Effects of advanced glycation end products and its receptor on oxidative stress in diabetic wounds / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the accumulation of advanced glycation end products (AGE) and the inflammatory response of skin and wound in diabetic patients, and to analyze their relationship in vitro.</p><p><b>METHODS</b>Histological staining and immunohistochemical staining was respectively performed on skin and wound tissue specimens collected from 10 patients with Type II diabetes mellitus (diabetes group) and 12 non-diabetic patients with skin injury (control group) to observe the arrangement of collagen and the distribution of inflammatory cells, and to determine the expression levels of AGE and its receptor (RAGE). Malondialdehyde (MDA) levels in skin and wound tissue homogenates were assayed by enzyme-linked immunosorbent assay. In vitro, human neutrophils were isolated and treated with RPMI-1640 culture medium or that containing AGE-human serum albumin in the concentration of 0.315, 0.625, 1.250 mg/mL, and they were identified as normal control (NC) group, low concentration (L) group, moderate concentration (M) group, and high concentration (H) group. Cell viability in each group was determined by MTT colorimetric assay, and the reactive oxygen species (ROS) in cell was measured with 2', 7'-dichlorofluorescein-diacetate. Data were processed with t test.</p><p><b>RESULTS</b>Compared with those of skin in control group, collagens of skin tissues in diabetes group atrophied and disorderly arranged. Inflammatory cells in wounds in diabetes group were dispersed, in which collagens arranged loosely and irregularly, as compared with those of wounds in control group. Expression levels of AGE and RAGE of skin in diabetes group were higher than those in control group. In diabetes and control groups, especially in diabetes group, the numbers of RAGE-positive cells in wound tissue were more than those in skin tissue. Large amount of inflammatory cells with positive expression of RAGE were observed in diabetes group. MDA level of skin and wound tissue in diabetes group was respectively (6.3 ± 1.0), (7.1 ± 2.4) nmol per milligram protein, which were obviously higher than those in control group [(2.9 ± 1.0), (3.6 ± 1.4) nmol per milligram protein, with t value respectively 8.017, 4.349, P < 0.05 or P < 0.01]. Cell viability and ROS levels in neutrophils were increased in L, M, and H groups [(59 ± 8)%, (77 ± 5)%, (67 ± 6)% and 1.67 ± 0.14, 2.13 ± 0.17, 3.48 ± 0.48] as compared with those in NC group [(34 ± 5)% and 0.58 ± 0.06, with t value respectively 7.195, 14.890, 11.130 and 20.195, 24.905, 16.864, P < 0.05 or P < 0.01].</p><p><b>CONCLUSIONS</b>Abnormal oxidative stress in diabetic skin leads to an atypical origin of wound repair. AGE-RAGE effect is a critical mediator for oxidative stress in diabetic wound tissue during wound healing.</p>

Effect of topical application of aminoguanidine cream on skin tissue of rats with diabetes / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of aminoguanidine cream on the proliferation of keratinocytes (KC), content of advanced glycosylation end products (AGE) and oxidative stress in skin tissue of rats with diabetes.</p><p><b>METHODS</b>Stearic acid, liquid paraffin, vaseline, lanolin, isopropyl myristate fat, glycerol, 50 g/L alcohol paraben, aminoguanidine hydrochloride etc. were mixed in certain proportion to make aminoguanidine cream, and cream without aminoguanidine was used as matrix. The dorsal skin of normal rats were harvested and treated by aminoguanidine cream with dose of 5, 10 g/L, or 5 g/L together with 10 g/L azone. The transdermal effect was respectively measured at post treatment hour 2, 4, 7, 10, 12, 24. Thirty SD rats were divided into normal control (NC, n = 6), diabetes (D, n = 8), aminoguanidine cream-interfered (AI, n = 8), matrix cream-interfered groups (MI, n = 8) according to the random number table. Diabetes was reproduced by intraperitoneal injection of STZ (65 mg/kg) in rats of D, AI, and MI groups, and rats in NC group were injected with 0.05 mmol/L citrate buffer as control. One week later, dorsal skin of rats in AI and MI groups were respectively treated with 10 g/L aminoguanidine cream and matrix cream by external use for 4 weeks. AGE content was determined with fluorescence detection from skin collagen extract. KC cell cycle was detected by flow cytometry. Skin tissue specimens were obtained for determination of levels of superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and total antioxidant capacity. Data were processed with t test.</p><p><b>RESULTS</b>Transdermal effect of aminoguanidine cream with dose of 10 g/L was better than that with 5 g/L or 5 g/L + 10 g/L azone cream. One rat was not induced successfully in MI group. Four weeks after model reproduction, 4 rats died in D group and 1 rat died in AI group. The AGE content in D group was obviously higher than that in NC group [(36.8 +/- 2.6), (24.6 +/- 2.7) U per milligram hydroxyproline, respectively, t = 7.2, P < 0.01], and that in AI group [(28.6 +/- 3.7) U per milligram hydroxyproline] was also lower as compared with that in D group (t = -3.9, P < 0.05). There was no significant difference in AGE content between MI [(32.2 +/- 5.2) U per milligram hydroxyproline] and D groups (t = 1.6, P > 0.05). The percentage of KC in S phase was obviously lower in D group than in NC group [(5.3 +/- 0.6)%, (7.6 +/- 0.9)%, respectively, t = 4.50, P < 0.01], while that in MI group [(9.2 +/- 1.5)%] was higher as compared with that in D group ( t = 4.90, P < 0.01). It was more higher in AI group than in D group on KC percentage in S and G2/M phase (with t value respectively 6.80, 3.17, P values all below 0.01). The oxidative stress indexes of skin tissue in D group were all higher than those in NC group, in which levels of MPO and SOD showed statistical difference (with t value respectively 4.4, 3.7, P values all below 0.05). The oxidative stress indexes were all lower in AI group than in D group, especially in SOD level (t = -1.4, P < 0.05). Levels of MAD, MPO in MI group were significantly lower than those in D group (with t value respectively 2.6, 2.9, P values all below 0.05).</p><p><b>CONCLUSIONS</b>Aminoguanidine cream can promote KC proliferation and appropriately reduce oxidative stress through inhibiting AGE formation to a certain extent in skin tissue of rats with diabetes. Signal use of matrix cream can also reduce oxidative stress in skin tissue of rats with diabetes.</p>

Relationship between cutaneous glycometabolic disorders and cutaneous neuropathy in diabetic rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To analyze the relationship between cutaneous glycometabolic disorders and cutaneous neuropathy in diabetic rats, and to look for the mechanism of neuropathy and impaired wound healing.</p><p><b>METHODS</b>Eighty male SD rats were randomly divided into the normal control group (NC, n = 20), diabetic group (D, n = 20), aminoguanidine-interfered group (AI, n = 20), and insulin-interfered group (II, n = 20) by drawing lots. Diabetes was reproduced in rats of D, AI, and II groups with intraperitoneal injection of streptozotocin (STZ). Then, rats in AI group were fed with 100 mg×kg(-1)×d(-1) aminoguanidine, while rats in II group were subcutaneously injected with insulin for satisfactory control of blood glucose. Changes in mechanical and heat pain thresholds of pad of hind limb were measured at post injection week (PIW) 2, 4, 8. Skin specimens were collected during PIW 2-8 from pads for determination of contents of glucose, advanced glycation end product (AGE), substance P (SP), calcitonin gene-related peptide (CGRP), and observation of distribution and ultrastructure of skin nerve fibers. Data were processed with t test.</p><p><b>RESULTS</b>The mechanical and heat pain thresholds in D group at PIW 2 [(6.3 ± 1.5) g, (6.0 ± 0.9) s, respectively ] were obviously lower than those in NC group [(13.0 ± 3.2) g, (10.3 ± 1.2) s, with t value respectively 2.71, 3.42, P values all below 0.05]. Contents of glucose and AGE in skin tissue in D group were significantly increased when compared with those in NC group, especially at PIW 8 [(2.85 ± 0.33) mg/g, (31.7 ± 3.2) U/mg of hydroxyproline vs. (0.82 ± 0.22) mg/g, (22.2 ± 1.9) U/mg of hydroxyproline, with t value respectively 1.65, 6.47, P values all below 0.01]. The myelinated nerve fibers were edematous and degenerated, with axons compressed, while the unmyelinated nerve fibers were vacuolated, with microfilament and microtubule disorderly arranged. Content of SP in skin tissue in D group was lower as compared with that in NC group, especially at PIW 2 [(16.8 ± 3.4) pg/g vs. (28.5 ± 5.0) pg/g, t = 2.42, P < 0.01]. There was no obvious difference in content of CGRP between NC and D groups, and also in content of glucose in skin between D and AI groups. Compared with those in D group, content of AGE in AI group at PIW 8 was decreased markedly [(27.2 ± 1.4) U/mg of hydroxyproline, t = 3.38, P < 0.05]; contents of glucose and AGE in II group at PIW 8 were significantly decreased [(1.42 ± 0.38) mg/g, (23.6 ± 1.3) U/mg of hydroxyproline, with t value respectively 1.74, 8.17, P < 0.05 or P < 0.01]. Compared with that in D group, contents of SP in AI and II groups were increased, with a delay in time of trough value. Content of CGRP showed no obvious difference among D, AI, and II groups.</p><p><b>CONCLUSIONS</b>High glucose and accumulation of AGE are key mediators of cutaneous neuropathy and impaired wound healing in diabetes mellitus, which confirms that diabetic wound takes an atypical footing during wound repairing. Aminoguanidine and insulin can reduce contents of glucose and AGE in diabetic skin tissue, and ameliorate diabetic cutaneous neuropathy.</p>

Influence of advanced glycosylation end products on wound healing of burn rats with diabetes / 中华烧伤杂志

<p><b>OBJECTIVE</b>To understand the influence of accumulation of advanced glycosylation end products (AGE) on wound healing of burn rats complicated with diabetes.</p><p><b>METHODS</b>Seventy-five SD rats were divided into control, diabetes, and aminoguanidine-interfered groups in completely randomized method, with 25 rats in each group. All rats were subjected to deep partial-thickness scald. Diabetes was reproduced in rats of diabetes and aminoguanidine-interfered groups. Rats in aminoguanidine-interfered group were fed with 100 mg x kg(-1) xd (-1) aminoguanidine. Rats were sacrificed on post-scald day (PSD) 0, 3, 7, 14, and 21, and portrait of the wounds were taken. Full-thickness skin tissue specimens were obtained for determination. Specimens of epidermis from back of SD rats were obtained for KC cultivation and verification. Wound healing rate, glucose content in skin tissue, morphologic change in wound tissue, AGE distribution in skin tissue, influence of AGE on proliferation and apoptosis of KC were observed.</p><p><b>RESULTS</b>Wound healing rate of rats was respectively lower in diabetes group than that in control group on PSD 7, 14, and 21 (P < 0.01), but it was obviously higher in aminoguanidine-interfered group than that in the former 2 groups (P < 0.01). Glucose content of rat skin in diabetes group was (2.62 +/- 0.19) mmol/g, and it was (2.58 +/- 0.07) mmol/g in aminoguanidine-interfered group, both higher than that in control group [(1.04 +/- 0.09) mmol/g, P < 0.01]. In control group, limited intensive infiltration of inflammatory cells was found in the wound with necrotic tissue formation which fell off in time, and with no obvious delay of wound healing. In diabetes group, infiltration of inflammatory cells in wounds of rats appeared slowly, but diffusely and persistently; necrotic tissue formed and fell off late in time, with obvious delay of wound healing. In aminoguanidine-interfered group, intensive infiltration of inflammatory cells was observed in time, and the time of necrotic tissue formation and sloughing, and wound healing were respectively earlier than that in diabetes group. Sporadic disposition of small amount of AGE was found in rats in control group. AGE accumulation increased significantly in rats in diabetes group. AGE content decreased significantly in rats in aminoguanidine-interfered group after administration of aminoguanidine. KC proliferation decreased significantly in concentration dependent manner 48 hours after AGE stimulation. Absorbance value of AGE decreased in each AGE-interfered group (P < 0.01). Early Annexin-V positive apoptotic KC rate was obviously higher in 100 ug/mL AGE-interfered group (15.1 +/- 2.3)% than that in control group [(11.2 +/- 1.2)%, P < 0.05]. There was no statistical significance between 100 ug/mL AGE-interfered group (14.3 +/- 3.5)% and control group (15.2 +/- 2.4)% in respect of the rate of double-positive cells apoptosis at final stage (P > 0.05).</p><p><b>CONCLUSIONS</b>Hyperglycemia may inhibit proliferation of repairing cells such as KC through AGE accumulation, thus impedes wound healing. Reduction of AGE accumulation could ameliorate wound healing delay due to diabetes.</p>

A potential mechanism for impaired wound healing--cutaneous environmental disorders in diabetes mellitus / 中华烧伤杂志

Impaired wound healing in diabetes is a significant clinical problem which is thought to be associated with neuropathy and angiopathy previously . The present study indicates that accumulation of glucose and glycometabolic products in skin tissue, as the result of glycometabolic disorders, which contributes to cutaneous environmental alterations in diabetes mellitus, and subsequently induces the abnormal cell behaviors, cytokine alteration and matrix modification. Thus, diabetic neuropathy and angiopathy might be regarded as the pathological outcome of cutaneous environmental alterations. In conclusion, glycometabolism disorders could be described as one of the initial events for the alteration involving in the underlying cutaneous disorder which impair healing process. The related research focuses on the initial event of controlling disorders in wound healing and therefore contribute to providing the strategy of treatment as based on these approaches.

Effect of advanced glycosylation end products on cell cycle of epidermal keratinocyte and the role of signal pathway / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effect of advanced glycosylation end products (AGE) on cell cycle of epidermal keratinocyte and its possible signal pathway.</p><p><b>METHODS</b>150 mg/L AGE-human serum albumin (AGE-HSA) was prepared in vitro. Primary cultured keratinocytes in logarithmic growth phase were harvested and divided randomly into: A group [with treatment of defined keratinocyte-SFM (DK-SFM) serum-free medium], B group (with treatment of DK-SFM medium including 150 mg/L AGE-HSA), C group (with DK-SFM medium after treatment of U0126) and group D (with D K-SFM medium including 150 mg/L AGE-HSA after treatment of U0126). Cell cycle distributions were analyzed by flow cytometer. The protein levels of cyclin D1, cyclin B1, CDK4 and p44/42 MAPK were measured by Western blot.</p><p><b>RESULTS</b>Compared with those of A group, the percentage of S-phase and G2/M-phase keratinocytes were decreased obviously in B group, the percentages of G2/M -phase keratinocytes showed the same tendency in C and D groups [(9.7 +/- 1.1)% , (9.8 +/- 0.7)%, respectively, P <0.05]. Compared with that of A group, the expression of cyclin D1 were decreased significantly in other groups, among which a weak expression was showed in D group. There was no obvious difference between A and B groups in CDK4, or cyclin B1 and p44/42 MAPK protein levels ,which were significantly higher than those in C and D groups.</p><p><b>CONCLUSION</b>AGEs inhibit the progress of cell cycle of keratinocytes by downregulation of cyclin D1 expression.</p>

Study on the proliferation of epidermal cells of wound edge in deep partial thickness scald injury in rat / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the rule and possible mechanism of epidermal proliferation in wound edge of deep partial thickness scald injury in rat.</p><p><b>METHODS</b>Twenty-four Sprague-Dawley rats inflicted with deep partial thickness scald were randomized into pre-scalding, 3 post-scalding day (PSD), 7PSD and 14PSD groups, with 6 rats in each group. Skin specimens from the wound edge were harvested for the observation of the histological characteristics of the epidermis. Cell cycles of epidermal cells were analyzed with flow cytometry. The expressions of cyclin D1, cyclin B1, cdk4 and the histone H1 kinase activity of MPF in epidermal cells were determined by Western blotting.</p><p><b>RESULTS</b>Augmentation of nuclei and nucleoli was found in the epidermal cells from the wound edge in 3PSD group, while increased number of epidermal cells with obviously augmented nuclei and nucleoli were found in 14PSD group. The percentage of the cells in S phase increased in 14 PSD group. The percentage of epidermal cells in G2/M phase began to increase in 3PSD group, and that in 7PSD (4.5 +/- 0.6) and 14PSD (5.4 +/- 1.0) groups were obviously higher than that in pre-scalding group (2.9 +/- 1.1, P < 0.05). The expression of cyclin D1 increased significantly in 3PSD group. The expression of cdk4 decreased in 3PSD group, but began to increase in 14PSD group. There was no difference in the expression of cyclin B1 among groups. The MPF activity was significantly increased in 14PSD group.</p><p><b>CONCLUSION</b>There was enhanced DNA synthesis and mitosis in epidermal cells of rats with deep partial scald during early post-scald stage, and active proliferation of epidermal cells was observed on 14PSD. The expression of cyclinD1/cdk4 complex and the activity of MPF increased since 14PSD, indicating that there was a special regulative pattern during wound healing.</p>

The influence of L-arginine on the angiogenesis in burn wounds in diabetic rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the possible mechanism of L-arginine supplementation on the angiogenesis of burn wounds in diabetic rats.</p><p><b>METHODS</b>One hundred male Sprague-Dawley (SD) rats were used in the study and were randomly divided into A (scalding control, n = 25), B (scalding of the rats with diabetes, n = 25), C (L-glycine control, n = 25) and D (L-arginine supplementation, n = 25) groups. Diabetes was produced by intra-peritoneal injection of streptozotocin (STZ) in B, C and D groups. The rats in C and D groups were gavaged with L-glycine and L-arginine in dose of 200 mg.kg(-1).d(-1), respectively. The glucose content of the back skin tissue was determined for five rats in each group eight weeks after STZ administration. Deep partial thickness scalding of 20% TBSA was engendered on the back in the other 80 rats. The wound area, wound healing rate, and microvascular density with CD34 immunohistochemistry staining were determined on 3rd, 7th, 14th, and 21st post scalding days (PSDs), In addition, the amount of nitric oxide (NO) released from the wound tissue and the tissue contents of vascular endothelial growth factor (VEGF) and transforming growth factor beta1 (TGF-beta1) from wound were determined at the above time points.</p><p><b>RESULTS</b>Compared to those in group B, the wound healing rate in group D increased significantly since the 7th PSD [(44.10 +/- 3.50)%, P < 0.05], and the wound MVD value was increased significantly at all postburn time points. Furthermore, the levels of VEGF, NO and TGF-beta1 in the wound tissue was also increased significantly, while the glucose content in the cutaneous tissue was decreased to (1.380 +/- 0.120) mg/g.</p><p><b>CONCLUSION</b>L-arginine supplementation could be beneficial to the angiogenesis in the burn wound of the rats with diabetes, as well as to wound healing by increasing the synthesis and the release of VEGF, NO and TGF-beta1 from burn wound and by decreasing the glucose content in the cutaneous tissue of diabetic rats.</p>

Effects of advanced glycation end-products on skin keratinocytes by NF-?B activation / 中华创伤杂志

Objective To investigate the effects of advanced glycation end-products(AGEPs)on the function of normal keratinocytes in vitro so as to explore the role of AGEPs in impaired wound healing. Methods Normal rat keratinocytes were incubated with different concentrations of AGEPs.After 48 hours of culturing,the cell proliferation rates were measured by MTT colorimetric determination.The cell cycle distributions and apoptosis were analyzed with flow cytometry,and the migration was investigated by 24-well fluorimetric cell migration assay kit by exposing to 100?g/ml AGEPs.Nuclear extracts from these cells were examined for binding of nucleotides containing NF-?B consensus by immunocytochemistry and EMSA in vitro.Results The proliferations of normal keratinocytes were significantly arrested and many cells were induced to early apoptosis compared with control ones(P＜0.05)by exposing to AGEPs for 48 hours. Meanwhile AGEPs also irritated keratinocytes migration compared with control ones(P＜0.05).Inhibiting the activation of NF-?B could partly recover the proliferation of keratinocytes,reverse apoptosis and attenu- ate migration.Conclusion AGEPs are correlated with the migration,proliferation and apoptosis of kera- tinocytes by NF-?B.