摘要
目的探讨三黄生肤油对糖尿病大鼠末梢循环障碍和足溃疡的作用及相关机制。方法（1）取25只Wistar大鼠按照随机数字表法分为非糖尿病组、糖尿病假治疗组、二甲双胍组、三黄生肤油组、联合治疗组，每组5只。非糖尿病组注射柠檬酸钠缓冲液，其余4组大鼠注射10mg／mL链脲佐菌素诱导建立糖尿病模型。注射后3周，5组大鼠均行足部低温处理制备末梢循环障碍模型。注射后第9—12周，非糖尿病组和糖尿病假治疗组大鼠均以生理盐水灌胃，双后足底涂抹麻油；二甲双胍组大鼠以稀释的二甲双胍灌胃，双后足底涂抹麻油；三黄生肤油组大鼠以生理盐水灌胃，双后足底涂抹三黄生肤油；联合治疗组大鼠以稀释的二甲双胍灌胃，双后足底涂抹三黄生肤油。注射后第9周治疗前（以下称治疗前）及治疗1、2、3周，采用红外测温仪及足底测试仪分别测定大鼠双后足底温度和热痛阈值。（2）另取25只大鼠同前分组并建立糖尿病模型（非糖尿病组除外），注射后第9周再行左后足底水蒸气烫伤制备足溃疡模型，并给予相应药物治疗。治疗3、7、21、35d，观察创面大体情况及面积变化。治疗35d处死大鼠，取创面组织行HE染色观察组织形态学及免疫组织化学染色检测环氧化酶2（COX-2）及血管内皮生长因子（VEGF）的表达。对数据行重复测量方差分析、单因素方差分析、Bonferroni事后检验。结果（1）糖尿病末梢循环障碍实验。除治疗2周时联合治疗组及治疗3周时二甲双胍组、三黄生肤油组、联合治疗组（t值为0．258～2．647，P值均大于0．05），各时相点4组糖尿病大鼠足底温度均明显低于非糖尿病组（t值为2．811—6．066，P值均小于0．05）。除治疗2、3周联合治疗组大鼠足底温度明显高于糖尿病假治疗组（t值分别为3．419和2．863，P值均小于0．05），各时相点二甲双胍组、三黄生肤油组、联合治疗组大鼠足底温度与糖尿病假治疗组相近（t值为0．128～1．654，P值均大于0．05）。治疗前4组糖尿病大鼠及治疗1周时三黄生肤油组大鼠足底热痛阈值明显高于非糖尿病组（t值为2．836～4．456，P值均小于0．05），各时相点二甲双胍组、三黄生肤油组、联合治疗组大鼠足底热痛阈值与糖尿病假治疗组相近（t值为0．312～1．611，P值均大于0．05）。（2）糖尿病足溃疡实验。治疗3d，各组大鼠创面均水肿。治疗7d，各组大鼠创面继续扩大，红肿明显并结痂。治疗21d，非糖尿病组、三黄生肤油组、联合治疗组大鼠创面结痂基本脱落；糖尿病假治疗组大鼠创面皮肤依然肿胀；二甲双胍组大鼠创面结痂尚未脱落，创缘皮肤暗红。治疗35d，非糖尿病组、三黄生肤油组、联合治疗组大鼠创面基本愈合；糖尿病假治疗组、二甲双胍组大鼠创面依然肿胀，创面结痂尚未完全脱落。治疗3、7d，4组糖尿病大鼠创面面积与非糖尿病组相近（t值为0．111～1．476，P值均大于0．05）。治疗21d，糖尿病假治疗组大鼠创面面积明显大于非糖尿病组（t=5．502，P〈0．01），其余3组糖尿病大鼠创面面积与非糖尿病组相近（t值为0．544～1．676，P值均大于0．05）；二甲双胍组大鼠创面面积与糖尿病假治疗组相近（t=1．895，P〉0．05），三黄生肤油组、联合治疗组大鼠创面面积明显小于糖尿病假治疗组（￡值分别为5．809、3．426，P〈0．05或P〈0．01）。治疗35d，糖尿病假治疗组和二甲双胍组大鼠创面面积明显大于非糖尿病组（t值分别为8．495、4．108，P值均小于0．01），三黄生肤油组和联合治疗组大鼠创面面积与非糖尿病组相近（t值分别为0．291、2．195，P值均大于0．05）；二甲双胍组大鼠创面面积与糖尿病假治疗组相近（t=0．897，P〉0．05），三黄生肤油组和联合治疗组大鼠创面面积明显小于糖尿病假治疗组（t值分别为6．923、6．583，P值均小于0．01）。治疗35d，与糖尿病假治疗组和二甲双胍组相比，非糖尿病组、三黄生肤油组、联合治疗组大鼠创面皮肤结构较为完整，真皮层炎性细胞浸润较少，创缘处胶原纤维排列较整齐。治疗35d，与糖尿病假治疗组[COX-2表达为（222±89）％，VEGF表达为（55±12）％]比较，二甲双胍组大鼠创面COX-2、VEGF表达[分别为（137±24）％、（94±36）％]无明显差异（t值分别为3．046、2．653，P值均大于0．05）；非糖尿病组、三黄生肤油组及联合治疗组大鼠创面COX-2表达[分别为（100±35）％、（91±42）％、（109±17）％]明显下调（t值为4．039～4．653，P值均小于0．01），VEGF表达[分别为（100±28）％、（143±12）％、（120±13）％]明显上调（t值为3．363～5．905，P〈0．05或P〈0．01）。结论三黄生肤油可改善糖尿病末梢循环障碍引起的大鼠足部皮温降低及痛觉迟钝；且可通过下调创面组织中COX-2表达、上调创面组织中VEGF表达，促进大鼠糖尿病足溃疡的愈合。
Objective To observe the effects of San-huang-sheng-fu oil （S） on peripheral circulatory disorders and foot ulcers in diabetic rats and the relevant mechanisms. Methods （ 1 ） Twenty-five Wistar rats were divided into non-diabetes （N） , diabetes and sham treatment （DS） , metformin （ M ） , S, and combined treatment （CT） groups according to the random number table, with 5 rats in each group. Rats in group N were injected with sodium citrate buffer solution, while rats in the other 4 groups were injected with 10 mg/mL streptozotocin to induce diabetes. In post injection week （PIW） 3, feet of rats in all the 5 groups received an ice-cold stimulation to induce peripheral circulatory disorders. From PIW 9 to 12, rats in groups N and DS were gavaged with saline and applied with sesame oil on pelma of both hind limbs ; rats in group M were gavaged with diluted M and applied with sesame oil on pelma of both hind limbs; rats in group S were garaged with saline and applied with S on pelma of both hind limbs; rats in group CT were gavaged with diluted M and applied with S on pelma of both hind limbs. In PIW 9 before treatment （ hereinafter referred to as before treatment） and post treatment week （PTW） 1, 2, and 3, plantar temperature and hot pain threshold of rats were detected by infrared thermometer and foot tester respectively. （2） Another 25 rats were divided and induced with diabetes （expect for group N） as above. In PIW 9, rats in the 5 groups were inflicted with foot ulcer in the left pelma of hind limb by steam and received the corresponding treatment. On post treatment day （PTD） 3, 7, 21, and 35, the general condition and area of wounds were observed and measured respectively. All the rats were sacrificed on PTD 35, and wound tissue was collected for histomorphological observation and determination of expressions of cyclooxygenase-2 （COX-2） and vascular endothelial growth factor （VEGF） using HE staining and immunohistochemical staining respectively. Data were processed with analysis of variance for repeated measurement, one-way analysis of variance, and Bonferroni post hoc test. Results （ 1 ） The experiment of peripheral circulatory disorders in diabetes. Compared with the plantar temperature of rats in group N, except for that in group CT in PTW 2 and groups M, S, and CT in PTW 3 （ with t values from 0. 258 to 2. 647, P values above 0.05 ） , the plantar temperature of rats with diabetes in the 4 groups at each time point was lowered significantly （ with t values from 2.811 to 6. 066, P values below 0.05 ）. Compared with the plantar temperature of rats in group DS, except for that in group CT in PTW 2 and 3 significantly increased （ with t values respectively 3. 419 and 2. 863, P values below 0.05 ） , the plantar temperature of rats in groups M, S, and CT showed no significant difference at each time point （ with t values from 0. 128 to 1. 654, P values above 0.05 ）. The plantar hot pain threshold of rats was significantly decreased in group N than in the other 4 groups before treatment and group S in PTW 1 （with t values from 2. 836 to 4. 456, P values below 0.05 ）. The plantar hot pain thresholds of rats in groups M, S, and CT were close to the hot pain threshold in group D S （with t values from 0.312 to 1.611, P values above 0.05 ）. （2） The experiment of diabetic foot ulcers. Edema existed in all the wounds of rats on PTD 3. The wound areas of all the rats continued to increase with swelling and scar formation on PTD 7. On PTD 21, the scar of rats in groups N, S, and CT fell off; the wounds of rats in group DS were still swollen; scar of rats did not fall off with dark red in the skin around the wound in group M. On PTD 35, wounds of rats in groups N, S, and CT were nearly healed; while wounds of rats in groups DS and M were still swollen and the scar around the wound failed to fall off. On PTD 3 and 7, the wound areas of rats with diabetes in the 4 groups were close to those in group N （ with t values from 0.111 to 1. 476, P values above 0.05 ）. On PTD 21, the wound area of rats in group DS was significantly larger than that in group N （ t = 5. 502, P 〈 0.01 ） , while the wound areas of rats with diabetes in the other 3 groups were close to the area in group N （ with t values from 0. 544 to 1. 676, P values above 0.05 ）. On PTD 21, the wound area of rats in group M was close to that in group DS （ t = 1. 895, P 〉 0.05 ） , while the wound areas of rats in groups S and CT were significantly smaller than the area in group DS （with t values respectively 5. 809 and 3. 426, P 〈 0.05 or P 〈 0.01 ）. On PTD 35, the wound areas of rats in groups DS and M were significantly larger than the area in group N （ with t values respectively 8. 495 and 4. 108, P values below 0.01 ） , while the wound areas of rats in groups S and CT were close to the area in group N （with t values respectively 0. 291 and 2. 195, P values above 0.05 ）. On PTD 35, the wound area of rats in group M was close to that in group DS （ t =0.897, P 〉0.05）; while the wound areas of rats in groups S and CT were significantly smaller than the area in group DS （ with t values respectively 6. 923 and 6. 583, P values below 0. 01 ）. On PTD 35, the structures of wound tissue were in better integrity with less inflammatory cells and more regularly arranged collagen fibers around the wounds of rats in groups N, S, and CT than in groups DS and M. On PTD 35, the expression levels of COX-2 and VEGF in the wounds of rats in group DS [respectively （222 ± 89）% and （55 ± 12）% ] were close to those in group M [ respectively （ 137 ± 24） % and （94 ± 36） % , with t values respectively 3. 046 and 2. 653, P values above 0.051] On PTD 35, the expression level of COX-2 in the wounds of rats in group DS was significantly higher than the expression levels of COX-2 in groups N, S, and CT [ respectively （ 100 ± 35 ） % , （91±42）% , and （109 ± 17）% , with t values from 4. 039 to 4. 653, P values below 0.01 ] , while the expression level of VEGF in the wounds of rats in group DS was significantly lower than the expression levels of VEGF in groups N, S, and CT [ respectively （100 ±28） % , （143± 12） % , and （120 ± 13） % , with t val- ues from 3.363 to 5.905, P 〈0.05 orP 〈0.01]. Conclusions S can improve the plantar temperature decrease and pain dysesthesia of rats caused by diabetic peripheral circulatory disorders. It also can promote wound healing of diabetic foot ulcers in rats with down-regulation of COX-2 and up-regulation of VEGF.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2016年第3期168-175,共8页
Chinese Journal of Burns
基金
国家自然科学基金（81373842）
湖北省自然科学基金（2013CFB451、2015CFB496）
关键词
糖尿病
糖尿病足
伤口愈合
末梢循环障碍
三黄生肤油
Diabetes mellitus
Diabetic foot
Wound healing
Peripheral circulatory disorders
San-huang-sheng-fu oil
