| Diabetes is a high incidence disease around world,about 20% of patients appear wound healing delay with leg or foot ulcers becoming the most common wound.Repair difficulties caused by the diabetic foot is one of the most serious complications leading to disability.However,the pathogenesis is not exactly clear.The mainstream view thinking that blood lipids and blood glucose are closely related to metabolic disorders,most treatments focus on controlling blood sugar debridement and external using drugs in the present,but the effect are very limited.Our previous study found that there was a link between the adenosine and diabetic wound in the body,adenosine level is mainly adjusted by adenosine deaminase(ADA).It is not clear whether ADA has an effect on wound healing disorders in diabetes mellitus.In this study,we set up DM2 model with mice to study the effect and mechanism of ADA on wound healing disorder.At first,we transferred the recombinant plasmid with mouse ADA c DNA into Ecoli BL21(DE3)to express.Then the recombinant ADA was purified by GST column and specific enzyme digestion.The enzyme activity was determined to be 106 U/mg.ADA was modified by m PEG-SPA to obtain PEG-ADA so that the metabolic time of PEG-ADA was increased to 4-6days in mice.We use db/db mice as well as mice fed with high fat food before induced STZ as type 2 diabetes model.The experimental group was injected PEG-ADA with dose of 1.5 U/g intraperitoneally,the control group was injected with PBS every 4 days,and the wound area was photographed and measured on day0,day2,day5,day7,day14.Experiment demonstrated that the rate of repair in processed mice was about 15% higher than the contrast mice within 14 days.The level of adenosine and adenosine precursor adenosine(AMP)in the wound tissue of the treatment group was lower than that of the control group by HPLC,and control group epidermis of the wound tissue grew slower than the treatment,and increased depositional collagen was observed,and the expression level of cytokines TNF-α,IL-1,IL-6,IL-12,MCP-1 in the treatment group was lower detected by fluorescence quantitative PCR.We observed AMP inhibited the fibroblasts migration by means of external cell experiments.Transcriptome analysis showed more gene differences in the AMP-treat group cells compared with the control group(normal saline treatment group),and the Focal adhesion pathway which controlled cell adhesion was included in the first 20 enrichment pathways by KEGG pathway analysis.In addition,the expression of PAK1～PAK4 which control cell adhesion and migration are also down-regulated as well as the AMP treatment group with the addition of PAK1 activator FTY720 effectively relieved the inhibition of AMP on the migration of fibroblasts.These studies have shown that high adenosine level in type 2 diabetes wounds will inhibit Focal adhesion pathway by inhibiting PAK gene expression,thus inhibiting cell adhesion and fibre cell migration will delay wound healing.However,adenosine deaminase can reduce diabetic wound adenosine level,promote the PAK gene expression level,accelerate the migration of fibre cell,promote skin collagen deposition,reduce inflammation factor expression,and accelerate wound healing in the end,suggesting that adenosine deaminase has the potential to be developed as a new drug to treat diabetic wound disorder. |
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