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目的 探讨外泌体抑制剂(GW4869)对2型糖尿病(T2DM)大鼠血管平滑肌细胞(VSMC)钙化的抑制作用机制。方法 采用高糖高脂饲料联合腹腔注射链脲佐菌素(STZ,30 mg/kg)建立T2DM大鼠模型，成模后随机分为对照组、T2DM组、T2DM+GW组(GW4869,1.25 mg/kg),给药12周；细胞分为对照组、高糖组(35 mmol/L HG)、GW组(5μmol/L GW4869)、高糖+GW组(35 mmol/L HG+5μmol/L GW4869);茜素红染色评估钙盐沉积；MTT确定安全浓度；分离外泌体，透射电镜观察形态，纳米颗粒示踪分析检测数量与粒径，Western blot检测肿瘤易感基因101(TSG101)蛋白、CD9、CD63及骨形态发生蛋白2(BMP2)、肌节同源盒基因同系物2(MSX2)、α-平滑肌肌动蛋白(α-SMA)水平；获取血管钙化靶点和收集外泌体靶点进行基因本体论(GO)富集分析；划痕实验评估VSMC迁移能力，碱性磷酸酶(ALP)染色与甲基百里香酚蓝法检测成骨与钙含量。结果 外泌体呈约120 nm球状囊泡，TSG101、CD9、CD63阳性，蛋白浓度在对照组、高糖组及高糖+GW组中分别为0.632、1.345、0.224 g/L(P<0.01);GO功能富集发现外泌体与血管钙化密切相关；GW4869减少T2DM大鼠主动脉钙盐沉积、抑制BMP2并增加α-SMA蛋白表达(P<0.01);GW4869抑制HG诱导VSMC迁移能力、降低ALP活性、减少钙盐沉积与细胞钙含量、抑制MSX2并增加α-SMA mRNA和蛋白表达(P<0.01)。结论 外泌体抑制剂GW4869可抑制T2DM大鼠主动脉VSMC钙化，其机制与调节VSMC成骨表型转化过程有关。
Abstract：Objective To investigate the inhibitory effect of the exosome inhibitor(GW4869) on vascular smooth muscle cell(VSMC) calcification in type 2 diabetes mellitus(T2DM) rats. Methods A T2DM rat model was established using a high-sugar, high-fat diet combined with intraperitoneal injection of streptozotocin(STZ, 30 mg/kg). After successful modeling, the rats were randomly divided into a Control group, a T2DM group, and a T2DM+GW group(GW4869, 1.25 mg/kg), and treated for 12 weeks. Cells were divided into a Control group, a high glucose(HG) group(35 mmol/L HG), a GW group(5 μmol/L GW4869), and a HG+GW group(35 mmol/L HG+5 μmol/L GW4869). Calcified salt deposition was assessed by alizarin red staining. A safe concentration was determined by MTT assay. Exosomes were isolated, their morphology was observed by transmission electron microscopy, their quantity and particle size were analyzed by nanoparticle tracking analysis, and the levels of tumor susceptibility gene 101(TSG101) protein, CD9, CD63, bone morphogenetic protein 2(BMP2), msh homeobox 2(MSX2), and α-smooth muscle actin(α-SMA) were detected by Western blot. Targets associated with vascular calcification and exosomes were identified for gene ontology(GO) enrichment analysis. VSMC migration ability was evaluated by a scratch assay. Osteogenic differentiation and calcium content were assessed by alkaline phosphatase(ALP) staining and the methylthymol blue method, respectively. Results Exosomes exhibited spherical vesicles approximately 120 nm in diameter and were positive for TSG101, CD9, and CD63, with protein concentrations of 0.632, 1.345, and 0.224 g/L, respectively(P<0.01). GO functional enrichment analysis revealed a close association between exosomes and vascular calcification. GW4869 reduced aortic calcified salt deposition in T2DM rats, inhibited BMP2 protein expression, and increased α-SMA protein expression(P<0.01). GW4869 inhibited HG-induced VSMC migration ability, decreased ALP activity, reduced calcified salt deposition and cellular calcium content, inhibited MSX2 expression, and increased the mRNA and protein expression of α-SMA(P<0.01). Conclusion The exosome inhibitor GW4869 inhibits aortic VSMC calcification in T2DM rats by regulating the osteogenic phenotypic transdifferentiation process of VSMCs.
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基本信息:
DOI：10.19367/j.cnki.2096-8388.2026.04.004
中图分类号:R587.2
引用信息:
[1]张闯,白德祚,李依桐,等.外泌体抑制剂GW4869对2型糖尿病大鼠血管平滑肌细胞钙化的作用及机制[J].贵州医科大学学报,2026,51(04):499-508.DOI:10.19367/j.cnki.2096-8388.2026.04.004.
基金信息:
国家自然科学基金项目(82060775); 贵州省卫生健康委科学技术基金项目(gzwkj2025-543)
2026-04-13
2026-04-13