Non-healing wounds which affect 15% of persons with diabetes is associated with persistence of inflammation and increased expression of matrix metalloproteinases in particular MMP-9. NGAL (Lipocalin-2) expressed by neutrophils, is known to bind MMP-9 to prevent its degradation. We hypothesized that increased inflammatory cell NGAL may play a role in the increased MMP-9 activities in diabetic wounds. We examined the effect of diabetes on NGAL and MMP-9 activity in cells from our rodent diabetic implant healing model. Whether insulin treatment could prevent these changes was also studied.
Diabetes was induced in Sprague-Dawley rats (D:n=12) with STZ (65mg/kg), half of the diabetic animals were then treated with insulin (D+Ins:10IU/day). Non-diabetic age matched rats (C:n=6) acted as control. Six weeks later animals were anesthetized and PVC sponges (4x1cm2) were implanted subcutaneously. After six days all animals were sacrificed and sponges were collected and the cells and fluids separated for analysis. The mRNA levels of NGAL, MMP-9 and MMP-8 (expressed only by neutrophils) was measured by RT-qPCR, MMP-9 (pro and active forms) and NGAL-MMP-9 protein complex was determined by zymography. Whether insulin treatment could normalise wound healing rate was examined in a parallel study.
Diabetes induced inflammatory cell NGAL (C: 1.00±0.12 vs D: 2.81±0.57) and MMP-8 (C: 1.00±0.12 vs D: 3.21±0.52) mRNA as well as the wound fluid NGAL-MMP-9 complex and total MMP-9 activity (all P<0.05). These changes were prevented by insulin treatment. Inflammatory cell MMP-9 mRNA was not altered by diabetes. Insulin treatment partially improved wound healing rate (C:69.9±2.1 vs D:49.8±2.9, D+Ins 60.5±3.4).
These results suggest that higher NGAL levels in diabetes are related to higher wound fluid MMP-9 activity. The role of persistence of neutrophils evidenced by increased MMP-8 in this process remains to be investigated. Therapies targeting NGAL may have utility in diabetic wound healing. Supported by IPRS and APA.