Changes in biological behaviors of rat dermal fibroblasts induced by high expression of MMP9.

BACKGROUND: The high level of matrix metalloproteinase 9 (MMP9) is thought to slow down the healing of diabetic foot ulcers. Whether it can influence the biological behaviors of skin fibroblasts and affect wound healing is still unclear. The present study aimed to observe changes in the biological behaviors of rat dermal fibroblasts induced by high expression of MMP9 and to clarify the possible mechanisms of wound healing for diabetic foot. METHODS: A cell model of skin fibroblast with high expression of MMP9 was established by co-culture of high glucose (22.0 mmol/L) and homocysteine (100 µmol/L). A control group was incubated with normal glucose (5.5 mmol/L). Realtime PCR, ELISA and gelatin zymography were used to detect the MMP9 mRNA, protein expression and activity of MMP9. Flow cytometry, CCK-8, ELISA assay, scratch test and transwell were used to detect cell proliferation, viability, collagen (hydroxyproline) secretion, horizontal migration and vertical migration of cells. The data were expressed as mean±SD. P value less than 0.05 was considered statistically significant. RESULTS: The expression of MMP9 mRNA, protein levels and the activity of MMP9 were much higher in the high MMP9 group than in the control group (7.05±1.02 vs. 1.00±0.00, 206.9±33.6 pg/mL vs. 40.4±5.9 pg/mL, and 1.47±0.13 vs. 0.57±0.12, respectively, P<0.01). The proportion of S-phase cells, proliferation index, cell viability, collagen (hydroxyproline) secretion, horizontal migration rate and the number of vertical migration cells were lower in the high MMP9 group than in the control group (P<0.01). CONCLUSION: Fibroblasts with a high expression of MMP9 decreased proliferation, activity, secretion and migration of collagens, suggesting that MMP9 may inhibit the biological behaviors of fibroblasts.

The biological behaviors of rat dermal fibroblasts can be inhibited by high levels of MMP9.

AIMS: To explore the effects of the high expression of MMP9 on biological behaviors of fibroblasts. METHODS: High glucose and hyperhomocysteine were used to induce MMP9 expression in skin fibroblasts. Cell proliferation was detected by flow cytometry and cell viability by CCK-8. ELISA assay was used to detect collagen (hydroxyproline) secretion. Scratch test was employed to evaluate horizontal migration of cells and transwell method to evaluate vertical migration of cells. RESULTS: The mRNA and protein expressions of MMP9 and its protease activity were significantly higher in cells treated with high glucose and hyperhomocysteine than those in control group. At the same time, the S-phase cell ratio, proliferation index, cell viability, collagen (hydroxyproline) secretion, horizontal migration rate, and the number of vertical migration cells decreased in high-glucose and hyperhomocysteine-treated group. Tissue inhibitor of metalloproteinase 1 (TIMP1), which inhibits the activity of MMP9, recovered the above biological behaviors. CONCLUSIONS: High expression of MMP9 in skin fibroblasts could be induced by cultureing in high glucose and hyperhomocysteine medium, which inhibited cell biological behaviors. Inhibitions could be reversed by TIMP1. The findings suggested that MMP9 deters the healing of diabetic foot ulcers by inhibiting the biological behaviors of fibroblasts.

[Effects of matrix metallopeptidase 9 on the proliferation, apoptosis, type I and III procollagen synthesis of rat dermal fibroblasts].

OBJECTIVE: To explore the effects of matrix metallopeptidase 9 (MMP-9) on the proliferation, apoptosis, type I and III procollagen synthesis of rat dermal fibroblasts. METHODS: Lipopolysaccharide (LPS) at 0.1 and 1.0 µg/ml was used to stimulate fibroblasts to up-regulate the expression of MMP-9 for 18 and 48 h. And then RNA interference was employed to inhibit the high expression of MMP-9. Cell proliferation was tested by CCK-8, cell apoptosis by flow cytometry and type I and III procollagen expressions by quantitative reverse transcriptase PCR (qRT-PCR). RESULTS: The MMP-9 expression of fibroblasts increased after the stimulation of LPS. And the 1.0 µg/ml LPS stimulation of 48 hours was 14.25 times of mRNA expression and 2.31 times of protein expression versus that of the normal group (both P < 0.05). The RNA interference obviously inhibited the high expression of MMP-9. The mRNA expression was 1/8 and protein expression 1/3 (both P < 0.05) as compared with the control group. Cell proliferation decreased with the rising expression of MMP-9 to some extent [(1.08 ± 0.08) vs (1.18 ± 0.09), P < 0.05] and improved after the inhibition of high expression of MMP-9 [(1.78 ± 0.17) vs (1.53 ± 0.15), P < 0.01]. There was no change of apoptosis accompanied with high expression of MMP-9 (P > 0.05). Apoptosis decreased after the inhibition of high expression of MMP-9 for 48 hours (3.53% ± 0.22% vs 4.47% ± 0.46%, P < 0.05). The synthesis of type I procollagen was the same no matter up-regulation or down-regulation of MMP-9 expression (P > 0.05). As the expression of MMP-9 increased, the synthesis of type III procollagen decreased (P < 0.01), but not increased by 1.02 folds after the inhibited expression of MMP-9 (P > 0.05). CONCLUSION: MMP-9 can affect the biological behaviors of rat dermal fibroblasts.

Increased level of soluble syndecan-1 in serum correlates with myocardial expression in a rat model of myocardial infarction.

A candidate marker for ventricular remodeling after myocardial infarction (MI), syndecan-1 (Sdc1), has been shown to be upregulated in myocardial tissues. However, the clinical potential of this marker depends on the ability to obtain samples safely and noninvasively. Therefore, we investigated the expression of soluble Sdc1 in the serum of rats after MI. Anterior descending coronary arteries of Sprague-Dawley rats were ligated, and MI was confirmed by morphologic and physiologic methods. Rats that underwent surgery without ligation served as the control group. We analyzed the expression of Sdc1 mRNA by quantitative reverse-transcription polymerase chain reaction and that of Sdc1 protein by western blot in heart tissue from the MI border and compared it to the expression of soluble Sdc1 in serum. The myocardial levels of expression of Sdc1 mRNA and protein were very low in the sham group but increased significantly in the MI group (P<0.01). The expression of myocardial Sdc1 reached a peak at day 3 and declined gradually thereafter, although the levels at 14 days remained significantly higher than those in the sham group. The expression of soluble Sdc1 in the sera of the rats in the MI group followed a similar pattern and was linearly correlated with the expression of Sdc1 protein in the MI border zone (r=0.952, P<0.01). Soluble Sdc1 was also detected at low levels in normal rat serum. These results may facilitate additional exploration of the utility of serum Sdc1 as a biomarker for MI in humans.

[The effects of glucose fluctuation on resistin].

OBJECTIVE: To explore the effect of glucose fluctuation on resistin. METHODS: The phorbol-12-myristate-13-acetate(PMA)-activated and differentiated U937 cells were exposed to experimental condition for 3 days, three groups of cells were formed, each one receiving the following fresh medium every 6 hours, respectively: (1) continuous 11.1 mmol/L glucose concentration medium (Con group), (2) continuous 22.2 mmol/L glucose concentration medium (CHG group), (3) alternating 11.1 mmol/L glucose concentration and 22.2 mmol/L glucose concentration medium every 6 hours (IHG group). The supernatants of cell median at the last 6 hours were collected to test resistin concentration. Besides, 92 subjects were selected and classified into three groups according to the results of oral glucose tolerance test: normal glucose tolerance group (NGT group, n=30), impaired glucose tolerance patients (IGT group, n=31) and newly diagnosed type 2 diabetes patients (T2DM group, n=31). Blood glucose and serum resistin levels were measured at 0 h and 1 h during oral glucose tolerance test (OGTT) to compare the glucose fluctuation (ΔGlu1-0) and the change of serum resistin level (ΔlnRes1-0) among the three groups. RESULTS: Resistin concentration in the Con, CHG and IHG group was (73.62±5.07) ng/L, (97.78±7.00) ng/L and (212.49±28.81) ng/L respectively and in IHG group it was higher as compared with the other two groups (P<0.05). ΔGlu1-0 in NGT, IGT and T2DM group was (2.31±2.30) mmol/L, (5.70±2.08) mmol/L and (8.41±2.63) mmol/L respectively; ΔGlu1-0 increased gradually in all the three groups (P<0.05). Serum resistin level from 0 h to 1 h in the NGT group was 6.41 (1.52-15.76) µg/L to 6.96 (1.52-22.70) µg/L, in the IGT group 5.47 (1.49-24.09) µg/L to 9.12 (1.27-21.94) µg/L and in the T2DM group 5.77 (1.11-30.10) µg/L to 9.27(1.02-48.15) µg/L. In the IGT and T2DM group serum resistin level increased from 0 h to 1 h (P<0.05), but no difference was observed in the NGT group (P>0.05). ΔlnRes1-0 in these 3 groups was (0.05±0.05) µg/L, (0.25±0.04) µg/L and (0.37±0.03) µg/L respectively and the change in the T2DM group was significant as compared with that in the NGT group, ΔlnRes1-0 was positively correlated with ΔGlu1-0 (r=0.23, P=0.02). CONCLUSION: Glucose fluctuation induced monocyte/macrophage to secrete resistin, greater the glucose fluctuation, greater the change of amplitude of serum resistin.