A Study About The Correlation Between Irisin And Healing Of Diabetic Foot Ulcer

Objective Clinical study was conducted to explore the difference of serum irisin concentration among normal subjects,patients with type 2diabetes mellitus(T2DM)and patients with diabetic foot(DFD).Experiments were conducted to explore whether irisin could promote diabetic wound healing and whether it is related to fibroblasts and the homing of fibrocytes.Methods Clinical study included 53 patients with DFD,53 patients with T2 DM and 53 healthy subjects.Age,gender and laboratory indicators were collected.The concentration of irisin was examined by enzyme-linked immunosorbent assay.Baseline data and irisin concentration in the three groups were compared,subgroup analysis was performed according to gender.Simple correlation analysis was used to explore the relationship between irisin concentration and various variables,and multinomial Logistic regression was conducted.Patients with DFD were divided into two groups according to the median of irisin concentration,and the differences of hospital stays and infection indexes were compared between groups.As for animal experiments,diabetes wound model was established in SD rats,and irisin was injected subcutaneously around the wound edge.The wound healing rate on the third and seventh days was recorded.Fibrocytes which expresses CD45+ and type 1 collagen(COI-1)in the wound tissue were detected by immunofluorescence,and it was quantitatively described by Pearson correlation coefficient(PCC)which calculated by Image J.Scratch experiment of human fibroblasts was conducted to observe the wound healing at 0h,4h,8h and 24h in the intervention group and control group.Image J was used to calculate the wound healing area.Proteomics was conducted between fibroblasts treated with irisin and without irisin.The differential proteins between two groups were screened,and differential proteins were analyzed by cluster analysis,GO analysis and KEGG pathway analysis.Results Clinical study showed that the concentration of irisin in healthy subjects,patients with T2 DM and patients with DFD were gradually decreased,and irisin concentration among these groups was not equal(64.6ng/ml vs 50.0ng/ml vs 45.3ng/ml,p=0.000).Pairwise comparison showed irisin concentration of normal people and that of patients with T2DM(64.6ng/ml vs 50.0ng/ml,p=0.000)was significantly different.The concentration of irisin in normal people and that in patients with DFD(64.6ng/ml vs 45.3ng/ml,p=0.000)was significantly different too.Multinomial Logistic regression showed that compared with healthy subjects,the concentration of irisin was associated with diabetes(OR=0.929,95% CI=0.887-0.973,p=0.002)after adjustment for gender,age,hemoglobin,absolute value of neutrophils,low density lipoprotein cholesterol and urea nitrogen.Compared with healthy subjects,the concentration of irisin was associated with diabetic foot after adjustment of above factors(OR=0.899,95% CI=0.848-0.953,p=0.000).Among patients with DFD,the hospital stay in the lower irisin concentration group was significantly longer than that in the higher irisin concentration group(30.0days vs 19.5 days,p=0.036).Animal experiment showed that the quartile of wound healing rate in the intervention group and the control group on day 3 was 27.54%(12.82%,41.29%)and 1.45%(-5.01%,20.57%),respectively.The quartile of wound healing rate on day 7 was 52.12%(42.17%,60.52%)and 48.78%(41.72%,63.94%),respectively.The wound healing rate of the intervention group was significantly faster than that of the control group on the third day(p=0.028).Immunofluorescence showed that the co-localization area of CD45+ and COI-1 was increased in diabetic wound tissue in intervention group.On day 3,the quantile of PCC in the intervention group and the control group were-0.23(-0.29,0.13)and-0.07(-0.19,0.18),respectively,the difference was not statistically significant(p=0.240).On day 7,the quantile of PCC in the intervention group and the control group were 0.21(-0.12,0.33)and-0.09(-0.25,0.13),respectively,and the difference was statistically significant(p=0.041).The scratch assay showed that the healing rates of control group and intervention group were2.8%(-14.4%,5.8%)and 0%(-4.3%,3.8%)at 4h,11.6%(3.1%,17.7%)and 4.7%(-4.3%,16.8%)at 8h,19.8%(8.4%,35.2%)and 11.4%(-1.6%,18.2%)at 24 h,respectively.There was no difference in healing rates between intervention group and control group.Proteomic showed that there were 48 differential proteins meet the criteria(fold change more than 2times and p-value less than 0.05),among which 29 differential proteins were up-regulated and 19 differential proteins were down-regulated.GO analysis showed that the differentially expressed proteins were involved in actin-myosin filament sliding,ubiquitin-protein transferase and microfilament motor activity.KEGG pathway analysis suggested that differential proteins were involved in the regulation of actin cytoskeleton and MAPK signaling pathway related to proliferation,differentiation,and migration of cells.Conclusion Clinical studies showed that low irisin concentration is a risk factor for T2 DM and DFD.Low irisin concentration may related to the wound healing of diabetic foot.Experiments showed that irisin may promote diabetic wound healing by promoting the homing of circulating fibrocytes.The effect of irisin on the migration of fibroblasts needs further investigation.According to proteomic,irisin may affect the proliferation,differentiation and migration of fibroblasts,and ubiquitination of protein may also play a certain role.Specific mechanism of the effect of irisin on fibroblasts needs to be further explored.