| The typical wound healing process includes inflammation, angiogenesis and granulation tissue formation, re-epithelialization and repair and reconstruction of connective tissues, among which the proliferation of keratinocytes and re-epithelization are the central links of wound plugging. Around this key point of promoting the proliferation of keratinocytes, a variety of local treatment methods and drugs have been derived. Products and dressings of silver, copper, zinc and other metals have been playing an important role in the local treatment on burn wound for a long time. In order to explore their cell biological effects, with the effects of silver nitrate and copper nitrate on the proliferation of human keratinocytes as an example, the related research work was carried out.At a sub-cytotoxic concentration, Ag+ and Cu2+ show no toxicity on cell proliferation, but intracellular reactive oxygen species(ROS) increase significantly. The cell biological effects of increased intracellular ROS are unclear yet. As it is widely known, ROS not only participate with the cell apoptosis and death process but also play roles in cell signal transductions, transcription activation and stimulation of cell proliferations and differentiations. In addition, we discovered that Ag+ and Cu2+ at certain sub-cytotoxic concentration can stimulate keratinocytes proliferation and induce intracellular ROS production through previous tests.Based on the above, we hypothesized different Ag+ and Cu2+ concentrations have various influences on inducing intracellular ROS production of keratinocytes. These influences can further affect the proliferation activities of keratinocytes, and Ag+ and Cu2+ have bidirectional dose-response relations with ROS. This study started with investigation of impacts of different Ag+ and Cu2+ concentrations on Ha Ca T cell proliferations, and then reactive oxygen scavenger N-Acetyl Cysteine(NAC) was applied to search and confirm the mutual relationship of Ag+ and Cu2+ in intracellular ROS upregulation. Besides, different proteins among groups were found through i TRAQ techniques, and their function rich features were further analyzed by bioinformatics analysis to confirm the potential key targets of keratinocytes proliferation with effects of Ag+ and Cu2+.1. ObjectiveThrough the observation of different concentrations of Ag+ and Cu2+ on theproliferation of human keratinocytes, the ability to produce intracellular ROS was detected.The active oxygen scavenger NAC was used to verify the role of ROS on the proliferationof human keratinocytes cocultured with sub-cytotoxic concentrations of Ag+ and Cu2+.Functional annotation and enrichment analysis were carried out on different proteinsscreened with the high-throughput i TRAQ technology. And the key molecules related tothe cell proliferation and ROS generation were found for providing an experimental basisfor the further study on the mechanism of Ag+ and Cu2+ on the proliferation of humankeratinocytes.2. Method2.1 Effects of Ag+ on keratinocytes proliferation and intracellular ROS production.2.1.1 The effects of Ag+ at different concentrations and/or NAC on keratinocytesproliferation were determined by CCK-8; Brd U, PCNA and cell cycle were detected atspecific sub-cytotoxic concentrations of Ag+ to verify the change of cell proliferationactivities;2.1.2 The effects of Ag+ at different concentrations and/or NAC on intracellular ROSproductions in keratinocytes were determined by DCFH-DA fluorescence analysis.2.1.3 i TRAQ protein quantification techniques were used to select different proteinsof keratinocytes with effect of Ag+ at sub-cytotoxic concentrations; selected differentproteins were processed with functional annotation and enrichment analysis.2.2 Effects of Cu2+ on keratinocytes proliferation and full-thickness skin defectswounds healing in mice.2.2.1 The effects of Cu2+ at different concentrations and/or NAC on keratinocytesproliferation were determined by CCK-8;2.2.2 The effects of Cu2+ at different concentrations and/or NAC on intracellularROS productions in keratinocytes were determined by DCFH-DA fluorescence analysis.2.2.3 i TRAQ techniques were used to select different proteins of keratinocytes with effect of Cu2+ at sub-cytotoxic concentrations; selected proteins were processed with functional annotation and enrichment analysis.2.2.4 The influence of Cu2+ at different concentrations on wound healing is evaluated through full-thickness skin wounds defects model in mice.3. Results3.1 Effects of Ag+ on keratinocytes proliferation and intracellular ROS production.3.1.1 At concentrations of 10-6 M and 10-5 M, Ag+ can significantly promote Ha Ca T cell proliferation at 24 h, 48 h and 72h; at each of those time point, Ag+ concentrations of 10-8 Mand 10-7 M have no obvious effect on Ha Ca T cell proliferation, while Ag+ concentrations of 10-4 M can cause a rapid cell death. At concentrations of 10-6 M and 10-5 M, Ag+ can cause increasing of Brd U positive cell number at 48 h, as well as strengthening of PCNA immuno-fluorescence; Ag+ concentrations of 10-5 M can also significantly increase cell proliferation index(PI). This proliferative effect at 10-6 M and 10-5 M Ag+ was neutralized when 5 m M of NAC co-culturing was added.3.1.2 The Ha Ca T cells show a significant increase of intracellular ROS production from 5 min to 60 min after processed with 10-6 M and 10-5 M Ag+; this positive influence on intracellular ROS production was neutralized when 5 m M NAC co-culturing was added.3.1.3 A subdivision screening in a concentration range of 10-6-10-4 M of Ag+ was applied, and the result shows that 7.5×10-6 M Ag+ has the highest proliferative effect. Proteomic analysis was carried out on different groups of Ha Ca T cell samples processed with 0 M, 1×10-7 M and 7.5×10-6 M of Ag+ for 48 hours. According to the result by bioinformatic analysis, many carbohydrate metabolic pathways(for instance glycolysis/ gluconeogenesis, citric acid cycle, pentose phosphate pathway, biosynthesis of secondary metabolites, glyoxylic acid and dicarboxylic acid metabolism) were significantly enriched at 7.5×10-6 M group. Among those five pathways, the upregulation rate of different protein expression is 73.24%(52/71); ratio of different protein enrichment is 15.15% in ribosome pathway, and all of different proteins were upregulated.3.2 Effects of Cu2+ on keratinocytes proliferation and full-thickness skin defects wounds healing in mice.3.2.1 Ha Ca T cells processed with 10-6 M and 10-5 M Cu2+ for 48 h show relative cell proliferation rate remarkably increase to 116.19%(P=0.027),113.37%(P=0.036) when compared with negative control, 5 m M of NAC has an antagonistic effect against the proliferative effect.3.2.2 Ha Ca T cells processed with 10-6 M and 10-5 M Cu2+ for 30 min illustrates 127.47%(P=0.001) and 124.28%(P=0.031) of intracellular ROS production when compared with negative control, and this intracellular ROS production further increased to 140.47%(P=0.002) and 134.29%(P=0.007) at 60 min. Cells processed with 10-8 M and 10-7 M Cu2+ for 60 min cause 110.99%(P=0.016) and 117.34%(P=0.016) of intracellular ROS productions. 5 m M of NAC has an antagonistic effect against the positive effect.3.2.3 The proteomic analysis was carried out on different groups of Ha Ca T cell samples processed with 0 M, 1×10-7 M and 1×10-6 M of Cu2+ for 48 hours. According to the results by bioinformatic analysis, a concentration of 1×10-6 M Cu2+ processed Ha Ca T cell had the highest ratio of protein difference enrichment(19.42%, 74/381). Many energy metabolic pathways were significantly enriched(for example glycolysis / gluconeogenesis, citric acid cycle, pentose phosphate pathway, pyruvate metabolism and fatty acid metabolism). The upregulating rate of different protein expression among those five pathways was 50%(25/50), and the upregulating rate of different protein expression among glutathione metabolism and cysteine, methionine metabolic pathways was 88.89%(8/9); the ratio of protein difference enrichment of 1×10-6 M Cu2+ group was 12.07% in ribosome pathway, and all of them were upregulated.3.2.4 Compared with control group, hydrogel dressings of 10-6 M Cu2+ could significantly promote the mice full-thickness skin defects wounds healing rates at 6, 8 and 10 day. Group of 10-8 M Cu2+ also showed remarkable increase of wound healing rate at 8 and 10 day. Group of 10-4 M Cu2+ showed no significant increase of wound healing rate at every time point, but there were significant differences between 10-6 M Cu2+ group and 10-4 M Cu2+ group at 8 and 10 day.4.Conclusions4.1 The concentrations of Ag+ and Cu2+ ≥10-4 M are cytotoxic to Ha Ca T cells.4.2 10-6-10-5 M Ag+ and Cu2+ can stimulate Ha Ca T cell proliferation. Meanwhile, 7.5×10-6 M Ag+ and 1×10-6 M Cu2+ has the highest proliferative effect among 10-6-10-5 M, respectively.4.3 Intracellular ROS production tends to increase with the concentrations of Ag+ and Cu2+ in in a range of sub-cytotoxic concentration. The elimination of ROS using NAC suppresses proliferation that was induced by sub-cytotoxic concentrations of Ag+.4.4 According to proteomic analysis results, 7.5×10-6 M Ag+ in sub-cytotoxic concentration may induce increase of carbohydrate metabolisms and protein synthesis in Ha Ca T cells, and this change can further cause a rise of intracellular ROS production through biological oxidation process. The part of increased ROS may promote the proliferation of human keratinocytes by activating ERK1/2 and JNK pathway. In 1×10-6 M Cu2+ group, changes of many energy metabolic pathways(glycolysis / gluconeogenesis, citric acid cycle, pentose phosphate pathway, pyruvate metabolism, fatty acid metabolism and so on) cannot clearly reflect the overall metabolic changing trend. The upregulation of metabolisms of sulphydryl compounds such as glutathione, cysteine and methionine may indirectly represent an increase of ROS production. The overall upregulation of ribosome pathway may go with the increase of protein synthesis. However, based on proteomic analysis results, there were still no enough direct proof for primary classic pathways like cell proliferations and ROS productions, which is required to be verified based on upstream and downstream of existing pathway change in the next phase.4.5 Full-thickness skin defects wounds in mice suggests that Cu2+ dressing in sub-cytotoxic concentration can promote wound healing in a specific period of time. |
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