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

doi:10.3969/j.issn.2095-4344.1838

Abstract

Abstract:

BACKGROUND: Previous studies have shown that bone marrow mesenchymal stem cells can certainly promote wound healing, and considerably accelerate the recovery of wound skin. However, relevant research on gene and drug regulation has yet to make breakthrough progress.
OBJECTIVE: To analyze the effects of ginsenoside-induced bone marrow mesenchymal stem cells on skin healing of rats with diabetic skin ulcer 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 stem cells, 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, 2.0×10⁹/L) and ginsenoside-induced cell treatment group (treated with 1 mL of ginsenoside-induced bone marrow mesenchymal stem cell suspension via tail vein, 2.0×10⁹/L). Animal models of diabetic skin ulcer were made in all groups except the blank control group. Treatments in each group were performed at 6 hours after modeling. The study protocol was approved by the Animal Ethics Committee of the 251st Hospital of PLA with approval No. 20161211 in December 2016.
RESULTS AND CONCLUSION: Compared with the blank control group, the serum levels of interleukin-1, high-sensitivity C-reactive protein and tumor necrosis factor-α increased and the serum levels of platelet-derived growth factor, vascular endothelial growth factor and basic fibroblast factor decreased in the model group. Compared with the model group, the serum levels of interleukin-1, high-sensitivity C-reactive protein and tumor necrosis factor-α decreased significantly and the serum levels of platelet-derived growth factor, vascular endothelial growth factor and basic fibroblast factor increased significantly in the single cell treatment and ginsenoside-induced cell treatment groups. Compared with the single cell treatment group, the serum levels of interleukin-1, high-sensitivity C-reactive protein and tumor necrosis factor-α decreased significantly and the serum levels of platelet-derived growth factor, vascular endothelial growth factor and basic fibroblast factor increased significantly in the ginsenoside-induced cell treatment group. Compared with the blank control group, the number of fibroblasts and density of newly born capillaries decreased in the model group. Compared with the model group, the number of fibroblasts and density of newly born capillaries increased in the two cell treatment groups. Compared with the single cell treatment group, the number of fibroblasts and density of newly born capillaries increased in the ginsenoside-induced cell treatment group. Compared with the blank control group, the expression of Wnt4 and β-catenin mRNA increased in the model group. Compared with the model group, the expression of Wnt4 and β-catenin mRNA decreased in the two cell treatment groups. Compared with the single cell treatment group, the expression of Wnt4 and β-catenin mRNA decreased in the ginsenoside-induced cell treatment group. These findings therefore indicate that ginsenoside-induced bone marrow mesenchymal stem cells considerably reduce the levels of serum inflammatory cytokines, increase the level of growth factors, and downregulate the expression of related proteins in the Wnt/β-catenin signaling pathway to accelerate wound healing.

Key words: diabetic skin ulcer, wound healing, bone marrow mesenchymal stem cells, ginsenosides, fibroblast number, capillary density, inflammatory cytokines, Wnt/β-catenin signaling pathway

CLC Number:

Ma Hongwei, Liu Panyun, Zhang Yaqiong. Ginsenoside-induced bone marrow mesenchymal stem cell intervention can affect healing and Wnt/beta-catenin signaling in rats with diabetic skin ulcer[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5300-5306.

Figures/Tables 3

2.1 骨髓间充质干细胞形态培养至第5天培养瓶底能够看到细胞分布，为三角形或者菱形，集落数量明显变多，胞体透亮。在培养10-14 d时，细胞集落融合为单层，集落细胞不断扩增，胞体细长，为放射状扩展，部分细胞为宽大扁平状况，表现是纤维细胞样，可看到突起多个长胞浆。伴随传代次数增多，倒置显微镜观察细胞形态可见贴壁细胞分布均匀，形态基本一致，主要为梭形，其胞体饱满，骨髓间充质干细胞一致性好，见图1。同时，流式细胞术检测其纯度显示，CD45为阴性，阳性率为0.615%，稳定表达CD29、CD90，其阳性率均在97%以上，见图2。 2.2 人参皂苷对骨髓间充质干细胞增殖的影响 MTT检测结果显示，随人参皂苷含量增加，细胞数量也逐步变大，在人参皂苷质量浓度为120 mg/L时，细胞增殖最为显著，见图3。因此后续选用120 mg/L的人参皂苷进行骨髓间充质干细胞诱导。 2.3 实验动物数量分析在实验过程中各组大鼠未发生意外死亡等事件，各组大鼠均进入结果分析。 2.4 人参皂苷诱导的骨髓间充质干细胞对糖尿病皮肤溃疡大鼠肉芽组织病理形态的影响苏木精-伊红染色结果显示，空白组有大量成纤维细胞，核仁深染，胞浆多，炎性细胞较少；糖尿病皮肤溃疡组存在大量淋巴细胞、中性粒细胞等炎性细胞，新生毛细血管及成纤维细胞较少；人参皂苷诱导骨髓间充质干细胞组成纤维细胞较多，核仁着色明显，胞浆多，炎性细胞较少；骨髓间充质干细胞组成纤维细胞较人参皂苷诱导骨髓间充质干细胞组少，胞浆突起不明显，胞体外形小，炎性细胞少，见图4。 2.5 人参皂苷诱导的骨髓间充质干细胞对糖尿病皮肤溃疡大鼠血清生长因子及炎性因子的影响与空白组相比，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水平上升(P < 0.05)，血小板衍生生长因子、血管内皮生长因子、碱性成纤维因子水平下降(P < 0.05)；与糖尿病皮肤溃疡组相比，骨髓间充质干细胞组和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水平下降(P < 0.05)，血小板衍生生长因子、血管内皮生长因子、碱性成纤维因子水平上升(P < 0.05)；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-反应蛋白水平下降(P < 0.05)，血小板衍生生长因子、血管内皮生长因子、碱性成纤维因子水平上升(P < 0.05)，见表1。 2.6 人参皂苷诱导的骨髓间充质干细胞对糖尿病皮肤溃疡大鼠肉芽组织中成纤维细胞数和毛细血管密度量的影响在400倍高倍镜视野下观察结果显示，与空白组相比，糖尿病皮肤溃疡组大鼠肉芽组织中成纤维细胞数和新生毛细血管数密度下降(P < 0.05)；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肉芽组织中成纤维细胞数和新生毛细血管数密度上升(P < 0.05)；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肉芽组织中大鼠成纤维细胞数和新生毛细血管数密度上升(P < 0.05)，见表2，图5。 2.7 人参皂苷诱导的骨髓间充质干细胞对糖尿病皮肤溃疡大鼠肉芽组织内Wnt4及β-catenin mRNA表达水平的影响与空白组相比，糖尿病皮肤溃疡组大鼠肉芽组织内Wnt4及β-catenin mRNA表达水平上升(P < 0.05)；与糖尿病皮肤溃疡组相比，骨髓间充质干细胞组和人参皂苷诱导骨髓间充质干细胞组大鼠肉芽组织内Wnt4及β-catenin mRNA表达水平下降(P < 0.05)；和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肉芽组织内Wnt4及β-catenin mRNA表达水平下降(P < 0.05)，见表3。"

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