMSCs. Polyvinylpyrrolidone-coated spherical AgNPs (nanoComposix, San Diego, CA) were used in this study. AgNPs were characterized by transmission electron microscopy before performing the experiment (Figure 1a). The energy diverse X-ray spectra demonstrated the strong presence of Ag (Figure 1b). In accordance with a previous study (
), AgNPs increased cell proliferation in a dose-dependent manner at subtoxic concentrations and decreased cell proliferation at higher concentrations above 10 μg ml-1 (Figure 1c and e). As shown in Figure 1d and f, a marked increase in cell proliferation was observed after treatment with AgNPs. To determine the effect of AgNPs on IL-8 mRNA expression, we treated hMSCs with AgNPs for 24 hours and performed real-time reverse transcription-PCR to evaluate mRNA expression of IL-8. IL-8 mRNA expression was significantly increased in the hMSCs treated with AgNPs (1, 2.5, 5, and 7.5 μg) compared with the untreated hMSCs (P<0.05 for all comparisons; Figure 1g). ELISA analysis also showed that AgNP-induced IL-8 secretion from hMSCs in a dose-dependent manner (Figure 1h).
After exposure to AgNPs, the expression of HIF-1α protein in hMSCs increased in a dose-dependent manner (Figure 2a and b). To investigate the role of HIF-1α in AgNP-mediated upregulation of IL-8 and cell proliferation in hMSCs, we treated the cells additionally with HIF-1α siRNA. As shown in Figure 2c, expression of HIF-1α protein in AgNP-treated hMSCs was significantly reduced after HIF-1α siRNA treatment, confirming that HIF-1α siRNA effectively blocked the expression of HIF-1α in these cells. The results showed that AgNP-mediated hMSC proliferation was decreased in hMSCs treated with HIF-1α siRNA (Figure 2d). We measured AgNP-induced IL-8 expression in hMSCs that were transfected with HIF-1α siRNA by ELISA and demonstrated that IL-8 production was also reduced following treatment with HIF-1α siRNA (Figure 2e). Six-week-old female C57BL/6 mice (Orientbio, Sungnam, Korea) were maintained and handled under protocols approved by the Korea University Institutional Animal Care and Use Committee (KUIACUC-2013-243). These mice averaged 18 g. The mice were divided into four different groups, with six animals in each group. Wounds were made on the dorsal skin of each mouse using a 5 mm punch. According to their respective groups, the wound site was injected with phosphate-buffered saline, hMSCs, AgNP-treated hMSCs with HIF-1α siRNA and AgNP-treated hMSCs with control siRNA. Our preliminary studies have shown that immunosuppressive therapies were not needed in hMSCs treatment. The wound areas were evaluated using image processing techniques of Matlab (The MathWorks, Natick, MA). This study showed that AgNP-treated hMSCs could accelerate cutaneous wound healing in vivo, compared with untreated hMSCs (Figure 2f and g). In addition, our findings presented delayed wound closure in the AgNP-treated hMSCs with HIF-1α siRNA group compared with the AgNP-treated hMSCs with control siRNA group (Figure 2f and g). The present results suggest that accelerated wound closure by injecting with AgNP-treated hMSCs may be HIF-1α dependent. Histopathologic examination demonstrated that hair growth and scar formation might be major factors for the effects of AgNP-treated hMSCs in wound healing (Supplementary Figure S1 online).
demonstrated that HIF-1α protein stabilization increases hematopoietic stem cell quiescence and accelerates blood recovery after severe irradiation in vivo. In addition, a previous study showed that various aspects of stem cell biology, such as differentiation and mobilization, involve the HIF-1 signaling pathway (
demonstrated that AgNPs induced HIF-1α activation in Caenorhabditis elegans. However, to our knowledge, this is the first study to elicit that AgNPs might induce the expression of HIF-1α protein in hMSCs.
The present study suggests that HIF-1α may be a key factor in AgNP-induced cell proliferation and IL-8 expression in hMSCs. In view of these findings, HIF-1α and the related downstream molecules might be an important target of research and development to improve wound healing by using AgNP-treated hMSCs.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A1A2044085), and a grant from the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A101334).