人参皂苷诱导骨髓间充质干细胞干预糖尿病大鼠皮肤溃疡的愈合及Wnt/β-catenin信号表达

Ginsenoside-induced bone marrow mesenchymal stem cell intervention can affect healing and Wnt/beta-catenin signaling in rats with diabetic skin ulcer

背景:以往研究表明骨髓间充质干细胞在促进创面愈合方面已经取得了一定成效,能明显促进创面皮肤的恢复,但关于基因及药物调控的相关研究目前尚未取得突破性进展。目的:分析人参皂苷诱导骨髓间充质干细胞对糖尿病皮肤溃疡大鼠愈合作用及其对Wnt/β-catenin信号表达的影响。方法:选取SPF级Wistar雄性大鼠61只,其中1只行骨髓间充质干细胞培养,另外60只随机数字表法将模型大鼠分成4组,空白组、糖尿病皮肤溃疡组、骨髓间充质干细胞组及人参皂苷诱导骨髓间充质干细胞组,除空白组,其他各组大鼠制备糖尿病皮肤溃疡模型,造模后6h糖尿病皮肤溃疡组和空白组大鼠由尾静脉注入1 mL细胞培养液,骨髓间充质干细胞组注入1 mL骨髓间充质干细胞悬液(细胞浓度2.0×10^9 L^-1),人参皂苷诱导骨髓间充质干细胞组注入1 mL人参皂苷诱导分化骨髓间充质干细胞悬液(细胞浓度2.0×10^9 L^-1)。实验方案经已于2016年12月获得解放军二五一医院动物伦理学委员会批准开展进行,批准号:20161211。结果与结论:与空白组相比,糖尿病皮肤溃疡组大鼠血清白细胞介素1、超敏C-反应蛋白、肿瘤坏死因子α含量上升,血小板衍生生长因子、血管内皮生长因子、碱性成纤维因子含量下降;与糖尿病皮肤溃疡组相比,骨髓间充质干细胞组及人参皂苷诱导骨髓间充质干细胞组大鼠血清白细胞介素1、超敏C-反应蛋白、肿瘤坏死因子α含量下降,血小板衍生生长因子、血管内皮生长因子、碱性成纤维因子含量上升;与骨髓间充质干细胞组相比,人参皂苷诱导骨髓间充质干细胞组大鼠血清白细胞介素1、超敏C-反应蛋白含量下降,血小板衍生生长因子、血管内皮生长因子、碱性成纤维因子含量上升;与空白组相比,糖尿病皮肤溃疡组大鼠成纤维细胞数和新生毛细血管数密度下降;与糖尿病皮肤溃疡组相比,骨髓间充质干细胞组,人参皂苷诱导骨髓间充质干细胞组大鼠成纤维细胞数和新生毛细血管数密度上升;与骨髓间充质干细胞组相比,人参皂苷诱导骨髓间充质干细胞组大鼠成纤维细胞数和新生毛细血管数密度上升;与空白组相比,糖尿病皮肤溃疡组大鼠Wnt4及β-catenin mRNA表达上升;与糖尿病皮肤溃疡组相比,骨髓间充质干细胞组、人参皂苷诱导骨髓间充质干细胞组大鼠Wnt4及β-catenin mRNA表达下降;与骨髓间充质干细胞组相比,人参皂苷诱导骨髓间充质干细胞组大鼠Wnt4及β-catenin mRNA表达下降。提示人参皂苷诱导骨髓间充质干细胞可显著降低糖尿病皮肤溃疡大鼠血清炎性因子含量,提升生长因子含量,下调Wnt/β-catenin信号路径内相关蛋白表达加速创面愈合。

BACKGROUND: Previous studies have shown that bone marrow mesenchymal stem cells can certainly promote wound healing, andconsiderably accelerate the recovery of wound skin. However, relevant research on gene and drug regulation has yet to make breakthroughprogress.OBJECTIVE: To analyze the effects of ginsenoside-induced bone marrow mesenchymal stem cells on skin healing of rats with diabetic skinulcer and their effects on Wnt/β-catenin signaling.METHODS: Sixty-one SPF-level Wistar male rats were selected, one of them was used for the culture of bone marrow mesenchymal stemcells, and the other 60 were randomized into four groups: blank control group, model group (treated with 1 mL of culture medium via tail vein),single cell treatment group (treated with 1 mL of bone marrow mesenchymal stem cell suspension via tail vein, 2.0×10^9/L) andginsenoside-induced cell treatment group (treated with 1 mL of ginsenoside-induced bone marrow mesenchymal stem cell suspension via tailvein, 2.0×10^9/L). Animal models of diabetic skin ulcer were made in all groups except the blank control group. Treatments in each group wereperformed at 6 hours after modeling. The study protocol was approved by the Animal Ethics Committee of the 251st Hospital of PLA withapproval No. 20161211 in December 2016.RESULTS AND CONCLUSION: Compared with the blank control group, the serum levels of interleukin-1, high-sensitivity C-reactive proteinand tumor necrosis factor-α increased and the serum levels of platelet-derived growth factor, vascular endothelial growth factor and basicfibroblast factor decreased in the model group. Compared with the model group, the serum levels of interleukin-1, high-sensitivity C-reactiveprotein and tumor necrosis factor-α decreased significantly and the serum levels of platelet-derived growth factor, vascular endothelial growthfactor and basic fibroblast factor increased significantly in the single cell treatment and ginsenoside-induced cell treatment groups. Comparedwith the single cell treatment group, the serum levels of interleukin-1, high-sensitivity C-reactive protein and tumor necrosis factor-α decreasedsignificantly and the serum levels of platelet-derived growth factor, vascular endothelial growth factor and basic fibroblast factor increasedsignificantly in the ginsenoside-induced cell treatment group. Compared with the blank control group, the number of fibroblasts and density ofnewly born capillaries decreased in the model group. Compared with the model group, the number of fibroblasts and density of newly borncapillaries increased in the two cell treatment groups. Compared with the single cell treatment group, the number of fibroblasts and density ofnewly born capillaries increased in the ginsenoside-induced cell treatment group. Compared with the blank control group, the expression ofWnt4 and β-catenin mRNA increased in the model group. Compared with the model group, the expression of Wnt4 and β-catenin mRNAdecreased in the two cell treatment groups. Compared with the single cell treatment group, the expression of Wnt4 and β-catenin mRNAdecreased in the ginsenoside-induced cell treatment group. These findings therefore indicate that ginsenoside-induced bone marrowmesenchymal stem cells considerably reduce the levels of serum inflammatory cytokines, increase the level of growth factors, anddownregulate the expression of related proteins in the Wnt/β-catenin signaling pathway to accelerate wound healing.