基于Klotho调控的FGF23/Pit1信号通路研究健脾补肾活血化浊中药对糖尿病肾病血管钙化的干预机制

Cardiovascular complications are closely related to vascular calcification in diabetic nephropathy. calcium and phosphate metabolism is an important link in diabetic nephropathy vascular calcification,which is asscosiated with the abnormal expression of Klotho (Kl) gene and bone secreting fibroblast growth factor 23 (FGF23) in the kidney.Based on kidney stores essence theory and kidney essence deficiency and blood stasis pathogenesis of diabetic vascular calcification ,vascular calcification as a target, the establishment of diabetic nephropathy in a mousethy model of feeding with “strengthening spleen and tonifying the kidney and activating blood circulation to eliminate turbid ”Chinese medicine observed its effects on vascular calcification model.real-time quantitative PCR and Western blot detection of renal tissue Kl, serum FGF23, thoracic aorta FGF23, mediating the expression of aortic type III sodium-dependent phosphate cotransporter (Pit-1) and of cor-binding factor alpha-l(Cbfα-l ).The use of biological mass spectrometry in the identification of the thoracic aorta is combined with FGF23 protein; by Von Kossa calcium salt staining was used to detect the degree of calcification.In vitro, to establish vascular smooth muscle cells (VSMC) calcification model, respectively with different concentrations of drug serum expression of Kl group and model group, the detection of relevant indicators. To clarify the mechanism of invigorating spleen, tonifying kidney, promoting blood circulation and removing blood stasis on diabetic nephropathy and The study of the theory of the kidney essence was extended from the calcium and phosphorus metabolism regulated by Kl gene to the study of vascular calcification, which provided a theoretical basis for the prevention and treatment of diabetic nephropathy.

糖尿病肾病（DN）心血管并发症与血管钙化密切相关，钙磷代谢异常是导致DN血管钙化的重要环节，其发生与肾脏产生的klotho（kl）基因和骨骼分泌的成纤维细胞生长因子23（FGF23）表达异常相关。本研究基于肾藏精理论及DN血管钙化肾精亏虚、脉络瘀阻病机，以血管钙化为靶点，观察健脾补肾活血化浊中药对糖尿病肾病大鼠血管钙化的影响。体内试验检测肾脏Kl、血FGF23、胸主动脉FGF23，介导主动脉Ⅲ型钠磷协同转运子（Pit1）及成骨细胞标记物(Cbfα-l)的表达，质谱法鉴定主动脉被FGF23结合的蛋白。在体外建立血管平滑肌细胞（VSMC）钙化模型，分别以不同浓度含药血清干预Kl过表达组及模型组，检测相关指标。阐明健脾补肾活血化浊法对糖尿病肾病血管钙化的干预机制，将肾藏精理论从Kl基因调控的钙磷代谢延伸到对血管钙化的研究，为糖尿病肾病血管并发症的防治提供理论基础。

糖尿病肾病（DN）发病率逐年上升，其高致死率多年来未见明显改善。钙磷代谢紊乱所导致的血管钙化，与DN心血管并发症关系密切，是影响患者生存质量和生存时间的独立危险因素。肾脏生成的抗衰老基因Klotho和骨骼分泌的调磷因子FGF23表达异常，可导致钙磷代谢紊乱、骨代谢异常，激活钠磷共转运子Pit-1，诱导血管平滑肌细胞（VSMCs）向成骨细胞表型转化，发生血管钙化。因此，本研究基于肾藏精主骨理论及DN肾精亏虚脉络瘀阻的病机，以血管钙化为靶点，通过分析健脾补肾活血化浊中药肾元颗粒干预后，Klotho、FGF23及Pit1之间的相互作用，及其对血管钙化的影响，将肾藏精主骨延伸到血管，阐明肾元颗粒对DN血管钙化的作用机制。在体内高磷诱导db/db糖尿病肾病小鼠血管钙化，肾元颗粒干预后，胸主动脉血管钙化较前改善，血清FGF23、胸主动脉组织中Pit-1和Cbfα1表达明显降低，肾脏Klotho及胸主动脉SM22表达显著上调。在体外高糖高磷诱导大鼠胸主动脉VSMCs钙化，分别给予5%、10%、15%含药血清干预，与模型组相比，不同剂量含药血清组细胞钙含量均有下降，且高剂量组较中低剂量组下降显著；且含药血清组FGF23、Pit1及Cbfα1均表达减低，而SM22表达呈不同程度上升，其中中、高剂量组较明显。说明含药血清可抑制FGF23、Pit1及Cbfα1的表达，增强SM22的表达，以减轻细胞钙化。在此基础上给予慢病毒包装，过表达的Klotho可协同肾元颗粒含药血清增强对FGF23、Pit1及Cbfα1的拮抗作用，增强SM22表达，进一步改善细胞钙化。据此，在db/db糖尿病肾病小鼠钙化模型和大鼠胸主动脉VSMCs钙化模型中，阐明了健脾补肾活血化浊中药通过Klotho介导的FGF23/Pit1/Cbfα1信号通路改善血管钙化，为DN血管并发症的防治提供理论基础。