基于伏毒理论研究糖尿病大鼠皮肤溃疡难愈机制  

Study on Mechanism of Refractory Skin Ulcer in Diabetic Rats Based on Theory of Latent Toxins

作　　者：王双勋[1,2] 李大勇[2] WANG Shuangxu;LI Dayong(Liaoning University of Traditional Chinese Medicine,Shenyang 110847,Liaoning,China;The First Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110032,Liaoning,China)

机构地区：[1]辽宁中医药大学,辽宁沈阳110847 [2]辽宁中医药大学附属医院,辽宁沈阳110032

出　　处：《辽宁中医杂志》2023年第8期225-229,I0006,共6页Liaoning Journal of Traditional Chinese Medicine

基　　金：国家重大科技专项(2019YFC1709303);中国人口福利基金项目(FY202107140413)。

摘　　要：目的观察正常大鼠和糖尿病大鼠皮肤溃疡愈合过程中肉芽组织晚期糖基化终末产物(AGEs)、晚期糖基化终末产物受体(RAGE),Kelch样环氧氯丙烷相关蛋白-1(Keap1)/核因子E2相关因子(Nrf2)通路和Wnt/β-连环蛋白(β-catenin)通路的变化,以研究糖尿病皮肤溃疡难愈的机制。方法Wistar雄性大鼠采用链脲佐菌素腹腔注射结合喂食高脂高糖饲料方法造糖尿病模型。28 d后,正常对照组和糖尿病组造背部皮肤溃疡模型。分别在第7、14天取溃疡肉芽组织。采用苏木素-伊红(HE)染色法检测肉芽组织病理变化;酶联免疫吸附测定(ELISA)法检测肉芽组织AGEs、RAGE、Wnt1、β-catenin、糖原合成酶激-3β(GSK-3β)蛋白含量;蛋白质印迹法(Western blot)法检测肉芽组织Keap1、Nrf2蛋白表达;逆转录-聚合酶链反应(RT-PCR)法检测肉芽组织RAGE、Keap1、Nrf2、Wnt1、β-catenin、GSK-3βmRNA表达水平。结果相比于正常对照组,糖尿病组大鼠肉芽组织中炎症细胞较多,成纤维细胞、胶原纤维和毛细血管数量较少。第7天的Wnt1、β-catenin蛋白含量均数上有差异,但无统计学意义(P>0.05),第14天的Wnt1、β-catenin蛋白含量低(P<0.05)。第7、14天的Wnt1、β-catenin mRNA表达水平低(P<0.01)。Nrf2蛋白和mRNA含量较低(P<0.01)。AGEs、RAGE、Keap1、GSK-3β蛋白和mRNA含量较高(P<0.01)。结论具有伏毒性质的AGEs在组织中的沉积,可能抑制了Keap1/Nrf2通路和Wnt/β-catenin通路,这可能是导致了糖尿病大鼠皮肤溃疡难愈合的原因之一。Objective The changes of advanced glycation end products(AGEs),receptor for advanced glycation end products(RAGE),Kelch like epichlorohydrin related protein-1(Keap1)/nuclear factor E2 related factor(Nrf2)pathway and Wnt/β-catenin pathway in granulation tissue of normal and diabetic rats during skin ulcer healing were observed to study the mechanism of refractory diabetic skin ulcer.Methods Wistar male rats were given streptozotocin intraperitoneal injection combined with high-fat and high-sugar diet.After 28 days,the normal control group and diabetic group were set up the skin ulcer model.The granulation tissue was collected on day 7 and 14.The pathological changes of granulation tissue were detected by hematoxylin-eosin(HE)staining.The protein contents of AGEs,RAGE,Wnt1,β-catenin and glycogen synthase excite-3β(GSK-3β)in granulation tissue were determined by enzyme linked immunosorbent assay(ELISA).The protein expression of Keap1 and Nrf2 in granulation tissue was detected by Western blot.The mRNA expression levels of RAGE,Keap1,Nrf2,Wnt1,β-catenin and GSK-3β in granulated tissues were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Compared with the normal control group,the diabetic group had more inflammatory cells and less fibroblasts,collagen fibers and capillaries in granulation tissue.There were no significant differences in the contents of Wnt1 orβ-catenin protein on day 7(P>0.05),and low contents of Wnt1 andβ-catenin protein on day 14(P<0.05).The mRNA expression levels of Wnt1 andβ-catenin on day 7 and 14 were low(P<0.01).Nrf2 protein and mRNA contents were low(P<0.01).The levels of AGEs,RAGE,Keap1 and GSK-3βprotein and mRNA were higher(P<0.01).Conclusion The deposition of AGEs with latent toxin in tissues may inhibit the Keap1/Nrf2 pathway and Wnt/β-catenin pathway,which may be one of the reasons for the refractory healing of diabetic skin ulcer.

关 键 词：糖尿病皮肤溃疡 AGES RAGE Keap1/Nrf2通路 WNT/Β-CATENIN通路 

分 类 号：R587.2[医药卫生—内分泌]