Effects and mechanisms of PIT on wound healing of full-thickness skin defects in diabetic mice
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摘要:
目的 探讨聚乙烯醇/离子液体-单宁酸复合水凝胶（PIT）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 该研究为成组设计及重复测量设计实验研究。制备聚乙烯醇-4-（1H-乙烯基咪唑）-1-亚甲基苯甲酸与氧化透明质酸交联的离子水凝胶基质,通过铜离子螯合负载单宁酸,构建PIT。配制1,1-二苯基-2-三硝基苯肼（DPPH）溶液,分别与单宁酸和PIT反应24 h后,采用紫外分光光度计检测DPPH自由基清除率。取小鼠巨噬细胞RAW264.7,采用随机数字表法（分组方法下同）分为加入磷酸盐缓冲液（PBS）培养的PBS组,以及均经过氧化氢处理12 h后分别常规培养、加入PIT培养的过氧化氢组、PIT组,培养24 h后,采用2',7'-二氯二氢荧光素二乙酸酯荧光探针法检测细胞中活性氧水平。取大肠埃希菌ATCC 25922、金黄色葡萄球菌ATCC 25923、耐甲氧西林金黄色葡萄球菌（MRSA）BNCC 337371、人脐静脉内皮细胞（HUVEC）,均分为PBS组、单宁酸组、PIT组,分别加入PBS、单宁酸溶液、PIT培养,细菌培养12 h后,采用平板菌落计数法计数菌落；细胞培养24 h后,行细胞成管实验,并测算成管总长度、分支节点数、分支数。前述实验样本数均为3。取18只8周龄雄性昆明小鼠,分为PBS组、单宁酸组、PIT组（每组6只）,构建为糖尿病全层皮肤缺损创面模型,伤后0 d（即刻）,分别于PBS组、单宁酸组、PIT组小鼠创面滴加PBS、单宁酸溶液、PIT处理,之后每天换药。观察伤后0、4、8、12 d创面愈合情况并计算伤后4、8、12 d创面愈合率。伤后12 d,取创面组织,行苏木精-伊红染色,观察创面上皮再生情况,并测量新生上皮厚度；行Masson染色,观察创面胶原纤维沉积情况,并计算胶原纤维阳性面积占比。 结果 反应24 h后,PIT的DPPH自由基清除率显著高于单宁酸（t=16.35,P<0.05）。培养24 h后,过氧化氢组RAW264.7细胞中活性氧水平显著高于PBS组（P<0.05）,PIT组RAW264.7细胞中活性氧水平显著低于过氧化氢组（P<0.05）。培养12 h后,PIT组大肠埃希菌、金黄色葡萄球菌、MRSA菌落数均显著少于PBS组及单宁酸组（P<0.05）。培养24 h后,与PBS组及单宁酸组比较,PIT组HUVEC成管总长度显著延长,且分支节点数与分支数显著增多（P<0.05）。伤后0~12 d,3组小鼠创面均逐步愈合。伤后4、8、12 d,PIT组小鼠创面愈合率分别为（31.6±2.0）%、（51.8±2.5）%、（97.9±1.5）%,均显著高于PBS组的（18.6±0.6）%、（39.5±2.0）%、（74.6±2.0）%和单宁酸组的（21.5±1.1）%、（40.7±0.8）%、（85.3±2.1）%（P<0.05）。伤后12 d,PBS组小鼠创面再上皮化不完全,胶原纤维分布稀疏且排列紊乱；单宁酸组小鼠创面再上皮化程度高于PBS组,胶原纤维呈束状分布但排列仍较松散；PIT组小鼠创面再上皮化程度高于单宁酸组,胶原纤维呈致密有序的层状排列。伤后12 d,与PBS组及单宁酸组比较,PIT组小鼠创面新生上皮厚度显著增厚且胶原纤维阳性面积占比显著增大（P<0.05）。 结论 PIT通过金属离子螯合驱动的抗菌、抗氧化、促血管生成多重机制,显著加速糖尿病小鼠全层皮肤缺损创面的愈合,并提高组织修复质量。 Abstract:Objective To investigate the effects and mechanisms of polyvinyl alcohol/ionic liquid-tannic acid composite hydrogel (PIT) on wound healing of full-thickness skin defects in diabetic mice. Methods This study adopted a grouped design and repeated measurement design for experimental research. An ionic hydrogel matrix crosslinked by polyvinyl alcohol-4-(1H)-vinylimidazole-1-methylene benzoic acid and oxidized hyaluronic acid was prepared, and tannic acid was loaded via Cu2+ chelation to construct PIT. A 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution was prepared, which was reacted with tannic acid and PIT respectively for 24 hours. An ultraviolet spectrophotometer was used to detect the DPPH radical scavenging rate. According to the random number table method (the same grouping method below), mouse macrophage RAW264.7 cells were divided into a phosphate buffered saline (PBS) group cultured with PBS, as well as a hydrogen peroxide group and a PIT group, in which cells were first treated with hydrogen peroxide for 12 hours and then respectively cultured under routine condition and with PIT. After 24 hours of culture, the 2',7'-dichlorodihydrofluorescein diacetate fluorescent probe method was adopted to detect the intracellular reactive oxygen species (ROS) level. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus (MRSA) BNCC 337371, and human umbilical vein endothelial cells (HUVEC) were collected, and all of them were divided into PBS group, tannic acid (TA) group, and PIT group, which were cultured with PBS, TA solution, and PIT, respectively. After 12 hours of bacterial culture, the plate colony counting method was used to count bacterial colonies; after 24 hours of cell culture, the tube formation assay was performed to measure the total tube length, the number of branching nodes, and the number of branches. The sample size of all the above experiments was 3. Eighteen 8-week-old male Kunming mice were selected and divided into PBS group, TA group, and PIT group (with 6 mice in each group) to build a full-thickness skin defect wound model of diabetes. At post injury day (PID) 0 (immediately), the wounds of mice in PBS group, TA group, and PIT group were treated with PBS, TA solution, and PIT by dropping, respectively, and then the dressing was changed every day. The wound healing status was observed at PID 0, 4, 8, and 12, and the wound healing rates at PID 4, 8, and 12 were calculated. At PID 12, wound tissue was harvested. Hematoxylin-eosin staining was performed to observe the status of wound re-epithelialization and measure the thickness of newborn epithelium. Masson staining was conducted to observe the deposition of collagen fibers in wounds and calculate the proportion of collagen fiber-positive area. Results After 24 hours of reaction, the DPPH radical scavenging rate of PIT was significantly higher than that of TA (t=16.35, P<0.05). After 24 hours of culture, the ROS level of RAW264.7 cells in hydrogen peroxide group was significantly higher than that in PBS group (P<0.05), and the ROS level of RAW264.7 cells in PIT group was significantly lower than that in hydrogen peroxide group (P<0.05). After 12 hours of culture, the bacterial colony counts of Escherichia coli, Staphylococcus aureus, and MRSA in PIT group were significantly less than those in PBS group and TA group (P<0.05). After 24 hours of culture, compared with those in PBS group and TA group, the total tube length of HUVECs in PIT group was significantly prolonged, and the number of branching nodes and the number of branches increased significantly (P<0.05). From PID 0 to 12, the wounds of mice in all three groups healed gradually. At PID 4, 8, and 12, the wound healing rates of mice in PIT group were (31.6±2.0)%, (51.8±2.5)%, and (97.9±1.5)%, respectively, which were significantly higher than (18.6±0.6)%, (39.5±2.0)%, and (74.6±2.0)% in PBS group and (21.5±1.1)%, (40.7±0.8)%, and (85.3±2.1)% in TA group (P<0.05). At PID 12, the re-epithelialization of wounds in mice of PBS group was incomplete, and collagen fibers were sparsely distributed with disordered arrangement; the degree of wound re-epithelialization of mice in TA group was higher than that in PBS group, and collagen fibers were distributed in bundles with loose arrangement; the degree of wound re-epithelialization of mice in PIT group was higher than that in TA group, and collagen fibers were densely and orderly arranged in layers. At PID 12, compared with those in PBS group and TA group, the thickness of newborn epithelium in wounds of mice in PIT group increased significantly, and the proportion of collagen fiber-positive area enlarged significantly (P<0.05). Conclusions PIT relies on multiple metal ion chelation-mediated mechanisms, including antibacterial activity, antioxidant activity, and angiogenesis promotion. It significantly accelerates the wound healing of full-thickness skin defects in diabetic mice, and improves the quality of tissue repair. -
Key words:
- Diabetes mellitus /
- Hydrogels /
- Oxidative stress /
- Tannic acid /
- Antibacterial property /
- Angiogenesis /
- Wound repair /
- Ionic liquid hydrogel
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参考文献
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图 1 PIT的成胶情况及微观形貌。1A.PIT成胶后倾斜时为稳定不流动的凝胶状态；1B.PIT呈多孔网状结构 扫描电子显微镜 ×200
注：PIT为聚乙烯醇/离子液体-单宁酸复合水凝胶
图 2 3组小鼠L929细胞培养24 h后活细胞与死细胞分布情况 钙黄绿素乙酰氧基甲酯-碘化丙啶 ×20。2A、2B、2C.分别为PBS组、单宁酸组、PIT组,均可见大量活细胞（绿色）、少量死细胞（红色）
注：磷酸盐缓冲液（PBS）组、单宁酸组、聚乙烯醇/离子液体-单宁酸复合水凝胶（PIT）组细胞分别加入PBS、单宁酸溶液、PIT培养
图 3 3组小鼠RAW264.7细胞培养24 h后活性氧水平 2',7'-二氯二氢荧光素二乙酸酯-Hoechst 33342 ×40。3A、3B、3C.分别为PBS组、过氧化氢组、PIT组,图3B的活性氧水平显著高于图3A、3C
注：磷酸盐缓冲液（PBS）组细胞加入PBS培养；过氧化氢组、聚乙烯醇/离子液体-单宁酸复合水凝胶（PIT）组细胞均经过氧化氢处理12 h后,分别行常规培养、加入PIT培养；活性氧阳性染色呈绿色,细胞核阳性染色呈蓝色
图 4 各3组大肠埃希菌ATCC 25922、金黄色葡萄球菌ATCC 25923与MRSA BNCC 337371培养12 h后的生长情况。4A、4B、4C.分别为PBS组、单宁酸组、PIT组大肠埃希菌,图4B、4C中菌落数相较于图4A明显减少；4D、4E、4F.分别为PBS组、单宁酸组、PIT组金黄色葡萄球菌,图4E、4F中菌落数相较于图4D明显减少；4G、4H、4I.分别为PBS组、单宁酸组、PIT组MRSA,图4H、4I中菌落数相较于图4G明显减少；4J.菌落数条图（样本数为3,
注：CFU为集落形成单位；磷酸盐缓冲液（PBS）组、单宁酸组、聚乙烯醇/离子液体-单宁酸复合水凝胶（PIT）组细菌分别加入PBS、单宁酸溶液、PIT培养；在图4J中,与PBS组比较,aP<0.05；与单宁酸组比较,bP<0.05；MRSA为耐甲氧西林金黄色葡萄球菌
图 5 3组人脐静脉内皮细胞培养24 h后成管情况 倒置相差显微镜 ×100。5A.PBS组成管数量少,无连续管状网络；5B.单宁酸组成管分支节点数、成管总长度明显优于图5A；5C.PIT组成管分支节点数、成管总长度明显优于图5B
注：磷酸盐缓冲液（PBS）组、单宁酸组、聚乙烯醇/离子液体-单宁酸复合水凝胶（PIT）组细胞分别加入PBS、单宁酸溶液、PIT培养
图 6 3组糖尿病小鼠伤后各时间点全层皮肤缺损创面愈合情况。6A、6B、6C.分别为PBS组伤后0、8、12 d创面,创面逐渐愈合；6D、6E、6F.分别为单宁酸组伤后0、8、12 d创面,图6F创面面积明显小于图6C；6G、6H、6I.分别为PIT组伤后0、8、12 d创面,图6H、6I创面面积明显小于图6E、6F；6J.创面愈合率折线图（样本数为6,
注：创面图中虚线方框为参照物,边长为10 mm；磷酸盐缓冲液（PBS）组、单宁酸组、聚乙烯醇/离子液体-单宁酸复合水凝胶（PIT）组创面分别滴加PBS、单宁酸溶液、PIT并每天换药；在图6J中,处理因素主效应,F=96.42,P<0.001；时间因素主效应,F=678.31,P<0.001；二者交互作用,F=24.57,P<0.001；与PBS组比较,aP<0.05；与单宁酸组比较,bP<0.05
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