小球藻提取物促进糖尿病小鼠皮肤创面愈合的作用研究

Study on the effect of Chlorella extract on promoting skin wound healing in diabetic mice

目的 探讨小球藻提取物(CE)对糖尿病小鼠皮肤创面的愈合作用。方法细胞毒性实验设置空白对照组(CON组),CE低(1 mg/L)、中(10 mg/L)和高(100 mg/L)剂量组;抗氧化实验分组增加了H_(2)O_(2)(100μmol/L)组,各实验组增加了100μmol/L H_(2)O_(2);抗炎实验分组增加了脂多糖(1 mg/L),各实验组增加了1 mg/L脂多糖干预。采用MTT法检测CE的细胞毒性;荧光探针法检测CE的抗氧化作用;荧光定量PCR检测CE的抗炎作用。设置空白CON组、CE低(0.5 g/L)、中(5 g/L)和高(50 g/L)剂量组,采用平板菌落计数法检测CE的抗菌能力。腹腔注射链脲佐菌素法诱导糖尿病小鼠模型,并采用随机数字表法分为CON组、CE低(0.5 g/L)、中(5 g/L)和高(50 g/L)剂量组,每组12只。在小鼠脊柱两侧对称性各做一个直径6 mm的圆形创面进行给药处理,每日1次,连续14 d。分别进行拍照并取材,记录创面愈合情况并对皮肤组织进行组织学(7 d和14 d)及免疫荧光染色(7 d)检测。结果体外实验:CE各剂量组对NIH-3T3细胞都有良好的细胞相容性;与H_(2)O_(2)组相比,CE低、中和高剂量组的活性氧(ROS)荧光信号依次减弱;与脂多糖组相比,CE低、中和高剂量组CD86的表达降低,CD206的表达升高(P<0.05);与CON组相比,CE低、中和高剂量组对细菌生长的抑制作用依次增强(P<0.05)。体内实验:术后第7天和第14天,CE组糖尿病小鼠创面的闭合速率加快,创面愈合效果明显;对创面组织分别进行HE染色和Masson染色发现,与CON组相比,CE低、中和高剂量组糖尿病小鼠创面有更多的胶原沉积。免疫荧光检测结果显示,与CON组相比,CE治疗后的创面的α平滑肌肌动蛋白(α-SMA)、血小板-内皮细胞黏附分子(CD31)及巨噬细胞甘露糖受体(CD206)水平升高,而白细胞分化抗原86(CD86)表达水平降低。结论CE可有效促进糖尿病小鼠创面愈合,其机制可能与其抗氧化、抗炎和抗菌有关。

Objective To investigate the effect of Chlorella vulgaris extract(CE)on skin wound healing in diabetic mice.Methods The blank control group(CON group),CE low(1 mg/L),medium(10 mg/L)and high(100 mg/L)dose groups were set up in vitro cytotoxicity assay.The additional H_(2)O_(2)(100µmol/L)group was set up for antioxidant experiments,and the concentration of H_(2)O_(2)(100µmol/L)in each experimental group was also added.The additional lipopolysaccharide(1 mg/L)group was set up for the anti-inflammatory experiments,and the concentration of lipopolysaccharide(1 mg/L)in each experimental group was also added.The cytotoxicity of CE was detected by MTT method.The antioxidant activity of CE was investigated by fluorescent probe assay.The anti-inflammatory effect of CE was detected by fluorescent quantitative PCR.The blank control group(CON group),CE low(0.5 g/L),medium(5 g/L)and high(50 g/L)dose groups were set up,and the antibacterial properties of CE were examined by plate colony counting method.The diabetic mouse model was induced by streptozotocin,and model mice were divided into the control group,the CE low(0.5 g/L),medium(5 g/L)and high(50 g/L)dose groups using the random number table method,with 12 mice in each group.After the model was successfully established,two 6 mm wounds were symmetrically created on each side of spine of mouse.The wounds were administered once daily for a total of 14 consecutive days.The wound healing was observed and photographed on days 0,7 and 14 respectively.The wound samples were taken and processed for histology(7 d and 14 d)and immunofluorescence(7 d).Results In vitro experiments:CE low,medium and high dose groups showed good cytocompatibility with NIH-3T3 cells compared to the control group,and CE low,medium and high dose groups showed weaker reactive oxygen species(ROS)fluorescence signal compared to the H_(2)O_(2) group.CE low,medium and high dose groups showed lower expression of CD86 and higher expression of CD206 compared to the control group(P<0.05).The inhibition of bacterial growth was enhanced in the CE low,medium and high dose groups compared to the control group(P<0.05).In vivo experiment results demonstrated that wound healing rates on the 7th and 14th day after operation were accelerated in the CE group,and the wound healing effect was obvious.Results of HE and Masson staining showed that there were more collagen deposition in wound in the CE low,medium and high dose groups compared to the control group.Results of the immunofluorescence assay showed that the higher expression levels ofα-smooth muscle actin(α-SMA),platelet endothelial cell adhesion molecule-1(CD31)and macrophage mannose receptor(CD206)after treatment,and the lower levels of leukocyte differentiation antigen 86(CD86)in wound compared to the control group.Conclusion CE can effectively promote wound healing in diabetic mice,and the mechanism may be related to its antioxidant,anti-inflammatory and antibacterial effects.