MDI 301, a synthetic retinoid, depressed levels of matrix metalloproteinases and oxidative stress in diabetic dermal fibroblasts

Abstract

Diabetic foot ulcerations could result in serious consequences such as amputations. The up-regulation of matrix metalloproteinases and down-regulation of TIMP1 were remarked as distinctive biological characteristics in the diabetic dermal fibroblast. The current study was performed in order to clarify the effect of high glucose on formation of diabetic dermal fibroblast cell. In addition, the effect of MDI 301 on ameliorating diabetic fibroblasts was investigated in this study. The mRNA and protein expression levels of MMPs, TIMP1 and catalase were evaluated against fibroblasts treated with high glucose (30 mM) using qRT-PCR, western blotting and zymography assays. Methods were also employed for investigating the biological effects of MDI 301 on high glucose-induced diabetic fibroblasts. In this study, we found that the unbalance of oxidative stress induced by high glucose concentration play an important role in the formation of diabetic dermal fibroblast from normal cells. In addition, MDI 301, a picolinic acid-substituted ester of 9-cis retinoic acid was employed in this study in order to ameliorate symptoms on diabetic dermal fibroblast induced by high glucose concentration. We found MDI 301 alleviate the effects of high glucose-induced skin damage by balancing the oxidative stress and regulating the MMPs and TIMP1 levels. Our finding indicated that MDI 301 offers the potential for repairing the faulty skin function arising from diabetes.

Keywords: MMPs; TIMP1; diabetic fibroblast cell; high glucose; retinoid acid.

Figure 1. Increased expression level of active MMPs in fibroblasts treated by high glucose (30 mM)

Legend: Lane 1 indicates the MMP zymography assay results from normal fibroblasts, and lane 2 shows the results from the fibroblasts treated with 30 mM glucose. These results indicated that the protein abundance levels of MMPs increased in glucose (30 mM) treated fibroblasts. The active forms of MMP2 and MMP9 increased significantly. Condition: The normal fibroblasts were cultured in DMEM cell culture containing 30 mM glucose. The cells were routinely cultured in a humidified atmosphere at 37°C, 5% (v/v) CO₂, 95% (v/v) air, and they were used for experiments between passages 3 and 5 (80% confluence). The cultured media were assayed for MMP-1, MMP-2 and MMP-9 by casein and gelatin zymography. Zymographic images were digitized and quantified by scanning densitometry. Quantitative values for MMP-1, MMP-2 and MMP-9 were obtained and normalized against MMPs normalization standards.

Figure 2. High glucose increased the mRNA expression levels of MMPs and decreased the expression level of TIMP1 on normal dermal fibroblasts

Legend: (A) The normalized mRNA expression level of MMP1 in the normal fibroblasts cultured in high glucose media. (B) The normalized mRNA expression level of MMP2 in the normal fibroblasts cultured in high glucose media. (C) The normalized mRNA expression level of MMP9 in the normal fibroblasts cultured in high glucose media. (D) The normalized mRNA expression level of TIMP1 in the normal fibroblasts cultured in high glucose media. These results suggested that high glucose increases MMP levels, P < 0.05. The presence of high glucose alters the ratios between MMPs and TIMP1 which is a standard diagnostic of diabetic foot ulcerations. Condition: The normal dermal fibroblasts were cultured in DMEM media containing high glucose (30 mM) for four days. The mRNA expression levels of MMPs and TIMP1 were investigated by qRT-PCR, and the 2^−ΔΔCT method was used to analyze the relative changes in gene expression.

Figure 3. Effects of MDI 301 on the mRNA expression levels of MMPs in high glucose treated-fibroblast cells

Legends: (A) The normalized mRNA expression level of MMP1 in high glucose treated-fibroblasts at the presence of 3 μM MDI 301. (B) The normalized mRNA expression level of MMP2 in high glucose treated-fibroblasts at the presence of 3 μM MDI 301. (C) The normalized mRNA expression level of MMP9 in high glucose treated-fibroblasts at the presence of 3 μM MDI 301. (D) The normalized mRNA expression level of TIMP1 in high glucose treated-fibroblasts at the presence of 3 μM MDI 301. These results showed a decrease in the mRNA expression levels of MMP1, MMP2 and MMP9 in the fibroblast cells treated with 3 μM MDI 301. After MDI 301 treatment, the mRNA expression level of MMP1 decreased by 67% (P < 0.05), MMP2 decreased by 51% (P < 0.05) and MMP9 decreased by 68% (P < 0.05). The mRNA expression level of TIMP1 was analyzed by qRT-PCR, and the data showed that the cells treated with MDI 301 had an increase of 72% (P < 0.05) compared to cells without MDI 301 incubation. Condition: The mRNA analysis kits for MMP1, MMP2 and MMP9 were supplied by Shanghai Sangon Institute of Biology, and the 2^−ΔΔCT method was used to analyze the relative changes in gene expression.

Figure 4. Effect of MDI 301 on the formation of active MMPs by MMP zymography assays

Legend: (A) MMP zymography assay for MMP1. (B) MMP zymography assay for MMP2 and MMP9. These results indicated that MDI 301 is capable of inhibiting the formation of MMPs in fibroblasts treated by 30 mM glucose. Condition: The normal fibroblasts were cultured in DMEM cell culture containing high glucose (30 mM). The cells were routinely cultured in a humidified atmosphere at 37°C, 5% (v/v) CO₂, and 95% (v/v) air and used for experiments between passages 3 and 5 (80% confluence). The cultured media were assayed for MMP-1, MMP-2 and MMP-9 by casein and gelatin zymography.

Figure 5. Effects of MDI 301 on procollagen I and III

Legend: (A left) Normalized protein abundance of procollagen I in high glucose-treated fibroblasts treated with MDI 301; (A right). Western blotting analysis of procollagen I in high glucose-treated fibroblasts treated with MDI 301. Lane 1 shows the level of procollagen I in the fibroblasts treated with MDI 301. Lane 2 shows the level of procollagen I in the fibroblasts not treated with MDI 301. β-actin was employed as a control in this experiment. (B left) The normalized protein abundance of procollagen III in high glucose-treated fibroblasts treated with MDI 301. (B right) Western blotting analysis of procollagen III in high glucose-treated fibroblasts treated with MDI 301. Lane 1 shows the level of procollagen III in the fibroblasts treated with MDI 301. Lane 2 shows the level of procollagen III in the fibroblasts not treated with MDI 301. β-actin was employed as a control in this experiment. The western blotting results of procollagen I and III suggest that MDI 301 induces higher expression levels of procollagen I and III in high glucose-treated fibroblasts upon the treatment of MDI 301. The images were digitized and quantified by scanning densitometry. Quantitative values for procollagen I and procollagen III were obtained and being normalized against the normalization standards. The data shows that MDI 301 induced an increase of procollagen I (103%) and of procollagen III (53%) in the high glucose-treated fibroblasts treated with MDI 301, P < 0.05. Condition: The high glucose-treated fibroblasts were incubated with 3 μM MDI 301 for four days in a humidified atmosphere at 37°C, 5 % (v/v) CO₂, and 95% (v/v) air and were used for experiments between passages 3 and 5 (80% confluence). The cultured media were assayed for procollagen I and procollagen III by western blotting assays.

Figure 6. Effects of MDI 301 on catalase and superoxide dismutase involved in oxidative stress

Legend: MDI 301 induced a 50% decrease in the mRNA expression level of catalase in high glucose-treated fibroblasts. In addition, MDI 301 induced a 64% decrease in the mRNA expression level of superoxide dismutase in high glucose-treated fibroblasts. These data indicate that the mRNA levels of both catalase and superoxide dismutase decrease in the presence of 3 μM MDI 301. Catalase and superoxide dismutase are involved in the induction of cellular oxidative stress which is attributed to the rise of MMP expression levels and activations. Condition: The cells were incubated with 3 μM MDI 301 for four days, and then the expression levels of catalase and superoxide dismutase were examined by qRT-PCR. The 2^−ΔΔCT method was used to analyze the relative changes in gene expression.