丹蛭降糖胶囊通过LncRNA TUG1/β-catenin信号通路保护线粒体功能减轻血管钙化的机制  

Mechanism of Danzhi Jiangtang capsule protecting mitochondrial function and reducing vascular calcification via LncRNA TUG1/β-catenin signaling pathway

作　　者：倪英群 林逸轩[2] 汪四海 卢芹[1] 骆金芝 吴春琴 方朝晖 NI Ying-qun;LIN Yi-xuan;WANG Si-hai;LU Qin;LUO Jin-zhi;WU Chun-qin;FANG ZHAO-hui(the First Clinical Medical College,Anhui University of Traditional Chinese Medicine,Hefei 230038,China;Dept of Endocrinology,the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine,Anhui Academy of Traditional Chinese Medicine,Hefei 230031,China;Institute of Traditional Chinese Medicine for Prevention and Treatment of Diabetes,Anhui Academy of Traditional Chinese Medicine,Hefei 230031,China;Key Laboratory of Xin’An Medicine,Ministry of Education,Hefei 230038,China)

机构地区：[1]安徽中医药大学第一临床医学院,安徽合肥230038 [2]安徽中医药大学第一附属医院内分泌科,安徽合肥230031 [3]安徽省中医药科学院中医药防治糖尿病研究所,安徽合肥230031 [4]新安医学教育部重点实验室,安徽合肥230038

出　　处：《中国药理学通报》2024年第5期899-906,共8页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 82274468);安徽中医药大学临床科研项目(No 2021yfylc07);安徽省高校优秀人才支持计划项目(No gxyqZD2021114);新安医学教育部重点实验室开放项目(No 2022XAYX06)。

摘　　要：目的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)成骨样分化是血管钙化(vascular calcification,VC)细胞学基础。线粒体功能障碍在VSMCs成骨样转化中起重要作用。本研究旨在探讨丹蛭降糖胶囊(Danzhi Jiangtang capsules,DJC)通过调节LncRNA TUG1/β-catenin信号通路保护VSMCs线粒体功能减轻血管钙化的机制。方法使用β-甘油磷酸盐来诱导血管平滑肌细胞的钙化模型,并采用维生素D 3+高脂饲料来诱导糖尿病大鼠的血管钙化模型。Von Kossa染色检测细胞和血管组织钙化;用邻甲酚酞络合物比色法测定钙含量;采用对硝基苯磷酸盐比色法测定碱性磷酸酶(alkaline phosphatase,ALP)活性;采用RT-qPCR分析VSMCs成骨样转化相关基因骨形态发生蛋白(bone morphogenetic protein2,BMP2)、平滑肌肌动蛋白(smooth muscle actin alpha,ɑ-SMA)及长链非编码RNA牛磺酸上调基因1(long noncoding RNA taurine up-regulated1,LncRNA TUG1)、β-连环蛋白(β-catenin)表达;Western blot分析BMP2、ɑ-SMA及β-catenin的蛋白表达;JC-1荧光探针检测线粒体膜电位;透射电镜观察线粒体结构。结果DJC降低β-甘油磷酸盐诱导的VSMCs中LncRNA TUG1的表达,下调β-catenin的表达,降低ALP活性和钙沉积,保护线粒体功能,恢复膜电位,减少VSMCs成骨样转化。重要的是,DJC通过下调糖尿病下肢血管组织LncRNA TUG1、β-catenin的表达,上调ɑ-SMA的表达来减轻糖尿病下肢VC。结论DJC对糖尿病下肢VC有明显的改善作用,机制是通过LncRNA TUG1/β-catenin信号保护线粒体功能减少VSMCs成骨细胞转化。Aim To explore how Danzhi Jiangtang capsules(DJC)safeguard the mitochondrial activity of vascular smooth muscle cells(VSMCs)by controlling the LncRNA TUG1/β-catenin signaling pathway to decrease vascular calcification(VC).Methods Vascular smooth muscle cell calcification models were induced withβ-glycerin and diabetic vascular calcification rat models were induced with vitamin D 3+high-fat diet.Von Kossa staining was applied to detect calcification of cells and vascular tissue.Colorimetric method of phthalein complex was used to determine calcium content.P-nitrobenzene phosphate colorimetry was employed to assess alkaline phosphatase(ALP)activity.RT-qPCR was used to analyze the expression of VSMCs’osteoblast transformation related genes bone morphogenetic protein2(BMP2),smooth muscle actin alpha(α-SMA),taurine up-regulated1,LncRNA Tug1(Lnc-RNA TUG1),andβ-catenin.Western blotting was utilized to detect the protein expression of BMP2,α-SMA andβ-catenin.The mitochondrial membrane potential was detected by JC-1 fluorescence probe.Mitochondrial structure was observed by transmission electron microscope.Results DJC reduced LncRNA TUG1 expression,down-regulatedβ-catenin expression,decreased ALP activity and calcium deposition,protected mitochondrial function,restored membrane potential,and decreased osteoblastic transformation of VSMCs induced by glycerin phosphate.Importantly,DJC attenuated diabetic lower limb VC by down-regulating the expression of LncRNA TUG1,β-catenin,and elevating the expression ofα-SMA.Conclusions DJC capsules significantly improved VSMCs by protecting mitochondrial function by LncRNA TUG1/β-catenin signaling to reduce VSMCs’osteoblast transformation.

关 键 词：LncRNA TUG1 Β-CATENIN 线粒体 糖尿病 成骨细胞转化 血管钙化 

分 类 号：R-332[医药卫生] R285.5R329.24R336R587.1