| BackgroundMatrix metalloproteinases(MMPs) are a family of Zinc-dependent endopeptidase that collectively degrade all extracellular matrix.Tissue inhibitors of metalloproteinases(TIMPs) are the endogenous inhibitors,the main function is specific inhibition for MMPs.They both work together,keep a dynamic balance,play an important pole in physiological and pathological process.Studies proved that the expression under control timely is necessary for the wound healing and tissue repair.The expression and activity of MMPs in wound of chronic diabetic foot ulcers increase significantly,and the expression and activity of TIMPs decrease significantly.so,the ulcers are difficult to heal.Tetracyclines are utilized in clinic generally as a family of broad-spectrum antibiotic, but studies show that they are multiple cellular functional drugs in the near future,they are potential inhibitors of MMPs.Additional studies showed that doxycycline can decrease the expression of MMPs in a certain concentration range to animal models or human body, inhibit the synthesis of MMPs,reduce the activity of MMPs.ObjectiveTo investigate the expression of matrix metalloproteinase-2(MMP-2) and tissue inhibitor of metalloproteinase-2(TIMP-2) in wound exudate of diabetic foot with bacteria infection.And to reveal the internal association between the expression of MMP-2 and TIMP-2 and diabetic foot ulcers with bacteria infection initially.And to illuminate the dependablity between the disequilibrium of MMP-2 and TIMP-2 with healing mechanism of diabetic foot ulcers with bacteria infection further by the variation of the MMP-2/TIMP-2 ratio.At the same time,we utilize doxycycline as a local therapy on diabetic foot ulcers,observe the effectiveness of the treatment,to inquire a new method for the therapy of diabetic foot ulcers.Method1. 20 patients with diabetic foot ulcers with bacteria infection(DF group),were type 2 Diabetes Mellitus,diagnosed by 1999 WHO criteria.All patients were classified by Wagner Classification,among them gradeⅡDF 8 cases,gradeⅢDF 12 cases.The ABI of affected limbs between 0.4-0.9,the duration of diabetic foot ulcers was over four weeks,removing venous ulcers,no disfunction of liver and renal,and no immunologic deficiency diseases.Confirmed concurrent infection with local secretion or deep necrotic tissue bacterial culture in ulcers:15 patients with single bacterial infection,5 patients with mixed bacterial infection.The control group included two groups,10 patients with diabetes but no infection(DM group),all were type 2 Diabetes Mellitus,diagnosed by 1999 WHO criteria;10 patients were normal humans(N group),confirmed no concurrent infection with local Secretion bacterial culture,there was no difference between diabetic foot ulcers with bacteria infection group and the control group in general conditions such as ages, sex.2. Specimen collection: to collect wound exudate from diabetic foot ulcers with bacteria infection and draining fluid from operation wounds.3. The concentrations of MMP-2 and TIMP-2 in ulcer exudate were assayed by ELISA, calculated the MMP-2/TIMP-2 ratios of each group,to made sure whether there would be a significant difference of the three groups.4. Treated the diabetic foot ulcers with bacteria infection with doxycycline locally(the drug concentration was 100mg/L),NS treatment as control.The time of granulation arising (GT) and the time of healing up (HT) were compared,to made sure whether there would be a significant difference of the two groups.Results1.The results of the concentration of MMP-2 ,TIMP-2 and the MMP-2/TIMP-2 ratio in the wound exudate of the three groups:In the diabetic foot ulcers with bacteria infection group,the concentration of MMP-2 was 83.58±15.09 ng/ml,the concentration of TIMP-2 was 12.88±2.59 ng/ml,and the MMP-2/TIMP-2 ratio was 6.84±2.33;In the diabetes but no infection group,the concentration of MMP-2 was 32.18±4.29 ng/ml,the concentration of TIMP-2 was 30.71±3.91 ng/ml, and the MMP-2/TIMP-2 ratio was 1.06±0.20;In the normal control group,the concentration of MMP-2 was 21.51±3.62 ng/ml,the concentration of TIMP-2 was 20.74±2.72 ng/ml, and the MMP-2/TIMP-2 ratio was 1.05±0.23.There was a significant difference of the concentration of MMP-2 between the DF group and the control group (P=0.000); The concentration of MMP-2 in the DF group was significantly higher than the DM group(P<0.01), and the concentration of MMP-2 in the DM group was higher than the N group(P<0.05).There was a significant difference of the concentration of TIMP-2 between the DF group and the control group (P=0.000);The concentration of TIMP-2 in the DM group was significantly higher than the N group(P<0.01),and the concentration of TIMP-2 in the N group was significantly higher than the DF group(P<0.01). There was a significant difference of the MMP-2/TIMP-2 ratio between the DF group and the control group(P=0.000),the MMP-2/TIMP-2 ratio of the DF group was significantly higher than the other groups(P<0.01),but there was no difference between the DM group and the N group(P>0.05).2.Subgroup comparison of diabetic foot ulcers with bacteria infection group:2.1 The comparison of different degree infection:in the single bacterial infection group,the concentration of MMP-2 was 79.26±13.77 ng/ml,the concentration of TIMP-2 was 12.98±2.80 ng/ml,and the MMP-2/TIMP-2 ratio was 6.51±2.48;in the mixed bacterial infection group,the concentration of MMP-2 was 96.51±11.82 ng/ml,the concentration of TIMP-2 was 12.58±2.09 ng/ml,and the MMP-2/TIMP-2 ratio was 7.85±1.62.There was a significant difference of the concentration of MMP-2 between the two groups (P=0.000), the concentration of MMP-2 in the mixed bacterial infection group was higher than the single bacterial infection group(P<0.05),but there was no difference of the concentration of TIMP-2 and the ratio between the two groups(P>0.05).2.2 The comparison of different grade DF:in the gradeⅡDF group,the concentration of MMP-2 was 73.0±8.56 ng/ml,the concentration of TIMP-2 was 13.56±2.82 ng/ml,and the MMP-2/TIMP-2 ratio was 5.59±1.33;in the gradeⅢDF group,the concentration of MMP-2 was 90.62±18.54 ng/ml,the concentration of TIMP-2 was 12.43±2.45 ng/ml, and the MMP-2/TIMP-2 ratio was 7.68±2.53.There was a significant difference of the concentration of MMP-2 and the ratio between two groups(P=0.007 and P=0.047),the concentration of MMP-2 and the ratio in the gradeⅢDF group were higher than the gradeⅡDF group(P<0.05),but there was no difference of the concentration of TIMP-2 between the two groups(P>0.05).3.The effects of doxycycline:In the doxycycline therapy group,the time of granulation arising (GT) in patients with gradeⅡ,ⅢDF were 3.5±1.3 days and 4.8±1.5 days respectively,the time of healing up (HT) in patients with gradeⅡ,ⅢDF were 22.8±3.3 days and 25.2±4.4 days respectively.GT and HT of the therapy group were 4.3±1.5 days and 24.2±4.0 days respectively.In NS control group,the time of granulation arising (GT) in patients with gradeⅡ,ⅢDF were 6.3±1.3 days and 7.3±1.5 days respectively,the time of healing up (HT) in patients with gradeⅡ,ⅢDF were 38.3±3.4 days and 46.5±5.0 days respectively.GT and HT of the control group were 6.9±1.5 days and 43.2±6.0 days respectively.There was a significant difference of GT and HT between the two groups(P<0.01),the GT and HT of the doxycycline therapy group were significantly shorter than the control group(P<0.01). Conclusions1. The concentration of MMP-2 increases significantly,the concentration of TIMP-2 decreases significantly,the MMP-2/TIMP-2 ratio increases significantly in diabetic foot ulcers with bacteria infection;2. Doxycycline can shorten the GT and HT of diabetic foot ulcers significantly,promote the healing of diabetic foot ulcers. |
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