摘要
目的:采用定量DCE-MRI评价外源性硫化氢(H2S)干预糖尿病重症肢体缺血兔骨髓血管内皮细胞氧化应激水平.方法:将15只糖尿病合并重症肢体缺血模型兔随机分为DM+CLI+Saline组(n=7)、DM+CLI+NaHS组(n=8),另设置空白对照组(NC组,n=6).连续3周予DM+CLI+NaHS组实验兔耳缘静脉注射NaHS溶液(30 μmol/kg/d).分别于造模后第0、1、2、3周对各组实验兔行MRI右侧股骨斜冠状位DCE-MRI序列扫描,通过后处理计算得到右股骨上段骨髓的血容量转移常数(Ktrans)、速率常数(Kep)和对比剂血管外细胞外间隙的容积(Ve)等定量参数.取第3周各组实验兔股骨上段行CD31免疫组化染色,计算骨髓微血管密度(MVD),并检测造模后第3周原代骨髓内皮祖细胞(EPCs)内活性氧(ROS)水平、骨髓组织超氧化物歧化酶(SOD)水平、血清可溶性细胞间粘附分子1(sICAM-1)、血管生成素-1(Ang-1)水平和骨髓来源EPCs迁移和成管功能.结果:外源性H2S干预第2、3周股骨上段骨髓Ktrans、Ve和Kep值较未干预组减低(P＜0.05),股骨上段MVD较未干预组增加(P＜0.05).干预第3周骨髓EPCs胞内ROS水平下降,骨髓组织SOD水平升高,血清sICAM-1显著降低,血清Ang-1浓度显著增加,且骨髓来源EPCs迁移和成管功能上调(P＜0.05).第3周股骨上段骨髓MVD与Ktrans值呈负相关(r=-0.780,P＜0.05)、与Kep值和Ve值呈正相关(r=0.808、0.889,P＜0.05),骨髓内皮祖细胞ROS阳性率与股骨上段骨髓Ktras值、Kep值、Ve值呈正相关(r=0.714、0.872、0.877,P＜0.05),而与骨髓组织 SOD 水平呈负相关(r=-0.754、-0.856、-0.880,P＜0.05).血清sICAM-1、Ang-1水平与股骨上段骨髓Ktrans、Kep、Ve值之间均无显著相关关系(P＞0.05).结论:利用DCE-MRI定量参数评价外源性H2S干预兔糖尿病重症肢体缺血骨髓氧化应激水平是可行的,外源性H2S通过降低骨髓血管内皮细胞氧化应激水平,降低骨髓微血管渗透性.
Abstract
Objective:To evaluate the effects of H2S on oxidative stress levels in bone marrow vascular endothelial cells using DCE-MRI in diabetic rabbits with critical limb ischemia.Methods:Fif-teen diabetic rabbits with severe limb ischemia were randomly divided into three groups:DM+CLI+Saline group(n=7),DM+CLI+NaHS group(n=8),and blank control group(NC group,n=6).Rabbits in the DM+CLI+NaHS group were injected with NaHS solution(30μmol/kg/d)through the auricular vein for 3 consecutive weeks.The right femur oblique coronal DCE-MRI sequential scanning was performed at 0,1,2 and 3 weeks after modeling.Quantitative parameters,including volume trans-fer constant(Ktrans),efflux rate constant(Kep),and extracellular extra-vascular volume fraction(Ve)of the right upper femoral bone marrow,were computed through post-processing.CD31 performed on the upper femur of each group at week 3.Bone marrow microvascular density(MVD)was calculated.The levels of reactive oxygen species(ROS)in primary bone marrow endothelial progenitor cells(EPCs),superoxide diastase(SOD)in bone marrow tissue,serum soluble inter-cellular adhesion mole-cule-1(sICAM-1),Angiopoietin-1(Ang-1),migration and tubular function of bone marrow-derived EPCs were measured at 3 weeks after model creation.Results:The values of Ktrans,Ve,and Kep of bone marrow in the upper femur were lower than those in the non-intervention group at the 2nd and 3rd weeks after exogenous H2S intervention(P＜0.05),and the MVD in the upper femur was increased compared with those in the non-intervention group(P＜0.05).At the 3rd week of intervention,the intracellular ROS level of bone marrow EPCs decreased,SOD level of bone marrow tissue increased,serum sICAM-1 decreased significantly,serum Ang-1 concentration increased significantly,and migra-tion and tubular function of bone marrow derived EPCs were up-regulated(P＜0.05).At the 3rd week,bone marrow MVD in the upper femoral segment was negatively correlated with Ktrans value(r=-0.780,P＜0.05),positively correlated with Kep value and Ve value(r=0.808,0.889,P＜0.05).The positive rate of ROS in bone marrow endothelial progenitor cells was positively correlated with Ktrans value,Kep value and Ve value in the bone marrow of the upper femur(r=0.714,0.872,0.877,P＜0.05),but negatively correlated with SOD level in the bone marrow(r=-0.754,-0.856,-0.880,P＜0.05).There was no significant correlation between serum sICAM-1 and Ang-1 levels and Ktrans,Kep and Ve values in bone marrow of upper femur(P＞0.05).Conclusion:It is feasible to use DCE-MRI quantitative MRI to evaluate the intevention of exogenous H2S on bone marrow oxidative stress level changes in diabetic rabbit with critical limb ischemia.Exogenous H2S reduces the bone marrow microvascular permeability by reducing the oxidative stress level of bone marrow vascular endothelial cells.
基金项目
国家自然科学基金(82171895)
国家自然科学基金(81871332)
武汉大学人民医院交叉创新人才项目(JCRCZN-2022-013)
万方数据