| BackgroundDiabetes foot ulceration is a common and severe complication of diabetes mellitus. Nowadays, the mainstays of therapy for diabetic foot ulcers are careful wound care, revascularization and surgical operation. However, these interventions always are limited. Therefore, there is an urgent need to study the underlying mechanisms of impaired wound healing in DFU and find new treatment means.Wound healing is a complicated and interplay between multifarious cell types, the extracellular matrix, growth factor, and cytokines. Fibroblasts, which are an important cell type in wound healing process, mediate numerous, essential repair processes in chronic wounds, such as cytokine secretion, production and remodeling of extracellular matrix. Fibroblasts will migration, differentiation, proliferation in the wound healing process, and secreting large amounts of FN and Col-1. Extracellular matrix will form the granulation tissue together with the new capillaries, promoting the wound closure.Transforming growth factor-β1 (TGF-β1) is important in wound healing process.The secretion of TGF-β1 at the early stage of the healing prompts recruitment of inflammatory cells into the wound site. Granulation tissues are gradually formed and TGF-β1 prompts the expression of ECM, such as FN and Col-1. Meanwhile, TGF-β1 promotes the migration, differentiation, proliferation, chemotaxis of fibroblasts strongly. TGF-β1 also accelerates the angiogenesis and inhibits the growth of epithelial cell, what’s more, stimulates fibroblasts synthesize ECM. During wound healing, TGF-β1 induce fibroblasts to differentiate into myofibroblasts enriched in α-SMA, which is primarily known for their important role in wound healing.The accumulation of advanced glycation end products (AGEs) has been recognized as pathogenic for DFU. AGEs reduce fibroblasts migration, proliferation, and extracellular matrix production. The persistent high expression of RAGE in the fibroblasts of diabetic patients will damage the function of fibroblasts.Early growth response-1 (Egr-1) is an immediate early response gene. With the deepening of research, it is proven that Egr-1 plays a crucial role in wound healing. In normal dermal fibroblasts, changes of Egr-1 expression altered over 600 genes involved in cell migration, proliferation, ECM synthesis, vascular development and wound healing. Besides, studies have confirmed that Egr-1 promoted angiogenesis and increased synthesis of ECM, accelerating wound healing.Egr-1, as an immediate early gene, plays a crucial role in wound healing. Studies indicated that TGF-β1 induced the Egr-1 expression and ECM production. However, how Egr-1 change in human dermal fibroblasts under the pathological condition of diabetes is unknown. Though numerous animal studies had indicated that AGEs delayed the wound healing, little molecular evidence had been proposed. We need to find out how the fibroblasts change under the pathological condition of diabetes. We also need to confirm that whether Egr-1 mediate ECM production as a key factor. In the present research, we studied human dermal fibroblasts to investigate TGF-β1-induced Egr-1 expression and extracellular matrix deposition with or without the incubation of AGEs. To verify the role of Egr-1 in TGF-β1-induced ECM production, we further used small interfering RNA (siRNA) and overexpression plasmid. At last, the expressions of Egr-1 and ECM in normal skin and DFU skin were detected.Part 1 TGF-β1-induced Egr-1 expression and extracellular matrix production in HDFs treated by AGEsObjectives1. To explore how TGF-β1-induced Egr-1 expression and extracellular matrix deposition in HDFs with or without the incubation of AGEs.2. To speculate the relations between Egr-1 and extracellular matrix.Methods1. Cell culture:The human primary dermal fibroblasts were cultured in the full-nutrient medium (high-glucose DMEM supplemented with 15% fetal calf serum, 100U/ml penicillin and 100ug/ml streptomycin). Only early passage (3-10) fibroblasts were studied.2. Preparation of AGEs:BSA (4.8mg/ml) with penicillin and gentamicin was incubated in PBS (pH 7.4), which containing D-glucose (0.8mol/l). The BSA control was incubated without D-glucose. After sterilization with filters, the solutions were incubated in the dark at 37℃ for 12 weeks. Both solutions were dialyzed against PBS for 24h after incubation.3. Grouping:control group; TGF-β1 group(10ng/ml TGF-β1); TGF-β1+A100 group(10ng/ml TGF-β1+100ug/ml AGEs); TGF-P1+A200 group(10ng/ml TGF-β1+200ug/ml AGEs); TGF-β1+A300 group(10ng/ml TGF-β1+300ug/ml AGEs); measured Egr-1 according time point(0min,15min,30min,60min,120min and 360min); AGEs group(measured Egr-1 with 300ug/ml AGEs); TGF-β1+AGEs group(measured Egr-1 with 10ng/ml TGF-β1+300ug/ml AGEs).4. Measuring the mRNA and protein expression of α-SMA after 24h of TGF-β1 (10ng/ml) stimulation by qRT-PCR and Western blot.5. We measured FN and Coll mRNA and protein expression in HDFs treated by TGF-β1(10ng/ml) and incubation of different concentrations of AGEs(100,200, 300ug/ml) for 24h. Cells were harvested and qRT-PCR and Western blot were proceeded.6. We measured Egr-1 mRNA and protein expression in HDFs treated by TGF-β1(10ng/ml) after Omin,15min,30min,60min,120min and 360min. Cells were harvested and qRT-PCR and Western blot were proceeded.7. We measured Egr-1 mRNA and protein expression in HDFs treated by TGF-β1(10ng/ml) at appropriate time with or without incubation of AGEs(300ug/ml).8. Statistical analysis:Data were presented as mean±SD of n independent experiments. Student’s t-test was used for analysis between two groups. One-way ANOVA of SPSS 13.0, followed by LSD-t test for multiple comparisons among groups, was used for all studies containing three or more groups. A value of P<0.05 was considered statistically significant.Results1. Effect of TGF-β1 on the expression of a-SMA at the transcriptional level in the HDFs.The α-SMA mRNA and protein expression level were increased after 24h of TGF-β1 induced(P<0.05).2. Effect of TGF-β1 on the production of ECM in the HDFs treated with different concentrations of AGEs.The FN and Coll mRNA expression rose obviously after 24h of TGF-β1 induced(P<0.05), while dramatically decreased with incubation of AGEs, especially the highest concentrations(300ug/ml) (P<0.05). Consistent with changes in mRNA level, TGF-β1 strikingly increased the expression of FN and Coll protein(P<0.05), and AGEs(300ug/ml) down-regulated them(P<0.05).3. Effect of TGF-β1 on the production of Egr-1 gene expression in HDFs.The results revealed that TGF-β1 induced a quick rise in Egr-1 mRNA levels in HDFs. We can see that a maximal 15-fold induction was observed at 30min(P<0.05), then followed by a progressive decrease and back to the basic levels by 360min. TGF-β1 strikingly increased the protein expression of Egr-1, which revealed a progressive time-dependent accumulation that peaked at 120min(P<0.05) and decline to basal levels by 360min. According to the results, we found out that which time point was the most significant of Egr-1 expression.4. How the Egr-1 expression change with the incubation of AGEs(300ug/ml).After 30min of TGF-β1 induction, Egr-1 mRNA levels increased obviously. However, if TGF-β1 was coexist with AGEs(300ug/ml), the Egr-1 mRNA levels dramatically decreased compare with the group without AGEs(P<0.05). After 120min of TGF-β1 induction, Egr-1 protein levels increased obviously. But when TGF-β1 was coexist with AGEs(300ug/ml), the Egr-1 protein levels dramatically decreased compare with the group without AGEs(P<0.05).Conclusions1. HDFs can transform into myofibroblasts phenotypes enrich in a-SMA after TGF-β1 induced, which procedure is important in wound healing.2. Under the impact of AGEs, the capacity of secreting ECM in the HDFs would be reduced.3. We found out that which time point was the most significant of Egr-1 mRNA and protein expression in HDFs.4. AGEs(300ug/ml) would affect the expression of Egr-1 induced by TGF-β1.5. Egr-1 expression was consistent with changes of ECM expression in HDFs after the stimulation of TGF-β1 and AGEs.Part 2 Relationship between TGF-β1-induced Egr-1 and extracellular matrixproduction in HDFsObjectives1. To explore the relationship between Egr-1 and extracellular matrix.2. To understand the up- and downstream relationship between Egr-1 and extracellular matrix.Methods1. Cell culture:As mentioned before.2. Grouping:blank group; mock group; Negative control group(NC); Negative control with TGF-β1(NC+TGF-β1); Egr-1-homo-1624 with TGF-β1(si1624+ TGF-β1); Egr-1-homo-749 with TGF-β1(si749+TGF-β1); Egr-1-homo-1508 with TGF-β1(si1508+TGF-β1); Egr-1-homo-353 with TGF-β1(si353+TGF-β1); pENTER group; pENTER-Egr-1 group; pENTER with TGF-β1 (pENTER+TGF-β1); pENTER-Egr-1 with TGF-β1(pENTER-Egr-1+TGF-β1).3. After NC, si1624, si749, si1508, si353 were transfected, we added 10ng/ml TGF-β1 to stimulate the HDFs. After 30min stimulation, Cells were harvested and qRT-PCR was proceeded to measure the mRNA expression of Egr-1. Next, after NC, si1624, si749 were transfected, we added 10ng/ml TGF-β1 to stimulate the HDFs. After 120min stimulation, Cells were harvested and western blot was proceeded to measure the protein expression of Egr-1.4. After NC, si1624, si749 were transfected, we added 10ng/ml TGF-β1 to stimulate the HDFs for 24h. Cells were harvested. The mRNA and protein expression of FN and Col-1 were measured.5. After overexpression plasmids were transfected, cells were harvested. The mRNA and protein expression of Egr-1 were measured.6. After overexpression plasmids were transfected, we added lOng/ml TGF-β1 to stimulate the HDFs for 24h. Cells were harvested. The mRNA and protein expression of FN and Col-1 were measured.7. Statistical analysis:As mentioned before.Results1. Effect of TGF-β1 on the Egr-1 mRNA and protein expression in HDFs transfected by siRNA.Egr-1 mRNA and protein expression was no difference between blank, mock and NC. When TGF-β1 was added to NC, the expression of Egr-1 mRNA and protein were increased(P<0.05). After si1624 and si749 were transfected, we added lOng/ml TGF-β1 to stimulate the HDFs. The expression of Egr-1 mRNA and protein were obviously lower than the NC+TGF-β1 group(P<0.05).2. Effect of TGF-β1 on the ECM mRNA and protein expression in HDFs transfected by siRNA.FN, Col-1 mRNA and protein expression was no difference between blank, mock and NC. When TGF-β1 was added to NC, the expression of their mRNA and protein were increased(P<0.05). After si 1624 and si749 were transfected, we added 10ng/ml TGF-β1 to stimulate the HDFs for 24h. The expression of FN and Col-1 were obviously lower than the NC+TGF-β1 group(P<0.05).3. Expression of the Egr-1 mRNA and protein expression in HDFs transfected by plasmids.Egr-1 mRNA and protein expression increased obviously after transfected for 24h(P<0.05).4. Effect of TGF-β1 on the ECM mRNA and protein expression in HDFs transfected by plasmids.After transfected by the overexpression plasmids, the expression of FN and Col-1 were increased(P<0.05). When the pENTER group and overexpression group were treated by TGF-β1, the expression of FN and Col-1 were also greater than the control group(P<0.05).Conclusions1. Egr-1 is an important mediator for TGF-β1-induced extracellular matrix production.2. Egr-1 was the upstream mediator of the extracellular matrix.Part 3 Expressions of Egr-1 and ECM in normal skin and DFU marginal cutaneous tissuesObjectivesTo explore whether Egr-1 and ECM are changed in DFU marginal cutaneous tissues.Methods1. Samples were obtained from the marginal cutaneous tissues of DFUs patients hospitalized in the Department of Endocrinology, Nanfang Hospital Affiliated with the Southern Medical University. In the control group, biopsies of normal dermal tissues were obtained from patients who underwent skin transplantation during surgery. Biopsies were obtained from 7 patients with DFUs (DFU group), and 5 with normal skin(control group). Expressions of Egr-1 and ECM were measured by qPCR and western blot.2. Statistical analysis:Data are presented by the median and inter-quartile range. As mentioned before.ResultsExpressions of Egr-1 and ECM in normal skin are greater than DFU marginal cutaneous tissues.ConclusionsEgr-1 expression and ECM production changed consistently, what’s more, the expression of Egr-1 and ECM were greater in normal condition. |
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