褐藻寡糖对创面的生物学效应:裂解程度依赖性

Biological effects of alginate oligosaccharides on wound:Degree of lysis dependence

目的探索不同裂解程度的褐藻寡糖对创面细胞和组织的生物学作用。方法采用不同孔隙的透析袋将褐藻寡糖分为分子量分别为<500、500~1000、1000~5000、5000~10000、10000~20000的5个组,通过分子量区分裂解程度,分别命名为实验组1~5。采用0.02%浓度前述褐藻寡糖与人角质形成细胞(Hum an keratinocytes,HaCat)、巨噬细胞共培养24 h,以正常培养的细胞为对照组,评估各组细胞增殖能力,免疫荧光检测Caspase 3评估细胞凋亡情况,活性氧类(ROS)试剂盒检测评估氧化应激情况。与巨噬细胞共培养24 h,ELISA检测IL-1β、IL-10、TNF-α,评估各组炎症因子释放情况,通过透射电镜观察细胞胞吞能力。通过微量肉汤法检测各组褐藻寡糖对金黄色葡萄球菌、大肠埃希菌、白色念珠菌的最低抑菌浓度(Minimum inhibitory concentration,MIC)和最低杀菌浓度(Minimum bactericidal concentration,MBC)。建立SD大鼠背部全层皮肤缺损创面模型,海藻酸钠溶液换药为阳性对照组,生理盐水换药为阴性对照组,0.02%浓度前述褐藻寡糖换药作为实验组,第6天和第12天观察创面愈合情况,取创面组织行HE染色观察组织学变化,并检测炎症因子、细胞凋亡和氧化应激情况。结果当褐藻寡糖分子量<500、500~1000时,细胞实验和动物实验(第6天)均提示其能有效促进细胞增殖和IL-10分泌,减少IL-1β、TNF-α分泌和细胞凋亡,相较于阴性对照组其可促进创面愈合,且当分子量<500时效果更明显,但促愈合效果弱于阳性对照组(P<0.05)。褐藻寡糖分子量>1000时,细胞实验提示前述作用均未与对照组出现明显差异(P>0.05),动物实验中当分子量处于5000~10000、10000~20000时创面愈合率增高(P<0.05),与阳性对照组相比,创面愈合率无明显差异(P>0.05),实验中观察到其在创面形成了薄膜结构,但其余指标无明显改变(P>0.05)。透射电镜未能观察到细胞吞噬能力增强表现。MIC和MBC检测各分子量褐藻寡糖在5%浓度下仍未能具有抑菌或杀菌作用。结论褐藻寡糖的生物学作用与分子量(裂解程度)严格相关,较低分子量的褐藻寡糖具有改善炎症因子分泌、促进细胞增殖、抑制细胞凋亡、改善氧化应激程度的作用,该作用并不依赖于褐藻寡糖是否进入细胞内;较高分子量的褐藻寡糖不具有明显的生物学作用,但具有成膜作用,间接促进创面愈合。但无论何种分子量的褐藻寡糖,均不具有抗菌作用。

Objective To explore the biological effects of alginate oligosaccharides with different degrees of lysis of wound cells and tissues.Methods Alginate oligosaccharides were divided into five groups with molecular weight ranges of<500,500-1000,1000-5000,5000-10000,and 10000-20000 using dialysis bags with pores of different sizes.The cleavage degree was distinguished according to molecular weight,and the categories named experimental groups 1-5 respectively.Brown algal oligosaccharide(0.02%concentration)was used to co-culture with human keratinocytes and macrophages for 24 h,taking normal cultured cells as the control group.The cell proliferation ability of each group was evaluated,Caspase3 was detected via immunofluorescence to evaluate cell apoptosis,and an ROS kit was used to evaluate oxidative stress.After co-culture with macrophages for 24 h,IL-1β,IL-10,and TNF-αwere detected using ELISA and the endocytosis ability of the cells was observed using transmission electron microscopy.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of fucoidan against Staphylococcus aureus,Escherichia coli,and Candida albicans were detected using the micro-broth method.SD rats with a full-thickness skin defect wound model on their backs were treated with sodium alginate solution as positive control group,normal saline dressing was used as negative control group,and 0.02%brown algal oligosaccharide as the experimental group.Wound healing was observed on days 6 and 12,the wound tissue was stained with HE to observe histological changes,and inflammatory factors,apoptosis,and oxidative stress were detected.Results At a fucoidan molecular weight of<500 or 500-1000,both the cell and animal experiments(on day 6)showed effective promotion of cell proliferation and IL-10 secretion,reduced IL-1β,TNF-α,and apoptosis levels.Compared with the negative control group,the effect of promoting wound healing was more obvious when the molecular weight was less than 1000,especially less than 500,but the effect of promoting wound healing was weaker than that of the positive control group(P<0.05).However,when the molecular weight of fucoidan was>1000,the cell experiments showed no significant differences from the control group(P>0.05).In the animal experiment,when the molecular weight was 5000-10000 or 10000-20000,the wound healing rate increased(P<0.05).Compared with the positive control group,there was no significant difference in wound healing rate(P>0.05).In the experiment,it formed a thin membrane structure in the wound,but the other indicators did not change significantly(P>0.05).Enhanced phagocytosis was not observed by transmission electron microscopy.MIC and MBC detection of alginate oligosaccharides with various molecular weights did not reveal antibacterial or bactericidal effects at 5%concentration.Conclusion The biological effects of fucoidan are strictly related to its molecular weight(cleavage degree).Low molecular weight fucoidan can improve the secretion of inflammatory factors,promote cell proliferation,inhibit cell apoptosis,and improve the degree of oxidative stress,which does not depend on whether fucoidan enters cells.High molecular weight fucoidan has no obvious biological effect,but forms a membrane and indirectly promotes wound healing.However,alginate oligosaccharide has no antibacterial effect no matter the molecular weight.