Effects and mechanism of baicalin on wound healing of full-thickness skin defects in diabetic mice
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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1 μg/mL内毒素/脂多糖（LPS）和相应终物质的量浓度黄芩素处理的0 μmol/L黄芩素组（不加黄芩素）、5 μmol/L黄芩素组、15 μmol/L黄芩素组、25 μmol/L黄芩素组、50 μmol/L黄芩素组、75 μmol/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1 μg/mL LPS处理的LPS组和用50 μmol/L黄芩素+1 μg/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50 μmol/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50 μmol/L黄芩素组巨噬细胞增殖活性明显高于0 μmol/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。 Abstract:Objective To investigate the effects and mechanism of baicalin on the wound healing of full-thickness skin defects in diabetic mice. Methods This 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 0 μmol/L baicalin group (no baicalin was added), 5 μmol/L baicalin group, 15 μmol/L baicalin group, 25 μmol/L baicalin group, 50 μmol/L baicalin group, and 75 μmol/L baicalin group treated with the corresponding final molarity of baicalin and 1 μg/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 1 μg/mL LPS and LPS+baicalin group treated with 50 μmol/L baicalin+1 μg/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 factor β1 (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 factor κB 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, 50 μmol/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 4th, 8th, and 12th day after injury. The wound tissue was sampled on the 8th 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. Results After treatment for 48 hours, only the proliferation activity of macrophages in 50 μmol/L baicalin group was significantly higher than that in 0 μmol/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 factor κB 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 4th and 8th 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 12th day after injury. On the 4th, 8th, and 12th 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 8th 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). Conclusions Baicalin 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. -
Key words:
- Diabetes mellitus /
- Macrophages /
- Reactive oxygen species /
- Baicalin /
- Wound repair /
- Inflammatory response
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参考文献
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图 1 6组小鼠巨噬细胞处理48 h后增殖活性比较（样本数为3,
注：以吸光度值表示细胞增殖活性；横坐标下方1、2、3、4、5、6分别指示0 μmol/L黄芩素组（不加黄芩素）、5 μmol/L黄芩素组、15 μmol/L黄芩素组、25 μmol/L黄芩素组、50 μmol/L黄芩素组、75 μmol/L黄芩素组,各组用相应物质的量浓度的黄芩素以及1 μg/mL内毒素/脂多糖处理；与0 μmol/L黄芩素组比较,aP<0.05
图 2 2组小鼠巨噬细胞处理48 h后活性氧表达情况 DCFH-DA-Hoechst 33342×100。2A.LPS组细胞核与DCFH-DA染色重叠图片,活性氧表达较多；2B.LPS+黄芩素组细胞核与DCFH-DA染色重叠图片,活性氧表达少于图2A
注：内毒素/脂多糖（LPS）组细胞经1 μg/mL LPS处理,LPS+黄芩素组细胞经50 μmol/L黄芩素+1 μg/mL LPS处理；DCFH-DA为2',7'-二氯二氢荧光素二乙酸酯；细胞核阳性染色为蓝色,活性氧阳性染色为绿色
图 3 蛋白质印迹法检测的2组小鼠巨噬细胞处理48 h后细胞核与细胞质中核因子κB表达情况。3A.细胞核；3B.细胞质
注：GAPDH为3-磷酸甘油醛脱氢酶,条带上方1、2均分别指示细胞经1 μg/mL内毒素/脂多糖（LPS）处理的LPS组、细胞经50 μmol/L黄芩素+1 μg/mL LPS处理的LPS+黄芩素组
图 4 2组小鼠巨噬细胞处理48 h后核因子2表达情况 4',6-二脒基-2-苯基吲哚-Alexa Fluor 488×100。4A、4B、4C.分别为LPS组细胞核染色、核因子2染色、细胞核与核因子2染色重叠图片,细胞核完整,核因子2的蛋白主要定位在细胞质中；4D、4E、4F.分别为LPS+黄芩素组细胞核染色、核因子2染色、细胞核与核因子2染色重叠图片,细胞核完整,核因子2的蛋白主要定位在细胞核中且表达多于图4A
注：内毒素/脂多糖（LPS）组细胞经1 μg/mL LPS处理,LPS+黄芩素组细胞经50 μmol/L黄芩素+1 μg/mL LPS处理；细胞核阳性染色为蓝色,核因子2阳性染色为绿色
图 5 2组糖尿病小鼠全层皮肤缺损创面伤后各时间点愈合情况。5A、5B、5C、5D.分别为生理盐水组伤后0（即刻）、4、8、12 d创面,逐渐愈合；5E、5F、5G、5H.分别为黄芩素组伤后0、4、8、12 d创面,图5E创面面积与图5A相同,图5F、5G、5H创面面积分别小于图5B、5C、5D
注：生理盐水组创面注射生理盐水,黄芩素组创面注射50 μmol/L黄芩素
图 6 2组糖尿病小鼠全层皮肤缺损创面伤后8 d表皮新生及炎症细胞浸润情况 苏木精-伊红×400。6A.生理盐水组炎症细胞多,有少量表皮再生；6B.黄芩素组炎症细胞较图6A少,新生表皮较图6A完整
注：生理盐水组创面注射生理盐水,黄芩素组创面注射50 μmol/L黄芩素
图 7 蛋白质印迹法检测的2组糖尿病小鼠全层皮肤缺损创面伤后8 d组织中CD31蛋白表达
注：GAPDH为3-磷酸甘油醛脱氢酶；条带上方1、2分别指示创面注射生理盐水的生理盐水组、创面注射50 μmol/L黄芩素的黄芩素组
Table 1. 2组小鼠巨噬细胞处理48 h后炎症因子分泌水平比较（pg/mL,
组别 样本数 IL-1β IL-6 IL-23 IL-10 IGF TGF-β1 LPS组 3 15.2±1.4 21.6±1.6 18.8±2.0 2.1±1.1 2.4±1.6 2.6±1.0 LPS+黄芩素组 3 2.6±0.7 3.2±1.1 2.1±1.3 15.6±2.5 19.3±1.8 18.0±1.7 t值 14.26 15.95 12.23 8.49 11.98 13.84 P值 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 注：内毒素/脂多糖（LPS）组细胞经1 μg/mL LPS处理,LPS+黄芩素组细胞经50 μmol/L黄芩素+1 μg/mL LPS处理；IL为白细胞介素,IGF为胰岛素样生长因子,TGF-β1为转化生长因子β1 
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Table 2. 2组糖尿病小鼠全层皮肤缺损创面伤后各时间点残余创面面积百分比比较（%,
组别 样本数 4 d 8 d 12 d 生理盐水组 6 75.6±2.2 44.6±4.1 13.6±2.9 黄芩素组 6 50.5±4.1 23.9±3.0 0 t值 13.29 10.08 11.72 P值 <0.001 <0.001 <0.001 注：生理盐水组创面注射生理盐水,黄芩素组创面注射50 μmol/L黄芩素；处理因素主效应,F=1.26,P=0.300；时间因素主效应,F=2 961.00,P<0.001；两者交互作用,F=56.06,P<0.001
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