基于AKAP1/Drp1信号通路探讨当归补血汤对糖尿病肾病大鼠足细胞线粒体分裂及凋亡的影响  

Danggui Buxuetang Inhibits Mitochondrial Fission and Apoptosis of Podocytes in Rat of Diabetic Kidney Disease via AKAP1/Drp1 Signaling Pathway

作　　者：顾悦 郑琳琳 郭登洲 GU Yue;ZHENG Linlin;GUO Dengzhou(Hebei University of Chinese Medicine,Shijiazhuang 050091,China;The First Affiliated Hospital of Hebei University of Chinese Medicine,Hebei Provincial Hospital ofTraditional Chinese Medicine,Shijiazhuang 050011,China)

机构地区：[1]河北中医药大学,石家庄050091 [2]河北中医药大学第一附属医院/河北省中医院,石家庄050011

出　　处：《中国实验方剂学杂志》2024年第15期38-45,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：河北省自然科学基金项目(H2022423367);河北中医学院研究生创新能力培养项目(XCXZZBS2022018)。

摘　　要：目的:观察当归补血汤对糖尿病肾病(DKD)大鼠足细胞线粒体分裂及凋亡的影响,探讨当归补血汤对DKD大鼠肾脏保护作用及机制。方法:SD大鼠随机分为造模组65只,喂养高糖高脂饲料;正常组10只,喂养普通饲料;6周后,造模组大鼠予腹腔注射链脲佐菌素(STZ)制备2型糖尿病(T2DM)模型。将制备成功的T2DM大鼠随机分为模型组、厄贝沙坦组(0.014 g·kg^(-1))、当归补血汤低、中、高剂量组(0.36、0.72、1.44 g·kg^(-1))。连续灌胃16周。治疗第16周末检测各组大鼠空腹血糖(FBG)、24 h尿蛋白(24 h UTP);苏木素-伊红(HE)、马松(Masson)染色观察大鼠肾组织病理;透射电镜(TEM)观察足细胞线粒体;二氢乙锭(DHE)法检测大鼠肾组织活性氧簇(ROS)表达水平;原位末端标记法(TUNEL)检测各组大鼠肾细胞凋亡情况;免疫组化法(IHC)检测大鼠肾组织中足细胞标志蛋白突触足蛋白(Synaptopodin)、膜蛋白(Podocin)及切割型胱天蛋白酶-3(cleaved Caspase-3)表达水平;蛋白免疫印迹法(Western blot)检测肾组织激酶锚定蛋白1(AKAP1)、磷酸化动力相关蛋白1(p-Drp1)、线粒体融合蛋白2(Mfn2)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达。结果:与正常组比较,模型组大鼠FBG、24 h UTP表达升高;可见肾小球内系膜增生,基底膜增厚;电镜下足细胞线粒体肿胀,线粒体嵴断裂、紊乱;ROS表达增加,TUNEL阳性细胞明显增多,Synaptopodin、Podocin表达降低,cleaved Caspase-3表达增加,AKAP1、p-Drp1表达增加,Mfn2、Bcl-2/Bax表达降低(P<0.01);与模型组比较,当归补血汤高剂量组能降低FBG、24 h UTP水平,肾组织病理损伤及足细胞线粒体损伤明显改善,ROS表达减少,肾组织TUNEL阳性细胞数明显减少,Synaptopodin、Podocin表达升高,cleaved Caspase-3表达降低,AKAP1、p-Drp1表达降低,Mfn2、Bcl-2/Bax表达增加(P<0.05,P<0.01)。结论:当归补血汤可通过调控AKAP1/Drp1通路抑制足细胞线粒体分裂及凋亡,减少蛋�Objective:To observe the effects of Danggui Buxuetang on the mitochondrial fission and apoptosis of podocytes in the rat model of diabetic kidney disease(DKD)and to explore the protective effect and mechanism of Danggui Buxuetang on DKD rats. Method: SD rats were randomized into a modeling group( n=65, fed with a high-sugar and high-fat diet) and a normal group( n=10, fed with a normal diet). After 6 weeks, the modeled rats were injected intraperitoneally with streptozotocin( STZ) for the modeling of type 2 diabetes mellitus (T2DM). Sixty T2DM rats were randomized into model, irbesartan (0.014 g·kg^(-1)), and low- , medium-, and high-dose( 0.36, 0.72, 1.44 g·kg^(-1), respectively) Danggui Buxuetang groups and administrated with corresponding drugs by gavage for 16 weeks. The levels of fasting blood glucose( FBG) and 24 h urinary protein( 24 h UTP) were determined at the end of the 16th week. The pathological changes of the renal tissue were observed by hematoxylin-eosin and Masson staining. The mitochondrial ultrastructure of rat podocytes was observed by transmission electron microscopy. The level of reactive oxygen species( ROS) in the renal tissue of rats was measured by the fluorescent probe labeling of dihydroethidium( DHE). The apoptosis of renal cells was detected by terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling( TUNEL). The expression levels of Synaptopodin, Podocin, and cleaved caspase-3 in renal podocytes were detected by the immunohistochemical method( IHC). Western blot was employed to determine the protein levels of A-kinase anchoring protein 1( AKAP1), phosphorylated dynamin-related protein 1( p-Drp1), mitofusin-2( Mfn2), B-cell lymphoma-2( Bcl-2), and Bcl-2-associated X protein( Bax). Result: Compared with the control group, the model group showed elevated levels of FBG and 24 h UTP, mesangial hyperplasia, basement membrane thickening, mitochondrial swelling, mitochondrial crista breakage and disorder, and increased ROS and TUNEL-positive cells. In addition, the model grou

关 键 词：糖尿病肾病 当归补血汤 线粒体分裂 足细胞凋亡 

分 类 号：R2-0[医药卫生—中医学] R24R33R289R587.1R692