载姜黄素静电纺丝纤维膜的制备及其抗菌与抗氧化性能
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Preparation and antibacterial and antioxidant properties of curcumin-loaded electrospun membranes
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通讯作者:
收稿日期: 2025-09-5 修回日期: 2025-12-23
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Received: 2025-09-5 Revised: 2025-12-23
作者简介 About authors
林晓静(2000—),女,硕士生。主要研究方向为生物医用纤维材料制备与功能评价。
为克服慢性伤口愈合中活性氧积累、细菌感染等问题,赋予纤维膜抗菌和抗氧化特性,采用静电纺丝技术成功制备了以聚乳酸-羟基乙酸共聚物/聚己内酯为纤维基质、负载不同质量分数姜黄素的载药纤维膜。借助扫描电子显微镜、红外光谱仪、X射线衍射仪、差示扫描量热仪、接触角测量仪,通过自由基清除法、振荡法等手段对其理化性能、表面浸润性、载药率与包封率、体外药物释放行为、抗氧化与抗菌性能进行系统表征。结果表明:所得静电纺纤维膜形貌良好,纤维直径分布均匀,且亲水性随载药量增加而显著提高;负载2.5%和5%姜黄素的静电纺纤维膜的包封率均高于97%,并展现出持续释药能力;姜黄素的引入赋予纤维膜广谱抗菌性和优异的抗氧化性,其中负载5%姜黄素的静电纺纤维膜对金黄色葡萄球菌抑菌率超过90%,抗氧化率高于80%,在伤口修复领域呈现巨大的应用潜力。
关键词:
Objective Chronic wounds are characterized by persistent bacterial infection and excessive reactive oxygen species accumulation, which impede the healing process. Poly(lactic-co-glycolic acid)/polycaprolactone (PLGA/PCL) electrospun membranes exhibit limited antibacterial efficacy despite demonstrating favorable dimensional stability and mechanical properties. This study addresses these limitations by fabricating a dual-functional fiber-based dressing with integrated antibacterial and antioxidant properties to promote chronic wound regeneration. Method Curcumin-loaded PLGA/PCL fiber membranes (FM-Cur) with varying curcumin (Cur) mass concentrations were fabricated using electrospinning technology. The morphology and fiber diameter distribution were analyzed by scanning electron microscopy. Chemical constitution and crystallinity were examined using Fourier transform infrared spectroscopy and X-ray diffraction, respectively. Thermal properties were assessed using differential scanning calorimetry. Surface wettability was determined through water contact angle measurements. Antioxidant activity was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays, while antibacterial efficacy was tested using colony counting methods. Results All FM-Cur samples exhibited well-defined fibrous morphologies with uniform diameter distributions. Hydrophilicity significantly increased with Cur loading, evidenced by water contact angle reduction from (134.0±0.5)° (FM) to (87.5±1.6)° (FM-10Cur). Quantitative analysis revealed that both absorbance and drug loading capacity (DLC) values in aqueous-organic solvent systems (PBS/ethanol) and pure ethanol increased proportionally with Cur content. Due to the inherent hydrophobicity of Cur, DLC and encapsulation efficiency values measured in PBS/ethanol systems were significantly lower than those in ethanol. Authentic drug loading parameters aligned closely with ethanol-based determinations, where FM-2.5Cur and FM-5Cur achieved encapsulation rate > 97%, confirming the fiber carrier's efficacy in enhancing Cur bioavailability while enabling sustained release, a critical feature for chronic wound management. The optimized FM-5Cur formulation demonstrated exceptional dual functionality, exhibiting over 90% antibacterial rate against S. aureus and over 80% DPPH radical scavenging capacity. This synergistic performance effectively mitigates persistent inflammation in chronic wounds, concurrently neutralizing bacterial infection and oxidative stress, thereby accelerating tissue regeneration processes. Conclusion Electrospun PLGA/PCL/Cur membranes are established as dual-functional wound dressings through a single-step fabrication process. The membranes demonstrate clinically relevant antibacterial and antioxidant capabilities, with encapsulation efficiency exceeding 97% ensuring optimal therapeutic delivery. Key performance metrics include over 90% antibacterial rate against S. aureus and over 80% DPPH scavenging capacity, directly addressing critical healing barriers in chronic wounds. Crucially, the fabrication method preserves structural integrity without compromising bioactivity. These results support clinical translation potential for diabetic ulcer and burn wound management, where concurrent infection and oxidative stress impede healing.
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本文引用格式
林晓静, 毛迎, 陈文兴, 吕汪洋.
LIN Xiaojing, MAO Ying, CHEN Wenxing, LÜ Wangyang.
慢性伤口不仅严重影响患者的生活质量,也给社会医疗系统带来沉重负担[1]。相关研究表明,活性氧(ROS)的过量积累以及持续存在的细菌感染,是导致慢性伤口难以愈合的关键因素[2]。为此,研究者致力于开发具有单一或多功能特性的纤维基静电纺敷料,通过选用不同性质与类型的聚合物,以适配伤口愈合不同阶段的需求[3]。此类敷料可模拟天然细胞外基质的结构与功能,为细胞增殖提供有利微环境,从而促进组织修复[4]。理想的伤口敷料除应具备屏障功能以抵御细菌入侵外,还应兼具清除自由基、诱导细胞迁移与增殖以及协助组织再生的能力[5-6]。在前期研究[7]中,已对聚乳酸-羟基乙酸共聚物(PLGA)/聚己内酯(PCL)复合基材(FM)进行了初步评价,证实其具有良好的尺寸稳定性和一定的力学支撑性能,然而该材料抗菌性能不足,难以满足慢性创面修复的实际应用需求[8]。
基于Cur的抗菌与抗氧化特性,为拓展其在慢性伤口修复中的应用潜力,本文采用静电纺丝技术制备了不同Cur含量的PLGA/PCL载药纤维膜(FM-Cur),系统表征了纤维膜的基本结构性能、药物负载与释放行为,并对其抗氧化及抗菌性能进行评价,为开发兼具抗菌与抗氧化功能的载药静电纺丝膜提供实验参考。
1 实验部分
1.1 实验材料
聚乳酸-羟基乙酸共聚物(PLGA,乳酸与羟基乙酸的量比为50∶50,平均分子量为110 000),济南岱罡生物材料有限公司;聚己内酯(PCL,平均分子量为80 000)、无水乙醇,上海麦克林生化科技有限公司;二氯甲烷(DCM),上海展云化工有限公司;N,N-二甲基甲酰胺(DMF)、姜黄素(Cur),上海阿拉丁生化科技有限公司;1,1-二苯基-2-三硝基苯肼(DPPH),翌圣生物科技(上海)股份有限公司;磷酸盐缓冲溶液(PBS),沃瑞达斯实验试剂耗材;金黄色葡萄球菌ATCC 6538、大肠埃希菌ATCC 8739,上海鲁微科技有限公司。
1.2 载姜黄素静电纺丝膜的制备
将一定量的Cur、PLGA以及PCL溶解于DCM/DMF(体积比为7∶3)混合溶剂中,制备质量分数为16%的纺丝液,其中PLGA与PCL质量比为5∶5,Cur的质量分数分别为0%、2.5%、5%、10%(基于聚合物(PLGA/PCL)总质量)。利用TL-Pro型静电纺丝机(深圳市通力微纳科技有限公司)将配制好的纺丝液制备成纳米纤维膜,并命名为FM、FM-2.5Cur、FM-5Cur、FM-10Cur。静电纺丝时,将规格为21G的针头连接到15 kV的正电压电源上,并以2 mL/h的恒定流速从注射器中推出溶液。将制备好的纳米纤维膜置于37 ℃烘箱中干燥24 h,以去除纤维膜中残留的有机溶剂。
1.3 测试与表征
1.3.1 理化性能表征
采用Geini 500型场发射扫描电子显微镜(德国CarL Zeiss公司)对纳米纤维膜的微观形貌进行观察。并利用图像分析软件Image J在SEM照片中随机选取100根纤维进行直径测量,统计分析纤维的平均直径及标准差。
采用Nicolet iS50型红外光谱仪(美国Thermo公司)对样品进行表征。选用ATR法测定样品的红外谱图,其中扫描范围为4 000~400 cm-1。并利用Empyrean型X射线衍射仪(荷兰Malvern Panalytical公司)进行X衍射图谱测试,扫描范围2θ为5°~40°。
采用TGA/DSC1型差示扫描量热仪(瑞士METTLER公司)测试纤维膜的热性能。将5~8 mg样品压入仪器坩埚内,于N2氛围下,以10 ℃/min升温速率由室温升温至250 ℃,得到升温曲线。
1.3.2 接触角测试
根据GB/T 30447—2013《纳米薄膜接触角测量方法》,使用DSA25型接触角测量仪(德国KRUSS公司)进行接触角测试。将体积为4 μL的水滴加至材料表面,并在液滴接触样品的瞬间测定样品的表面静态水接触角。
1.3.3 载药率和包封率测试
取适量样品裁剪为1 cm × 1 cm大小,精确称量。将样品分别分散于10 mL的PBS/乙醇(体积比为9∶1)混合溶剂和乙醇溶剂中,经超声波细胞破碎机处理30 min,离心后取上层清液。采用酶标仪于425 nm波长处测定吸光度值,并由Cur标准曲线计算溶液中的Cur质量浓度,样品载药率D(%)和包封率E(%)分别按下式计算:
式中:MDN为样品中Cur的实际质量,g;C为样品中Cur的理论含量,%;MN为载药静电纺丝膜的质量,g。
绘制Cur标准曲线。精密称量Cur标准品,经逐级稀释配制成0.5~10 μg/mL系列质量浓度标准溶液,测定425 nm波长下的吸光度,绘制标准曲线,如图1所示。
图1
对Cur体外药物释放的评价。将样品裁剪成10 mm × 30 mm大小,精确称量后置于50 mL PBS缓冲溶液(pH值为7.4)中,于37 ℃恒温振荡器中进行释放实验。在预设时间点5 min、10 min、20 min、30 min、1 h、2 h、3 h、5 h、7 h、9 h、12 h、1 d、2 d、3 d、5 d、7 d、14 d、21 d、28 d后分别取样2.5 mL,并补充等体积PBS缓冲溶液。取出的样品与无水乙醇按体积比9∶1混合,测定425 nm处吸光度,依据姜黄素标准曲线换算得到各时间点药物释放质量浓度,逐时间点累加计算累积释放量,结合实际载药量计算累积释放率。
1.3.4 DPPH自由基清除率计算
采用DPPH自由基清除法测定样品的清除率。将少量样品搅拌溶解在0.1 mmol/L的DPPH乙醇溶液中,在不同时间点(5、10、20、30、40、50、60、120 min)取样,测定溶液在528 nm下的吸光度。DPPH清除率Ds(%)通过下式计算:
式中,As和Ac分别为样品及对照样品的吸光度值。
1.3.5 抗菌性能测试
参照 GB/T 20944.3—2008《纺织品 抗菌性能的评价 第3部分:振荡法》测试样品的抗菌性能,按照下式计算抑菌率A(%):
式中,Ct和St分别为对照样和测试样活菌数的平均值,CFU/mL。采用标准平板计数法测定。
1.3.6 统计学分析
实验数据均以平均值 ± 标准差表示。组间的统计分析通过单因素方差确定。每组样本至少进行3次平行实验(n≥3)。*表示P<0.05、**表示P<0.01、***表示P<0.001,代表组间差异具有统计学意义。
2 结果与讨论
2.1 理化性能分析
图2示出载药静电纺丝膜的形貌和直径分布。与FM相比,负载Cur的静电纺丝膜呈现出明显的橙黄色,且其颜色随Cur负载量的增加而逐渐加深。这主要归因于Cur本身为橙黄色结晶粉末。所有载药静电纺丝膜均具备良好的纤维形态,未见串珠结构,纤维直径分布均匀,表面均未观察到其它异相结构,表明Cur在纺丝液中完全溶解且分散均匀。该结构赋予静电纺丝膜孔隙率高、比表面积大的特点,不仅便于与外界进行气体交换,更能有效促进药物的负载与释放。此外,制备的纳米纤维具备网状多孔结构,模拟了天然细胞外基质的结构特点,为细胞生长提供了适宜的微环境,有助于加速伤口愈合。FM、FM-2.5Cur、FM-5Cur和FM-10Cur的平均直径分别为(0.96±0.01)、(0.94±0.02)、(1.06±0.03)、(0.87±0.01) μm。
图2
图2
不同Cur含量载药静电纺丝膜形貌与直径分布
Fig.2
Morphology and diameter distribution of drug-loaded electrospun membranes with different Cur contents. (a) Macroscopic morphologies; (b) Microscopic morphologies; (c) Diameter distribution histogram
图3
图3
不同Cur含量载药静电纺丝膜的红外光谱图
Fig.3
FT-IR spectra of electrospun membranes with different Cur contents
图4(a)示出Cur粉末、FM及不同Cur含量的载药静电纺丝膜的XRD谱图。所有载药静电纺丝膜均在2θ为22°和22.7°处出现特征衍射峰,归属于PCL作为半结晶聚合物的结晶结构。随着Cur添加量的增加,载药静电纺丝膜的结晶度(Xc)逐渐降低,表明Cur的引入在一定程度上抑制了PCL的结晶能力。在载药静电纺丝膜的XRD谱中未观察到Cur的特征结晶衍射峰,说明药物在纤维基质中以无定形态分散。
图4
图4
不同Cur含量载药静电纺丝膜的XRD谱图及DSC曲线
Fig.4
XRD pattern (a) and DSC curves (b) spectra of electrospun membranes with different Cur contents
图4(b)示出Cur粉末、FM及不同Cur含量的载药静电纺丝膜的DSC曲线。除Cur外,所有样品在59 ℃附近均出现吸热峰,对应于PLGA的熔融转变。Cur在171 ℃处出现的吸热峰归因于其熔化并伴随脱水脱羟基过程;而在所有载药静电纺丝膜中该峰均未出现,表明在纺丝液制备过程中Cur已充分溶解,其原有结晶结构被破坏,导致药物以无定形形式存在于纤维中,这与XRD分析结果一致。
2.2 表面浸润性分析
表1示出纳米纤维膜的接触角测试结果。FM、FM-2.5Cur、FM-5Cur和FM-10Cur的接触角分别为(134.0±0.5)°、(125.4±0.9)°、(113.6±1.1)°和(87.5±1.6)°。对比不同Cur含量的纤维膜,FM表现出较强的疏水性;随着Cur添加量的提高,纤维膜的接触角逐渐降低,亲水性相应增强。Cur的引入改善了纤维膜的亲水性能,这主要归因于其化学结构中的氧原子作为质子供体,增强了与水分子的相互作用。纤维膜以PLGA和PCL为基材,二者均属疏水性聚合物,而Cur的加入在一定程度上调节了材料的表面润湿性。适宜的亲水性有助于促进载药静电纺丝膜与水性环境之间的相互作用,同时保留足够的疏水结构以维持力学稳定性和功能持久性。
表1 不同Cur含量载药静电纺丝膜接触角
Tab.1
| 样品编号 | 接触角/(°) |
|---|---|
| FM | 134.0±0.5*** |
| FM-2.5Cur | 125.4±0.9*** |
| FM-5Cur | 113.6±1.1*** |
| FM-10Cur | 87.5±1.6*** |
2.3 载药率与包封率分析
药物负载量与包封率是影响药物释放与吸收行为的关键参数[15]。Cur水溶性较差,但易溶于乙醇;在PBS/乙醇混合溶剂中,随着乙醇比例提高,其载药率与包封率均相应上升。表2示出不同Cur含量静电纺丝膜的载药率和包封率。在体积比为9∶1的PBS/乙醇混合溶剂中,随着药物投料量的增加,载药量逐渐升高,但包封率呈下降趋势。其中,FM-2.5Cur包封率为(6.2±0.003)%,FM-5Cur和FM-10Cur包封率均小于0.05%,该现象与Cur在PBS/乙醇体系中溶解度有限、接近饱和浓度有关。进一步测定乙醇溶液中载药静电纺丝膜的载药率与包封率,Cur在乙醇中的载药率随投药量增加而升高:FM-2.5Cur、FM-5Cur和FM-10Cur的载药率分别为(2.4±0.001)%、(4.9±0.001)%和(7.3±0.003)%。由于Cur在乙醇中溶解性显著优于混合溶剂,该体系所测得的载药率更接近理论值。FM-2.5Cur与FM-5Cur的包封率均高于97%,而FM-10Cur的包封率则出现明显下降。
表2 不同Cur含量静电纺丝膜的载药率和包封率
Tab.2
| 样品编号 | 载药率/% | 包封率/% | ||
|---|---|---|---|---|
| 溶剂a | 溶剂b | 溶剂a | 溶剂b | |
| FM-2.5Cur | 0.2±0.001 | 2.4±0.001 | 6.2±0.003 | 97.2±0.001 |
| FM-5Cur | 0.3±0.001 | 4.9±0.001 | 0±0.001 | 97.1±0.002 |
| FM-10Cur | 0.3±0.011 | 7.3±0.003 | 0±0.001 | 73.4±0.002 |
注:溶剂a为PBS/乙醇(体积比9∶1)混合溶剂;溶剂b为乙醇溶剂。
2.4 体外药物释放行为分析
载药静电纺丝膜在PBS缓冲溶液中的药物释放行为主要受Cur溶解特性及其在纤维中负载量的影响。图5示出不同Cur含量静电纺丝膜的药物释放质量浓度和药物释放率。可看出,FM-2.5Cur、FM-5Cur和FM-10Cur在1 h时累积释放的Cur质量浓度分别为0.10、0.15、0.22 mg/mL,累积药物释放率分别为2.26%、1.65%和1.15%;24 h时,释放的Cur质量浓度逐渐升高至0.20、0.35、0.68 μg/mL,药物释放率分别达到4.67%、4.00%和3.56%。在整个释放周期中,Cur呈现持续而缓慢的释放特征,药物释放质量浓度随负载量提高而增加,但药物释放率呈下降趋势,这与Cur在PBS中溶解度低有关,该现象与PBS/乙醇体系中载药行为一致。释放初期出现的突释效应可归因于静电纺纤维材料具有高比表面积与多孔结构,增大了药物与介质的接触面积,缩短扩散路径,从而促进初始快速释放[16]。随后静电纺丝纤维膜可实现药物的持续缓释,表明其作为载药敷料具备潜在的长效给药应用潜力。
图5
图5
不同Cur含量静电纺丝膜的释药行为
Fig.5
Drug release absorbance and drug release rate of electrospun membranes with different Cur contents. (a) Mass concentrations of drug release within 28 d; (b) Mass concentrations of drug release within 24 h; (c) Drug release rate within 28 d; (d) Drug release rate within 24 h
2.5 抗氧化性能分析
Cur是一种天然抗氧化剂,可有效清除DPPH。维持载药敷料的抗氧化能力并使其有效释放药物以清除DPPH自由基对载药敷料促进伤口愈合至关重要。图6示出用DPPH法测定的载药静电纺丝膜负载Cur前后的抗氧化能力。FM在24 h内的自由基清除率小于10%,几乎没有抗氧化性能。引入Cur后,静电纺丝膜出现抗氧化性能,且随着Cur含量的增加,静电纺丝膜的抗氧化活性逐步增强。FM-2.5Cur、FM-5Cur和FM-10Cur在24 h的自由基清除率均达到80%以上,具有显著差异性,表明载药静电纺丝膜的抗氧化性来源于Cur。Cur的烯醇结构和2个甲氧基苯酚环之间的庚二酮烷基链上都含有一个高活性碳原子,碳原子上的氢原子易被取代,并作为质子供体与DPPH自由基结合,使其形成稳定分子,从而中止自由基链式反应,这种结合作用是Cur抗氧化性的化学基础,使得载药静电纺丝膜具有抗氧化性。随着时间延长,载药静电纺丝膜的抗氧化率逐渐增加,2 h后抗氧化率趋于平缓,这是由于Cur从静电纺丝膜纤维基中的持续释放,并达到有效作用水平,其分子链上有可提供质子的羟基,羟基可与自由基反应,活性羟基的数目越多,可以提供的质子数量越多,导致抗氧化活性提高。载药静电纺丝膜具有抗氧化活性,且能够持续有效释放药物,清除DPPH,良好的抗氧化性能可以保护生物大分子不受细胞代谢的ROS的氧化损伤,从而促进伤口愈合,有望在慢性伤口愈合炎症阶段抑制ROS自由基的产生,缩短炎症反应时间,达到促愈合的目的。
图6
图6
不同Cur含量静电纺丝膜的自由基清除率
Fig.6
Antioxidant rate of eletrospun membranes with different Cur contents
2.6 抗菌性能分析
Cur是一种植物来源的多酚活性物质,其结构特征和抗氧化生成的产物可有效阻止细菌的生长,具有广谱抗菌特性[17]。其抗菌机制主要涉及活性分子从纤维基质中持续释放,并与细菌细胞壁的肽聚糖层结合,导致结构完整性破坏,干扰细菌正常生理活动。本研究采用菌落计数法,以金黄色葡萄球菌和大肠埃希菌为模型菌株,系统评价了不同含量载药静电纺丝膜的抗菌性能。
图7示出载药静电纺丝膜的抗菌性能检测结果。根据图7计算静电纺丝膜的抑菌率,结果如表3所示。可以看出,空白组与FM组在2种细菌的菌落数上均无显著差异,表明FM本身不具备明显抑菌作用,而载药静电纺丝膜FM-2.5Cur、FM-5Cur和FM-10Cur均表现出不同程度的抗菌活性。抑菌效果随载药量增加而增强,Cur分子可有效进入细菌细胞膜的磷脂双分子层中,破坏膜完整性,增加通透性。此外,金黄色葡萄球菌为革兰阳性菌,其细胞壁由厚而疏松的肽聚糖层构成,更易于姜黄素分子渗透并作用于细胞膜及肽聚糖合成靶点,而大肠埃希菌为革兰阴性菌,具有由脂多糖构成的外膜,形成了一道有效的亲水性屏障,限制了疏水性的姜黄素分子的穿透,从而导致其敏感性较低。这证实其抗菌机制与药物释放及肽聚糖靶向作用密切相关,且Cur对金黄色葡萄球菌的敏感性显著高于大肠埃希菌。上述结果表明,该载药静电纺丝膜具有良好的细菌抑制能力,可预防伤口处的细菌感染,并具有促伤口愈合的潜力。
图7
图7
不同Cur含量静电纺丝膜的抗菌检测结果
Fig.7
Antibacterial test results of eletrospun membranes with different Cur contents against S.aureus (a) and E.coli (b)
表3 不同Cur含量静电纺丝膜的抗菌性能
Tab.3
| 样品编号 | 抑菌率/% | |
|---|---|---|
| 对金黄色葡萄球菌 | 对大肠埃希菌 | |
| FM | 0.2±0.1*** | 0±0.1*** |
| FM-2.5Cur | 59.4±0.6*** | 7.5±2.0*** |
| FM-5Cur | 90.6±0.6*** | 16.3±1.1*** |
| FM-10Cur | 95.5±0.8*** | 42.6±2.1*** |
3 结论
利用静电纺丝技术,以聚乳酸-羟基乙酸共聚物/聚己丙酯(PLGA/PCL)为基材,通过调控姜黄素(Cur)的添加量,制备了不同Cur含量的载药静电纺丝膜,并对其理化性能、浸润性能、载药率与包封率、释药行为、抗氧化及抗菌作用进行了表征分析,主要得出以下结论。
1)所得载药静电纺丝膜均形成良好的纤维结构,纤维直径分布均匀。亲水性随Cur质量分数的增加而显著增强,质量分数为0%、2.5%、5%、10%的静电纺丝膜的静态水接触角分别为(134.0±0.5)°、(125.4±0.9)°、(113.6±1.1)°和(87.5±1.6)°。
2)在PBS/乙醇与乙醇溶剂中,载药静电纺丝膜的吸光度与载药量均随Cur含量增加而升高。由于Cur水溶性差,其在PBS/乙醇中的载药量与包封率显著低于乙醇体系。真实载药量更接近乙醇中的测定值,负载2.5%和5%姜黄素的静电纺丝膜的包封率均高于97%,表明纤维载体可有效提高Cur利用率,并具备持续释放能力,在伤口治疗方面应用前景良好。
3)优选载药5%的静电纺丝膜表现出优异的抗氧化与抗菌性能,对金黄色葡萄球菌抑菌率超过90%,DPPH自由基清除率高于80%,有望在慢性伤口炎症阶段通过抗感染与抗氧化作用缩短炎症时间,促进组织愈合。
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