摘要
目的:探讨肾元颗粒通过线粒体介导平滑肌细胞凋亡对糖尿病肾病小鼠血管钙化的影响。方法:将40只SPF级db/db小鼠随机分为模型组和肾元颗粒高、中、低剂量组,每组10只,10只同系背景野生型(WT)小鼠作为对照组。模型组、肾元颗粒各剂量组予以高磷饲料(含磷1.2%),对照组予以普通饲料,肾元颗粒高、中、低剂量组分别予以6.0、3.0、1.5g·kg^(-1)·d^(-1)肾元颗粒灌胃,对照组、模型组予以等体积的0.9%氯化钠溶液灌胃,干预12周后,检测血清钙(Ca)、磷(P)、血肌酐(CREA)、尿素氮(UREA)。Von Kossa染色观察小鼠胸主动脉病理形态变化以及钙盐沉积情况,透射电镜观察胸主动脉血管平滑肌细胞线粒体结构形态,RT-PCR法检测各组小鼠胸主动脉B淋巴细胞瘤-2(Bcl-2)、B淋巴细胞瘤-2关联X蛋白(Bax)、α-平滑肌蛋白(α-SMA)、成骨细胞特异性转录因子(Osterix)mRNA表达水平,Western blot、免疫组化法检测半胱氨酸天冬氨酸蛋白酶(Caspase)-3、Caspase-9、细胞色素C(Cyt-C)蛋白表达水平,ELISA法检测胸主动脉匀浆上Bcl-2、Bax蛋白表达含量。结果:透射电镜显示模型组平滑肌细胞呈合成型,线粒体结构肿胀,空泡变,线粒体基质熔解,嵴消失,而肾元颗粒各剂量组平滑肌细胞呈半合成型或收缩型,线粒体结构呈不同程度改善,肿胀减轻,嵴存在,膜较完整。Von Kossa染色显示模型组血管壁可见明显钙盐沉积。与对照组比较,模型组血P、CREA、UREA水平升高显著,血Ca水平显著下降,胸主动脉Osterix mRNA表达显著升高,α-SMA mRNA表达显著下降,Bcl-2表达显著下调,Bax、Caspase-3、Caspase-9、Cyt-C表达显著上调(P<0.01);与模型组比较,肾元颗粒各剂量组血P、CREA、UREA水平显著下降(P<0.01,P<0.05),血Ca水平显著升高(P<0.01)。肾元颗粒中、高剂量组胸主动脉Osterix、Bax、Caspase-3、Caspase-9、Cyt-C表达显著降低,α-SMA、Bcl-2表达显著升高(P<0.01,P<0.05)。结论:肾元颗粒可改善糖尿病肾病db/db模型小鼠的肾功能及血管钙化程度,其机制可能与改善线粒体介导的血管平滑肌凋亡有关。
Objective:To investigate the effects of Shenyuan Granules on vascular calcification in mice with diabetic nephropathy through mitochondria-mediated apoptosis of smooth muscle cells.Methods:Forty SPF db/db mice were randomly divided into model group and high-,medium-,and low-dose Shenyuan Granules groups,10 in each group,and 10 wild-type(WT)mice with the same strain as the control group.The model group and the Shenyuan Granules groups were given high-phosphorus feed(1.2%phosphorus),the control group was given normal feed,and the high-,medium-and low-dose Shenyuan Granules groups were given 6.0,3.0,1.5 g·kg^(-1)·d^(-1) Shenyuan Granules were given by intragastric administration,the control group and model group were given an equal volume of 0.9%normal saline.After 12 weeks of intervention,serum calcium(Ca),phosphorus(P),blood creatinine(CREA)and urea nitrogen were measured(UREA).Von Kossa staining was used to observe the pathological changes of mouse thoracic aorta and calcium salt deposition.Transmission electron microscope was used to observe the mitochondrial structure and morphology of thoracic aorta vascular smooth muscle cells.Real-time PCR method was used to detect thoracic aortic B lymphoma-2(Bcl-2),B lymphocyte tumor-2 associated X protein(Bax)α-smooth muscle protein(α-SMA),osteoblast-specific transcription factor(Osterix)mRNA expression level,Western blot,immunohistochemical detection Caspase-3,Caspase-9,Cytochrome C(Cyt-C)protein expression levels,detected by ELISA Bcl-2 and Bax protein expression levels in the thoracic aorta homogenate.Results:Transmission electron microscopy showed that the mitochondrial structure of the model group was swollen and arranged disorderly,while the mitochondrial structure of the Shenyuan Granules groups of kidney particles improved and the swelling was reduced.Von Kossa staining showed that there were obvious calcium deposits on the vessel wall of the model group.Compared with the control group,the blood P,CREA,and UREA levels of the model group increased significantly,the blood Ca level decreased significantly,the expression of Osterix mRNA in the thoracic aorta increased significantly,the expression ofα-SMA mRNA decreased significantly,and the expression of anti-apoptotic protein Bcl-2 significantly down-regulated,and the apoptotic protein Bax,Caspase-3,Caspase-9,Cyt-C expression was significantly up-regulated(P<0.01);compared with the model group,the levels of blood P,CREA,and UREA in the Shenyuan Granules groups were significantly decreased(P<0.01,P<0.05),the blood Ca level was significantly increased(P<0.01).The expressions of Osterix,Bax,Caspase-3,Caspase-9,Cyt-C in the thoracic aorta were decreased,and the expressions ofα-SMA and Bcl-2 were increased in the medium-and high-dose Shenyuan Granules groups(P<0.01,P<0.05).Conclusion:Shenyuan Granules can improve the renal function and the degree of vascular calcification in diabetic nephropathy db/db model mice.The mechanism may be related to the improvement of mitochondrialmediated apoptosis of vascular smooth muscle.
作者
邓丹芳
孙龙
林腊梅
金善善
郑毅
王岚
王小琴
DENG Dan-fang;SUN Long;LIN La-mei;JIN Shan-shan;ZHENG Yi;WANG Lan;WANG Xiao-qin(Hubei University of Chinese Medicine,Wuhan 430061,China;Hubei Provincial Hospital of Traditional Chinese Medicine,Wuhan 430074,China;Hubei Academy of Traditional Chinese Medicine,Wuhan 430061,China)
出处
《中华中医药杂志》
CAS
CSCD
北大核心
2021年第5期2641-2646,共6页
China Journal of Traditional Chinese Medicine and Pharmacy
基金
国家自然科学基金项目(No.81874439)
武汉市科技局应用基础前沿项目(No.2019020701011455)。
关键词
糖尿病肾病
肾元颗粒
血管钙化
线粒体
凋亡
Diabetic nephropathy
Shenyuan Granules
Vascular calcification
Mitochondria
Apoptosis
