Diabetic wounds are recalcitrant to healing. However, the mechanism causing this dysfunction is not fully understood. High expression of matrix metalloproteinase-9 (MMP-9) is indicative of poor wound healing. In this study, we show that specificity protein-1 (Sp1), a regulator of MMP-9, binds directly to its promoter and enhances its expression. Additionally, we demonstrated that Sp1 is the direct target of two microRNAs (miRNAs), miR-129 and -335, which are significantly downregulated in diabetic skin tissues. In vitro experiments confirmed that miR-129 or -335 overexpression inhibits MMP-9 promoter activity and protein expression by targeting Sp1, whereas the inhibition of these miRNAs has the opposite effect. The beneficial role of miR-129 or miR-335 in diabetic wound healing was confirmed by the topical administration of miRNA agomirs in diabetic animals. This treatment downregulated Sp1-mediated MMP-9 expression, increased keratinocyte migration, and recovered skin thickness and collagen content. The combined treatment with miR-129 and miR-335 induced a synergistic effect on Sp1 repression and MMP-9 downregulation both in vitro and in vivo. This study demonstrates the regulatory mechanism of Sp1-mediated MMP-9 expression in diabetic wound healing and highlights the potential therapeutic benefits of miR-129 and -335 in delayed wound healing in diabetes.
In order to investigate the inhibitory effects and possible mechanisms of Oolong tea polyphenols, (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) on pancreatic α-amylase, the inhibition, enzyme kinetics, ultraviolet (UV) absorption spectrum and fluorescence spectrum of α-amylase were investigated. The results showed that Oolong tea polyphenols, EGCG, and EGCG3″Me all exhibited inhibitory effects against α-amylase, and their half inhibitory concentration (IC50) values were 0.375, 0.350, and 0.572 mg/mL, respectively. The results of Lineweaver-Burk double reciprocal plot indicated that the inhibitory types of Oolong tea polyphenols and EGCG were competitive, whereas EGCG3″Me was in a noncompetitive pattern. Oolong tea polyphenols, EGCG, and EGCG3″Me all induced red-shift of UV absorbance and quenching of fluorescence of α-amylase, suggesting possible changes in the conformation of α-amylase. The differences of inhibitory effects and inhibition types for EGCG and EGCG3″Me might be due to their structural difference (the hydroxyl group at C-3 in D ring of EGCG substituted by methoxy group, forming EGCG3″Me).
Polyphenols and caffeoylquinic acid (CQA) derivatives (3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) were prepared from Ilex kudingcha C.J. Tseng, and their effects and mechanisms on the activities of α-glucosidase from Saccharomyces cerevisiae were investigated in the present study. As results, the IC50 values for CQA derivatives were 0.16-0.39 mg/mL, and the inhibition mode of CQA derivatives was noncompetitive. On the basis of fluorescence spectroscopy and circular dichroism spectroscopy data, the binding constants and number of binding sites were calculated to be 10(6)-10(8) M(-1) and 1.42-1.87, respectively. CQA derivatives could bind to the enzyme mainly through hydrophobic interaction, altering the microenvironment and molecular conformation of the enzyme, thus decreasing the catalytic activity. To the authors' knowledge, this is the first report on α-glucosidase inhibitory mechanism by CQA derivatives from I. kudingcha, and the findings suggest a potential use of kudingcha as functional foods for the prevention and treatment of diabetes and related symptoms.
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