Objective To evaluate the effects of turmeric volatile oil (TVO) combined with cisplatin on the proliferation and apoptosis of a human cutaneous squamous cell carcinoma cell line A431,and to explore their mechanisms.Methods Some cultured A431 cells at exponential growth phase were divided into several groups to be treated with 5,10,20,40 and 80 mg/L TVO,as well as high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 1％ dimethyl sulfoxide (DMSO,control group),respectively.After 24-hour treatment,cell counting kit 8 (CCK8) assay was performed to estimate the proliferative activity of A431 cells in the above groups.Some other A431 cells were divided into 4 groups:control group treated with high-glucose DMEM containing 1％ DMSO,TVO group treated with 40 mg/LTVO,cisplatin group treated with 10 mg/L cisplatin,and TVO + cisplatin group treated with 40 mg/L TVO and 10 mg/L cisplatin.After 24-hour treatment,CCK8 assay was performed to estimate the cellular proliferative activity,inverted microscopy to observe changes in cell morphology,fluorescence microscopy to detect cell apoptosis after acridine orange (AO)/ethidium bromide (EB) double-staining,colorimetry to evaluate the activity of Caspase-3 and Caspase-9,and Western blot analysis to determine the protein expression of Caspase-3 and p-glycoprotein.Results After 24-hour treatment with 5,10,20,40 and 80 mg/L TVO,the cell proliferation rates were inhibited by (12.83 ± 6.4)％,(16.27 ± 11.4)％,(21.61 ± 9.1)％,(33.11 ± 2.0)％ and (46.00 ± 3.3)％ respectively,and the inhibition rates were all significantly higher in these groups than in the control group (4.03％ ± 1.4％,all P ＜ 0.05).The 50％ inhibitory concentration (IC50) of TVO at 24 hours was (61.66 ± 1.03) mg/L.Compared with the control group,the proliferation inhibition rates significantly increased in the TVO group,cisplatin group and TVO + cisplatin group (all P ＜ 0.05),suggesting that the combination of TVO and cisplatin showed synergistic inhibitory effects with a combination index of 1.366.Moreover,A431 cells turned round to different extents and became apoptotic in the TVO group and cisplatin group,and the TVO + cisplatin group showed obviously decreased number of cells and a large number of cell debris.The TVO + cisplatin group also showed significantly increased activity of Caspase-3 (1.520 ± 0.115) and Caspase-9 (2.760 ± 0.297) as well as protein expression of Caspase-3 (1.482 ± 0.016) compared with the TVO group (Caspase-3 activity:1.117 ± 0.095;Caspase-9 activity:1.259 ± 0.059;Caspase-3 protein expression:1.156 ± 0.006,all P ＜ 0.01) and cisplatin group (Caspase-3 activity:1.381 ± 0.089;Caspase-9 activity:1.829 ± 0.171;Caspase-3 protein expression:1.296 ± 0.021,all P ＜ 0.01),but significantly decreased p-glycoprotein expression (0.528 ± 0.014) compared with the TVO group (1.311 ± 0.011,P ＜ 0.01) and cisplatin group (1.169 ± 0.012,P ＜ 0.01).Conclusion TVO combined with cisplatin can synergistically inhibit the proliferation of A431 cells and induce cell apoptosis,which may be associated with activation of the caspase system and decreased expression of pglycoprotein.

OBJECTIVETo 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.

METHODSHistological 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.

RESULTSCompared 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].

CONCLUSIONSAbnormal 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.

Aged ; Case-Control Studies ; Diabetes Mellitus, Type 2 ; metabolism ; pathology ; Female ; Glycation End Products, Advanced ; metabolism ; Humans ; Male ; Middle Aged ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; metabolism ; Serum Albumin ; metabolism ; Serum Albumin, Human ; Skin ; metabolism ; pathology ; Wound Healing

OBJECTIVETo 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.

METHODSStearic 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.

RESULTSTransdermal 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).

CONCLUSIONSAminoguanidine 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.

Administration, Cutaneous ; Animals ; Cell Proliferation ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; Glycation End Products, Advanced ; metabolism ; Guanidines ; administration & dosage ; pharmacology ; Keratinocytes ; drug effects ; Male ; Ointments ; administration & dosage ; pharmacology ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; Skin ; drug effects ; metabolism ; pathology

Objective·To investigate the influence of blocking receptor for advanced glycation end products (RAGE) on macrophages infiltration in diabetic wound healing. Methods·Ninety-six male C57BL/6J mice (8-week-old) were divided into diabetic group (n=72) and normal group (n=24) randomly. Diabetic mice were induced by streptozocin multiple intraperitoneal injection. One full-thickness excisional wound (diameter of 9 mm) was created by a sterilized punch. Diabetic mice were divided into 3 groups in which different topical treatments were applied to the wounds. Anti-RAGE antibody were applied in group R, rabbit IgG applied in group I, normal saline applied in group C. Normal mice were applied with saline topically (group N). All treatments were repeated on day 3 and day 7 after wounded. The wound healing conditions were observed. The wound and surrounding tissues from animals in each group were excised on day 1, 3, and 7 after wounded. Immunohistochemistry was utilized to investigate the changes of macrophages infiltration in quantity. Macrophages were also analyzed with respect to morphology by transmission electron microscopy (TEM). Results·① The wound closure ratio of group R was higher than those of group C and group I on day 14 after being wounded (P=0.000). ② On day 1, the numbers of macrophages in group R and group N were both bigger than those of group C and group I, but smaller on day 14 (P=0.000). ③ The morphological characters of macrophages also existed great differences under TEM. Conclusion·Number and morphology of macrophages are both abnormal in diabetic wound in a RAGE pathway depending manner. Based on macrophages, it suggests that impaired healing of diabetic wound is closely related to RAGE pathway.