补肾活血方调控外泌体miR-32介导PTEN/PI3K/AKT通路对CKD血管钙化的作用机制研究

Vascular calcification is an important pathological etiology for cardiovascular death in patients with chronic kidney disease (CKD). The phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblasts is the core in vascular calcification. Our previous studies have confirmed that BushenHuoxue Decoction can inhibit the phenotypic transformation of VSMCs into osteoblasts and vascular calcification of CKD can be inhibited as a result. However, its specific mechanism is still unknown yet. Some new studies have found that exosome miR-32 derived from calcified VSMCs in CKD plays an important role in vascular calcification by mediating PTEN/PI3K/AKT pathway. Based on these findings, it can be hypothesized that BushenHuoxue Decoction can inhibit vascular calcification by regulating exosome miR-32-mediated PTEN/PI3K/AKT pathway. In this study, a high-phosphorus diet combined with adenine gavage rats and high-phosphorus-induced VSMCs in vitro are used respectively to establish a model of vascular calcification of CKD in vivo and in vitro. Focused on miR-32 and PTEN/PI3K/AKT pathway, nanoparticle tracking analysis, siRNA interference technology, qRT-PCT and Western blot are used to explore the mechanism of BushenHuoxue Decoction in inhibiting vascular calcification of CKD. Additionally, theoretical and experimental basis is provided to interpret the treatment of vascular calcification of CKD by BushenHuoxue Decoction.

血管钙化是慢性肾脏病（CKD）患者发生心血管疾病死亡的重要病理基础，血管平滑肌细胞（VSMCs）向成骨细胞表型转化是血管钙化的核心环节。前期研究证实补肾活血方可抑制VSMCs向成骨细胞表型转化从而抑制CKD血管钙化，但具体机制不明；最新研究发现CKD钙化VSMCs来源外泌体miR-32介导的PTEN/PI3K/AKT通路激活在血管钙化中发挥重要作用。结合前期基础提出假说：补肾活血方可通过调控外泌体miR-32介导PTEN/PI3K/AKT通路抑制血管钙化。本研究采用高磷饮食联合腺嘌呤灌胃大鼠及体外高磷诱导VSMCs建立体内外CKD血管钙化模型，应用纳米粒子追踪、siRNA干扰、qRT-PCT、Western blot等方法，围绕钙化VSMCs外泌体miR-32这一靶点及PTEN/PI3K/AKT通路，探讨补肾活血方抑制CKD血管钙化的关键机制，为补肾活血方治疗CKD血管钙化提供科学依据。

CKD血管钙化显著增加全因死亡率和心血管死亡率。研究防治血管钙化的方法对改善CKD患者预后至关重要。前期研究提示补肾活血方可减轻CKD血管钙化，但具体作用机制尚不明确。结合目前该领域研究热点---外泌体miRNA，探讨相关机制。本研究为补肾活血方临床应用奠定分子学基础，有望为改善CKD患者远期结局提供手段。.目的：观察补肾活血方通过调节外泌体miR-32mRNA介导PTEN/PI3K/AKT信号通路对CKD血管钙化（VC）的抑制作用，探讨补肾活血方抑制CKD-VC的作用机制。方法：体内实验采用腺嘌呤灌胃联合高磷饮食建立CKD-VC大鼠模型。将大鼠分为对照组，模型组，低、中、高剂量补肾活血方组和司维拉姆组。给药8周后测血清肌酐、尿素氮、总钙、无机磷、全段甲状旁腺激素水平。观察肾脏组织和主动脉的病理变化，检测主动脉组织中钙含量和ALP含量。透射电镜观察外泌体形态，纳米流式检测仪（NanoFCM）检测外泌体粒径和的表面标记标记性蛋白。体外实验采用高磷培养基(2.4mM)诱导VSMCs成骨分化。将VSMCs分为正常组、高磷（HP）、中药血清组、模型+抑制剂组及中药血清+抑制剂组。使用PKH67和DAPI荧光染色观察正常组VSMCs对HP组VSMCs外泌体的摄取。Western blot法检测α-SMA、BMP-2、Runx2及PTEN/PI3K/AKT信号通路成分的水平。RT-PCR检测外泌体miR-32mRNA表达。结果：补肾活血方对CKD-VC大鼠肾功能、钙磷代谢指标、肾脏病理和主动脉病理有改善作用，能降低主动脉钙含量和ALP活性；上调主动脉α-SMA，下调BMP-2和Runx2蛋白表达；下调外泌体miR-32mRNA表达，上调PTEN蛋白表达以及下调p-PI3K/PI3K和p-AKT/AKT比值。荧光染色显示钙化VSMCs来源外泌体可被正常VSMCs摄取。补肾活血方含药血清能减少钙化VSMCs钙沉积，降低VSMCs钙含量和ALP活性；上调α-SMA，下调BMP-2和Runx2蛋白表达；下调外泌体miR-32 mRNA表达，上调PTEN蛋白表达以及下调p-PI3K/PI3K和p-AKT/AKT比值。结论：补肾活血方通过下调外泌体中miR-32表达，从而促进PTEN表达，抑制PI3K/AKT信号通路，减轻了CKD大鼠的VC和钙化VSMCs钙沉积。