Zhichuan Liu1, Ling Hu2, Tao Zhang2, Hang Xu2, Hailin Li2, Zhouqian Yang2, Mei Zhou2, Hendrea Shaniqua Smith2, Jing Li3, Jianhua Ran2, Zhongliang Deng4. 1. Department of Emergency, The Second Affiliated Hospital, Chongqing Medical University Chongqing 400010, China. 2. Department of Anatomy and Laboratory of Neuroscience and Tissue Engineering, Basic Medical College, Chongqing Medical University Chongqing 400016, China. 3. Laboratory of Stem Cell and Tissue Engineering, Department of Histology and Embryology, Chongqing Medical University Chongqing 400016, China. 4. Department of Orthopedics, The Second Affiliated Hospital, Chongqing Medical University Chongqing 400010, China.
Abstract
OBJECTIVE: To explore the role and mechanism of oxidative stress injury in the diabetic foot. METHODS: Immunohistochemistry and staining were used to detect changes in diabetic foot tissue, and the CCK-8 method was used to measure high glucose effect on cell viability. The DCFH-DA assay was used to detect the intracellular ROS content, and colorimetric methods were used to detect the activities of the CAT and SOD enzymes and the NO and MDA content in tissues and cells. In addition, the protein expression levels of PKCβ, p66shc, eNOS, ICAM-1 and NF-κB in tissues and cells were detected by Western blotting, and the distribution of p66shc and eNOS was observed by immunofluorescence. RESULTS: The results of clinical specimens experiments showed that the DFU group exhibited disordered morphology and increased glucose metabolism, decreased activities of the enzymes CAT and SOD in tissues, and increased MDA and NO contents compared to those in the CON group. Furthermore, protein levels of the p-PKCβ, p-p66shc, ICAM-1, and p-NF-κB were increased, and eNOS protein level was decreased; these results were consistent in clinical specimens and in vitro experiments. CONCLUSIONS: High glucose levels may induce oxidative stress injury in cells and tissues by activating the PKCβ-p66shc signaling pathway. AJTR
OBJECTIVE: To explore the role and mechanism of oxidative stress injury in the diabetic foot. METHODS: Immunohistochemistry and staining were used to detect changes in diabetic foot tissue, and the CCK-8 method was used to measure high glucose effect on cell viability. The DCFH-DA assay was used to detect the intracellular ROS content, and colorimetric methods were used to detect the activities of the CAT and SOD enzymes and the NO and MDA content in tissues and cells. In addition, the protein expression levels of PKCβ, p66shc, eNOS, ICAM-1 and NF-κB in tissues and cells were detected by Western blotting, and the distribution of p66shc and eNOS was observed by immunofluorescence. RESULTS: The results of clinical specimens experiments showed that the DFU group exhibited disordered morphology and increased glucose metabolism, decreased activities of the enzymes CAT and SOD in tissues, and increased MDA and NO contents compared to those in the CON group. Furthermore, protein levels of the p-PKCβ, p-p66shc, ICAM-1, and p-NF-κB were increased, and eNOS protein level was decreased; these results were consistent in clinical specimens and in vitro experiments. CONCLUSIONS: High glucose levels may induce oxidative stress injury in cells and tissues by activating the PKCβ-p66shc signaling pathway. AJTR
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