纳米细胞囊泡负载姜黄素促进糖尿病小鼠创面的愈合

Nanocellular vesicles loaded with curcumin promote wound healing in diabetic mice

背景:间充质干细胞来源的外泌体及姜黄素均能促进糖尿病难愈合创面的愈合,但外泌体存在产量低的问题,而姜黄素存在结构相对不稳定、溶解性差等问题,影响了修复效果。目的:观察负载姜黄素的纳米细胞囊泡对糖尿病小鼠难愈合创面的修复效果。方法:分离提取C57BL/6J乳鼠原代骨髓间充质干细胞,使用姜黄素溶液悬浮细胞,采用微型挤出机制备出负载姜黄素的纳米细胞囊泡,检测囊泡的包封率与载药量。①体外实验:采用细胞计数法检测不同质量浓度(0,10,20,40,80 mg/L)纳米细胞囊泡对成纤维细胞NIH-3T3增殖的作用,Transwell实验检测纳米细胞囊泡(40 mg/L)对成纤维细胞迁移能力的影响。检测姜黄素对脂多糖诱导的RAW 264.7巨噬细胞炎症因子——肿瘤坏死因子α、白细胞介素6 mRNA表达量的影响。②体内实验:取24只成年C57BL/6J小鼠,腹腔注射链脲佐菌素建立糖尿病模型,造模成功后于背部制作2个直径6 mm的创面,A组注射PBS,B组注射纳米细胞囊泡,C组注射姜黄素溶液,D组注射负载姜黄素的纳米细胞囊泡,每组6只,观察创面愈合情况与组织形态学变化。结果与结论:①囊泡的包封率为42%,载药率为2.3%;不同质量浓度的纳米细胞囊泡均可促进成纤维细胞的增殖,其中以40 mg/L效果最明显;40 mg/L的纳米细胞囊泡可促进成纤维细胞的迁移;姜黄素降低了脂多糖刺激的巨噬细胞的炎症反应;②创面造模14 d后,B、C、D组创面明显缩小,其中D组创面面积最小;苏木精-伊红与Masson染色显示,创面造模后7 d,B、C、D组创面有大量的肉芽组织形成与胶原纤维沉积,其中以D组最多;创面造模后14 d,B、C、D组创面的肉芽组织形成与胶原纤维沉积进一步增多,其中以D组最多;③结果表明,负载姜黄素的纳米细胞囊泡可发挥纳米细胞囊泡和姜黄素的协同作用,促进糖尿病创面的愈合。

BACKGROUND:Mesenchymal stem cells-derived exosome and curcumin can enhance the diabetic wound healing.However,the yield of exosome is low,while curcumin has relatively unstable structure and poor solubility,which affects the repair effect.OBJECTIVE:To observe the therapeutic effect of the nanocellular vesicles loaded with curcumin for the refractory wounds in diabetic mice.METHODS:Primary bone marrow mesenchymal stem cells were harvested from limbs of C57BL/6J suckling mice.Nanocellular vesicles loaded with curcumin were prepared via micro-extrusion mechanism.Encapsulation efficiency and drug loading of vesicles were detected.(1)In vitro,the effect of nanocellular vesicles on fibroblast NIH-3T3 proliferation at different mass concentrations(0,10,20,40,and 80 mg/L)was detected by cell counting method.The effect of nanocellular vesicles(40 mg/L)on fibroblast migration was detected by Transwell assay.The effect of curcumin on tumor necrosis factorαand interleukin-6 mRNA expression were detected in RAW 264.7 macrophages induced by lipopolysaccharide.(2)In vivo,twenty-four adult C57BL/6J mice were taken and injected intraperitoneally with streptozotocin to establish diabetic models.Skin wounds with two 6 mm diameter wounds were produced on the backs of diabetic mice after modeling.Group A was injected with PBS;group B was injected with nanocellular vesicles;and group C was injected with curcumin solution;group D was injected with curcumin-loaded nanocellular vesicles(n=6 per group).The wound healing and histomorphological changes were observed.RESULTS AND CONCLUSION:(1)The encapsulation rate of vesicles was 42%,and the drug loading rate was 2.3%.Nanocellular vesicles with different mass concentrations could promote the proliferation of fibroblasts.Of them,the concentration of 40 mg/L had the best proliferation effect.Nanocellular vesicles 40 mg/L promoted the migration of fibroblasts.Curcumin decreased the LPS-stimulated inflammatory response in macrophages.(2)After 14 days of wound modeling,the wounds in groups B,C,and D were significantly reduced,and the wound area in group D was the smallest.Hematoxylin-eosin staining and Masson staining showed that there was a large amount of granulation tissue formation and collagen fiber deposition in groups B,C,and D after 7 days,among which group D had the most.At 14 days after the wound modeling,the granulation tissue formation and collagen fiber deposition in groups B,C,and D further increased,and the changes were most remarkable in group D.(3)These results suggested that the nanocellular vesicles loaded with curcumin showed their synergistic role in promoting diabetic wound healing.