摘要
目的探讨黄芩素对糖尿病小鼠全层皮肤缺损创面愈合的影响及其机制。方法该研究为实验研究。从5只8~12周龄雄性C57BL/6J小鼠中分离单核细胞并诱导分化为巨噬细胞,进行后续实验。按照随机数字表法(分组方法下同),将高糖环境中的巨噬细胞分为采用1μg/mL内毒素/脂多糖(LPS)和相应终物质的量浓度黄芩素处理的0μmol/L黄芩素组(不加黄芩素)、5μmol/L黄芩素组、15μmol/L黄芩素组、25μmol/L黄芩素组、50μmol/L黄芩素组、75μmol/L黄芩素组,处理48 h后,用酶标仪检测细胞增殖活性。将高糖环境中的巨噬细胞分为采用1μg/mL LPS处理的LPS组和用50μmol/L黄芩素+1μg/mL LPS处理的LPS+黄芩素组,处理48 h后,采用免疫荧光法检测细胞中诱导型一氧化氮合酶(iNOS)与CD80双阳性细胞百分比、精氨酸酶1(Arg1)与CD206双阳性细胞百分比,用酶联免疫吸附测定法检测细胞的白细胞介素1β(IL-1β)、IL-6、IL-23、IL-10、胰岛素样生长因子(IGF)、转化生长因子β1(TGF-β1)分泌水平,用荧光探针法检测细胞中活性氧表达,用蛋白质印迹法检测细胞中核因子κB蛋白表达,采用免疫荧光法观察细胞中核因子2表达。细胞实验样本数均为3。取24只8周龄雄性db/db小鼠,于其背部制备全层皮肤缺损创面后将其分为黄芩素组和生理盐水组(每组12只小鼠),伤后3 d分别向小鼠创面注射50μmol/L黄芩素和生理盐水。于伤后4、8、12 d观察创面愈合情况并计算残余创面面积百分比;取伤后8 d创面组织,行苏木精-伊红染色观察表皮新生情况及炎症细胞浸润情况,采用蛋白质印迹法检测CD31的蛋白表达,采用酶标仪检测活性氧表达。动物实验样本数均为6。结果处理48 h后,仅50μmol/L黄芩素组巨噬细胞增殖活性明显高于0μmol/L黄芩素组(P<0.05)。处理48 h后,LPS+黄芩素组巨噬细胞中iNOS与CD80双阳性细胞百分比[(21.0±2.4)%]明显低于LPS组[(66.6±4.5)%,t=15.63,P<0.05],LPS+黄芩素组巨噬细胞中Arg1与CD206双阳性细胞百分比[(59.1±2.1)%]明显高于LPS组[(18.6±1.7)%,t=25.38,P<0.05];与LPS组比较,LPS+黄芩素组巨噬细胞的IL-1β、IL-6和IL-23分泌水平均明显降低(t值分别为14.26、15.95、12.23,P<0.05),IL-10、IGF和TGF-β1分泌水平均明显升高(t值分别为8.49、11.98、13.84,P<0.05);LPS+黄芩素组巨噬细胞中活性氧表达较LPS组明显降低(t=5.54,P<0.05);与LPS组相比,LPS+黄芩素组巨噬细胞的细胞核中核因子κB的蛋白表达明显降低(t=36.22,P<0.05),细胞质中核因子κB的蛋白表达明显升高(t=14.47,P<0.05),细胞核内核因子2表达增多。伤后4、8 d,黄芩素组小鼠创面面积均明显小于生理盐水组,且黄芩素组小鼠创面于伤后12 d完全愈合。伤后4、8、12 d,黄芩素组小鼠残余创面面积百分比均明显低于生理盐水组(t值分别为13.29、10.08、11.72,P<0.05)。伤后8 d,与生理盐水组比较,黄芩素组小鼠创面组织再上皮化明显,炎症细胞浸润减少,CD31蛋白表达明显增多(t=17.23,P<0.05),活性氧表达明显降低(t=5.78,P<0.05)。结论黄芩素通过降低细胞中的活性氧水平、推动巨噬细胞向M2型极化,从而减轻巨噬细胞的炎症反应,促进糖尿病小鼠全层皮肤缺损创面愈合。
ObjectiveTo investigate the effects and mechanism of baicalin on the wound healing of full-thickness skin defects in diabetic mice.MethodsThis study was an experimental research.Mononuclear cells were isolated from five male C57BL/6J mice aged 8-12 weeks and induced to differentiate into macrophages for conducting the subsequent experiments.According to the random number table(the same grouping method below),macrophages in a high-glucose environment were divided into 0μmol/L baicalin group(no baicalin was added),5μmol/L baicalin group,15μmol/L baicalin group,25μmol/L baicalin group,50μmol/L baicalin group,and 75μmol/L baicalin group treated with the corresponding final molarity of baicalin and 1μg/mL endotoxin/lipopolysaccharide(LPS).After treatment for 48 hours,the cell proliferation activity was detected using a microplate reader.Macrophages in a high-glucose environment were divided into LPS group treated with 1μg/mL LPS and LPS+baicalin group treated with 50μmol/L baicalin+1μg/mL LPS.After treatment for 48 hours,the percentage of double-positive cells for inducible nitric oxide synthase(iNOS)and CD80,as well as that for arginase 1(Arg1)and CD206 among the cells,were detected using immunofluorescence method,the secretion levels of interleukin 1β(IL-1β),IL-6,IL-23,IL-10,insulin-like growth factor(IGF),and transforming growth factorβ1(TGF-β1)by the cells were detected using enzyme-linked immunosorbent assay,the expression of reactive oxygen species in the cells was detected using a fluorescent probe method,the protein expression of nuclear factorκB in the cells were detected using Western blotting,and the expression of nuclear factor 2 in the cells was observed using immunofluorescence method.The number of cell experimental samples was 3.Twenty-four 8-week-old male db/db mice were selected.After preparing full-thickness skin defect wounds on their backs,they were divided into baicalin group and normal saline group(with 12 mice in each group).On the third day after injury,50μmol/L baicalin and normal saline were injected into the wounds of mice,respectively.The wound healing situation was observed and the percentage of the residual wound area was calculated on the 4 th,8 th,and 12 th day after injury.The wound tissue was sampled on the 8 th day after injury,hematoxylin-eosin staining was performed to observe the epithelial regeneration and inflammatory cell infiltration,the protein expression of CD31 was detected by Western blotting,and the expression of reactive oxygen species was detected by a microplate reader.The number of animal experimental samples was 6.ResultsAfter treatment for 48 hours,only the proliferation activity of macrophages in 50μmol/L baicalin group was significantly higher than that in 0μmol/L baicalin group(P<0.05).After treatment for 48 hours,the percentage of double-positive cells for iNOS and CD80 among the macrophages in LPS+baicalin group was(21.0±2.4)%,which was significantly lower than(66.6±4.5)%in LPS group(t=15.63,P<0.05);the percentage of double-positive cells for Arg1 and CD206 among the macrophages in LPS+baicalin group was(59.1±2.1)%,which was significantly higher than(18.6±1.7)%in LPS group(t=25.38,P<0.05);compared with those in LPS group,the secretion levels of IL-1β,IL-6,and IL-23 by the macrophages in LPS+baicalin group were significantly decreased(with t values of 14.26,15.95,and 12.23,respectively,P<0.05),while the secretion levels of IL-10,IGF,and TGF-β1 were significantly increased(with t values of 8.49,11.98,and 13.84,respectively,P<0.05);the expression of reactive oxygen species in the macrophages in LPS+baicalin group was significantly lower than that in LPS group(t=5.54,P<0.05);compared with those in LPS group,the protein expression of nuclear factorκB in the nucleus of the macrophages in LPS+baicalin group was significantly decreased(t=36.22,P<0.05),while that in the cytoplasm was significantly increased(t=14.47,P<0.05),and the expression of nuclear factor 2 in the nucleus was increased.On the 4 th and 8 th day after injury,the wound area of mice in baicalin group was significantly smaller than that in normal saline group,and the wounds of mice in baicalin group completely healed on the 12 th day after injury.On the 4 th,8 th,and 12 th day after injury,the residual wound area percentage of mice in baicalin group was significantly lower than that in normal saline group(with t values of 13.29,10.08,and 11.72,respectively,P<0.05).On the 8 th day after injury,compared with those in normal saline group,the wound tissue of mice in baicalin group showed significant re-epithelization,the infiltration of inflammatory cells was reduced,the expression of CD31 protein was significantly increased(t=17.23,P<0.05),and the expression of reactive oxygen species was significantly reduced(t=5.78,P<0.05).ConclusionsBaicalin alleviates the inflammatory response of macrophages by lowering the level of reactive oxygen species in cells and promoting the polarization of macrophages to the M2 type,thereby facilitating the healing of full-thickness skin defect wounds in diabetic mice.
作者
施彦
易亮
张伟强
刘妮可
文辉才
杨荣华
Shi Yan;Yi Liang;Zhang Weiqiang;Liu Nike;Wen Huicai;Yang Ronghua(Department of Plastic,Medical Center of Burn Plastic and Wound Repair,the First Affiliated Hospital,Jiangxi Medical College,Nanchang University,Nanchang330006,China;Department of Burn,Plastic Surgery and Wound Repair,the Second Affiliated Hospital(Guangzhou First People's Hospital),South China University of Technology,Guangzhou510180,China)
出处
《中华烧伤与创面修复杂志》
CAS
CSCD
北大核心
2024年第11期1085-1094,共10页
Chinese Journal of Burns And Wounds
基金
国家自然科学基金面上项目(82060350,82272276)
中国高校产学研创新基金(2021JH028)
广东省基础与应用基础研究基金(2022A1515110490,2022A1515012160)深圳市自然科学基金(JCYJ20220530152015036)。
关键词
糖尿病
巨噬细胞
活性氧
黄芩素
创面修复
炎症反应
Diabetes mellitus
Macrophages
Reactive oxygen species
Baicalin
Wound repair
Inflammatory response
