桑黄素下调KLF5表达改善糖尿病血管炎性病变  

Morin improves diabetic inflammatory angiopathy by down-regulating KLF5 expression

作　　者：周静[1] 杨高山 聂子元[3,4] 张龙 秦岩 ZHOU Jing;YANG Gaoshan;NIE Ziyuan;ZHANG Long;QIN Yan(Department of Endocrinology,Second Hospital of Hebei Medical University,Shijiazhuang 050000,Hebei,China;Department of Biochemistry and Molecular Biology,School of Basic Medical Sciences,Hebei University of Chinese Medicine,Shijiazhuang 050000,Hebei,China;Department of Hematology,Second Hospital of Hebei Medical University,Shijiazhuang 050000,Hebei,China;Hebei Provincial Key Laboratory for Hematology,Shijiazhuang 050000,Hebei,China;Central Laboratory,Affiliated Hospital of Hebei University,Hebei Provincial Key Laboratory for Radiotherapy and Chemotherapy Mechanisms and Regulations,Baoding 071000,Hebei,China)

机构地区：[1]河北医科大学第二医院内分泌科,河北石家庄050000 [2]河北中医学院基础医学院生物化学与分子生物学教研室,河北石家庄050000 [3]河北医科大学第二医院血液内科,河北石家庄050000 [4]河北省血液病重点实验室,河北石家庄050000 [5]河北大学附属医院中心实验室,河北省放化疗机制与规程研究重点实验室,河北保定071000

出　　处：《上海中医药大学学报》2020年第1期57-64,共8页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：河北省中医药管理局科研计划项目(2018127)。

摘　　要：目的:研究桑黄素对糖尿病血管炎性病变的作用及其分子机制。方法:①选取24只db/db小鼠,随机分为模型组(高糖高脂饲料)12只和桑黄素治疗组(高糖高脂饲料+腹腔注射40 mg/kg桑黄素)12只,另设12只C57BL/6J小鼠作为正常对照组(正常饲料)。各组均给予相应干预,连续12周。末次给药后,分离腹主动脉。HE染色后光镜下观察血管组织形态学变化,PCR检测血管组织Krüppel样因子5(KLF5)和白介素-6(IL-6)的mRNA表达水平。②采用不同浓度的葡萄糖(5.5、10、15、25 mmol/L)刺激血管平滑肌细胞(VSMCs)24 h,或高浓度葡萄糖(25 mmol/L)处理VSMCs不同时间(0、6、12、24 h),Western blot检测细胞KLF5和IL-6蛋白表达水平。③采用不同浓度(0、5、10、50、100、200μmol/L)桑黄素干预VSMCs细胞24 h后,MTT法检测并计算细胞生长抑制率。④将VSMCs细胞分为空白组、高糖组、桑黄素组和高糖+桑黄素组。先给予高糖组和高糖+桑黄素组细胞25 mmol/L葡萄糖刺激24 h后,再给予桑黄素组和高糖+桑黄素组细胞10μmol/L桑黄素干预24 h,Western blot检测各组细胞KLF5和IL-6蛋白表达水平。⑤应用转染技术构建KLF5过表达的VSMCs细胞,将细胞分为空白组、KLF5过表达组、桑黄素组和KLF5过表达+桑黄素组,细胞转染6 h后,桑黄素组和KLF5过表达+桑黄素组细胞给予10μmol/L桑黄素干预24 h,Western blot检测各组细胞KLF5和IL-6蛋白表达水平。结果:①与正常对照组相比,模型组小鼠出现明显的血管平滑肌细胞增生及紊乱,并有斑块形成;且血管组织KLF5和IL-6的mRNA表达量显著增加(P<0.01)。桑黄素能有效抑制糖尿病小鼠的血管病变,并显著下调KLF5和IL-6的mRNA表达量(P<0.01)。②葡萄糖可以诱导VSMCs细胞KLF5和IL-6蛋白表达增加(P<0.01),且呈浓度和时间依赖性。选择25 mmol/L葡萄糖干预24 h作为模型诱导条件。③随着桑黄素浓度增加,VSMCs细胞的生长抑制率显著增高(P<0.01)。选择10�Objective:To study the effects and molecular mechanisms of morin on diabetic inflammatory angiopathy. Methods:①Twenty-four db/db mice were randomly divided into two groups,12 mice in the model group(fed with high-sugar and high-fat diet) and 12 mice in the morin treatment group(fed with high-sugar and high-fat diet and treated with 40 mg/kg morin by intraperitoneal injection). Another twelve C57 BL/6 J mice were taken as the normal control group(fed with normal diet). Each group was treated with the corresponding intervention for 12 weeks. After the last administration, the abdominal aorta was isolated. The morphological changes of vascular tissue were observed under light microscope after HE staining. The mRNA expressions of Krüppel-like factor 5(KLF5) and interleukin-6(IL-6) in vascular tissue were detected by PCR. ②The vascular smooth muscle cells(VSMCs) were stimulated with glucose at different concentrations(5.5, 10, 15, 25 mmol/L) for 24 hours, or treated with glucose at high concentration(25 mmol/L) for different time(0, 6, 12, 24 h).The protein expressions of KLF5 and IL-6 were detected by Western blot. ③The VSMCs were treated with morin at different concentrations(0, 5, 10, 50, 100, 200 μmol/L) for 24 hours, the growth inhibition rate was detected and calculated by MTT assay. ④VSMCs were divided into the blank group, high glucose group, morin group and high glucose+morin group. The cells in the high glucose group and the high glucose+morin group were stimulated with 25 mmol/L glucose for 24 hours, and then the cells in the morin group and the high glucose+morin group were further treated with 10 μmol/L morin for 24 hours. The protein expressions of KLF5 and IL-6 in each group were detected by Western blot. ⑤KLF5 overexpressed VSMCs were constructed by transfection technique. The cells were divided into the blank group, KLF5 overexpression group, morin group and KLF5 overexpression+morin group. After transfection for 6 hours, the morin group and the KLF5 overexpression+morin group were treate

关 键 词：桑黄素 糖尿病 血管病变 KLF5 IL-6 DB/DB小鼠 血管平滑肌细胞 

分 类 号：R285.5[医药卫生—中药学]