上海海洋大学学报  2020, Vol. 29 Issue (4): 632-640    PDF    
牡蛎酶解产物促进皮肤软组织创伤愈合的作用
杨发明1, 林海生1,2,3,4,5, 秦小明1,2,3,4,5, 章超桦1,2,3,4,5, 曹文红1,2,3,4,5, 高加龙1,2,3,4,5     
1. 广东海洋大学 食品科技学院, 广东 湛江 524088;
2. 广东省水产品加工与安全重点实验室, 广东 湛江 524088;
3. 广东普通高等学校水产品深加工重点实验室, 广东 湛江 524088;
4. 国家贝类加工技术研发湛江分中心, 广东 湛江 524088;
5. 南海生物资源开发与利用协同创新中心, 广东 湛江 524088
摘要:通过动物实验,比较牡蛎酶解产物低、中、高3个灌胃剂量组与2个对照组在皮肤创伤愈合过程中的照片、创伤愈合率、瘢痕缩小率、IL-6、IL-10和TGF-β等指标。结果显示:第6、10天低、高剂量组创伤愈合率均高于两个对照组(P < 0.05),第14天,低、高剂量组的愈合率较阴性对照组显著提高(P < 0.05);牡蛎酶解产物能够有效抑制炎症因子IL-6生成,并促进IL-10分泌(P < 0.05);低剂量组FGF-2含量高于其他组,但无显著性差异(P>0.05),与阴性对照组TGF-β含量相比,其他组皆呈现显著差异(P < 0.05),3个剂量组CCND1含量皆显著高于2个对照组(P < 0.05),低、高剂量组EGF含量显著高于其他组(P < 0.05);第7天低剂量组羟脯氨酸含量较阴性对照组显著提高(P < 0.05),第14天,各给药组均能显著提高皮肤组织中的羟脯氨酸含量且低剂量组提高了瘢痕缩小率(P < 0.05)。牡蛎酶解产物能够加快小鼠软组织开放性创伤愈合,对浅表瘢痕增生具有一定的抑制作用。
关键词牡蛎    酶解产物    皮肤    创伤愈合    

牡蛎作为最容易获得的海洋生物资源之一,是第一批入选食药同源的经济贝类,含有丰富的优质蛋白质,是多种维生素和矿物质的良好来源[1]。本研究团队及其他学者对牡蛎肽进行了功能研究,发现其具有抗高血压[2]、抗氧化[3-4]、增强免疫力[5-6]、抗疲劳[7]、抗肿瘤[8]、抑菌[9]、改善记忆[10]和解酒护肝[11]等功能,但未见针对其深入药用价值研究的报道。因此,充分开发牡蛎的药用价值极具前景。

皮肤具有在内部器官和外部环境之间提供物理屏障、保护身体的作用[12-13]。创伤愈合是一个复杂的过程,包括三/四个重叠阶段:止血/炎症,细胞增殖和重塑期[14-15]。对创伤的治疗主要是自体皮肤移植、缝合、生理敷料和使用抗生素等方法[16],但容易造成增生性瘢痕[17]。目前,研究发现部分活性肽在皮肤组织创伤愈合方面具有很高的潜在应用价值[18-19]。酶水解是从蛋白质来源获得生物活性肽的有效方法,该研究采用酶法制备牡蛎酶解产物(enzymolysis products of oyster, EPO),依据皮肤创伤愈合的炎症、细胞增殖和组织重塑3个阶段,开展灌胃给药的动物实验,分别选择对应的关键指标进行其作用阶段的确定,并研究其促进小鼠皮肤软组织创伤愈合的作用,为进一步深入研究其机制提供理论依据。

1 材料与方法 1.1 EPO制备工艺

牡蛎肉购买自广西钦州,将牡蛎肉清洗、打浆,调pH至7.0,加1 000 U/g动物蛋白酶,酶解5 h,灭酶10 min,8 000 r/min离心,浓缩,-80 ℃保存24 h,冷冻干燥,保存于4 ℃冰箱备用[20];蛋白质含量测定采用凯氏定氮法[21];肽含量测定采用双缩脲法[22]

1.2 动物分组及创伤模型建立

SPF清洁级昆明雄性小鼠100只,体质量为(20±2)g。将小鼠按体质量随机分为5组,每组20只,分别为阴性对照组(生理盐水0.1 mL/10 g体质量)、阳性对照组(初元Ⅰ型产品主要成分为小麦低聚肽、海洋鱼皮胶原低聚肽0.1 mL/10 g体质量)、灌胃低(0.25 g/kg体质量,pH 5.29)、中(0.5 g/kg体质量,pH 5.40)、高(1 g/kg体质量,pH 5.44)剂量组。小鼠用10%水合氯醛腹腔麻醉后,背部剃毛,消毒。在小鼠背部剪去直径约0.8 cm的全层皮肤,造成全皮层创伤动物模型。每只小鼠均分笼饲养,每天灌胃0.1 mL/10 g体质量,阴性对照组每天灌胃生理盐水。每2天对创伤口面观察,拍照,测量伤口大小并记录结果。

1.3 愈合率及瘢痕缩小率

每隔2天用游标卡尺测量创面直径大小1次,计算创伤愈合率(wound healing rate, WHR)和瘢痕缩小率(scar reduction rate, SRR):

    (1)
    (2)

式中:RWH为创伤愈合率WHR,%;RSR为瘢痕缩小率SRR,%;D0为初始伤口形状的直径,cm;Dn为未愈合伤口形状的直径,cm。

1.4 创面组织中生化指标测定

小鼠创面组织匀浆液制备参考文献[23],分装保藏于-80 ℃,待测。IL-6(白介素-6)、IL-10(白介素-10)、TGF-β(转化生长因子β)、FGF-2(碱性成纤维细胞生长因子)、EGF(表皮细胞生长因子)、Cyclin D1(细胞生长周期素1)和羟脯氨酸含量检测按照南京建成试剂盒说明进行。

1.5 数据统计分析

每组实验至少重复3次, 采用SPSS 20软件进行统计学分析。组间比较采用LSD检验,以P < 0.05为差异有统计学意义。

2 结果与分析 2.1 EPO的基本性质

本实验自制的EPO的主要质量指标:加酶量为1 000 U/g蛋白的牡蛎酶解液水解度为26.33%,EPO的蛋白质含量为43.54 g/100 g,肽含量为25.26%。

2.2 创面形态变化观察

各组愈合情况如图 1所示,造模后第2天,各实验组动物皆已结痂,伤口周围出现红肿,无液体渗出,伤口收缩均不明显。造模后第4、6、8天,各组创面结痂不断变硬,创伤周围表面不平整,创缘皮肤收缩较明显,其中,各给药组结痂厚度优于对照组,局部创面呈现黑红色。造模后第10天,对照组伤口结痂部分脱落,但瘢痕面积变化小。各给药组:伤口明显缩小,并开始部分脱痂、缩痂明显。造模后第12天,各组皆已掉痂,愈合处开始长出毛发。各给药组进一步缩痂,中剂量组二次结痂。造模后第14天,对照组的多数伤口直径小于4 mm,各给药组伤口直径均小于2 mm,中、高剂量组呈现肉红色新生皮肤,新生肉色组织完整。各给药组较对照组愈合情况良好,无增生性瘢痕及瘢痕疙瘩生成。这表明EPO具有一定的促进小鼠皮肤创伤愈合的优势。

图 1 EPO对小鼠创面的影响 Fig. 1 Effect of EPO on wound of mice
2.3 创伤愈合率

表 1可以看出:造模后第6天,低、高剂量组的创伤愈合率均在25%以上,与阴性对照组相比,分别高出21%和12%(P < 0.05);造模后第10天,低、高剂量组与阴性对照组相比有显著性差异(P < 0.05);造模后第14天时,低、高剂量组较阴性对照组具有明显的愈合优势,并与阳性对照组无显著性差异(P < 0.05)。

表 1 EPO对小鼠皮肤创伤愈合的影响 Tab.1 Effect of EPO on skin wound healing in mice(X±SD)
2.4 炎症因子测定

该研究选择测定造模后第3天、第5天、第7天的IL-6与IL-10的含量,结果如图 23所示。

若同一天字母完全不同,则表示组间存在显著差异(P < 0.05),标有相同小写字母者表示组间无显著性差异(P>0.05),*表示与其他组存在极显著差异(P < 0.01) If the letters on the same day are completely different, there is a significant difference between the groups(P < 0.05), and the means with the same letters within the same line have no significant differences(P>0.05), * indicates that there is a very significant difference (P < 0.01) 图 2 EPO对创伤皮肤组织中炎症因子IL-6的影响 Fig. 2 Effect of EPO on inflammatory factors IL-6 in wound skin tissue
若同一天字母完全不同,则表示组间存在显著差异(P < 0.05),标有相同小写字母者表示组间无显著性差异(P>0.05),“*”表示与其他组存在极显著差异(P < 0.01) If the letters on the same day are completely different, there is a significant difference between the groups(P < 0.05), and the means with the same letters within the same line are not significant differences(P>0.05), "*" indicates that there is a very significant difference (P < 0.01) 图 3 EPO对创伤皮肤组织中抗炎因子IL-10的影响 Fig. 3 Effects of EPO on inflammatory factors IL-10 in wound skin tissue

图 2可知:第3天,低剂量组IL-6含量显著低于两个对照组(P < 0.05);第5天,各组IL-6含量无显著性差异(P>0.05);第7天,高剂量组IL-6含量极显著低于其他组(P < 0.01)。这表明EHM对小鼠创伤皮肤组织中炎症因子IL-6有一定的抑制效果。

图 3可知:第3天,阳性对照组、中、高剂量组IL-10含量极显著高于阴性对照组(P < 0.01);第5天,高剂量组IL-10含量高于其他组,但无显著性差异(P>0.05);第7天,阳性对照组,低剂量组IL-10含量显著高于其他组(P < 0.05)。

2.5 生长因子测定

表 2可以看出:低剂量组FGF-2含量高于其他组,但无显著差异(P>0.05);与阴性对照组TGF-β含量相比,其他组皆呈现显著差异(P < 0.05);各灌胃给药剂量组CCND1含量显著高于其他实验组(P < 0.05);低、高剂量组EGF含量显著高于其他组(P < 0.05)。

表 2 EPO对创伤皮肤组织中生长因子的影响 Tab.2 Effect of EPO on growth factors in wound skin tissue(X±SD)
2.6 羟脯氨酸含量及瘢痕缩小率测定

表 3可知:造模后7 d,高剂量组羟脯氨酸含量显著高于阴性对照组(P < 0.05);造模后14 d,各给药组皆显著高于阴性对照组(P < 0.05)。这表明灌胃给药EPO具有促进小鼠创面胶原蛋白的分泌、沉积及交联的作用,从而加快愈合速度,这与图片及愈合率结果一致。同时,由图 4结果可知,与阴性对照组相比,灌胃给药EPO低剂量组在第14天时可有效抑制疤痕残留(P < 0.05),这与照片和羟脯氨酸结果相符。

表 3 EPO对创伤皮肤组织中羟脯氨酸的影响 Tab.3 Effect of EPO on hydroxyproline in wound skin tissue(X±SD)
若字母完全不同,则组间存在显著差异(P < 0.05) If the letters in the same column are completely different, there is a significant difference between the groups(P < 0.05) 图 4 EPO对小鼠皮肤创伤瘢痕生成的影响 Fig. 4 Effect of EPO on scar formation of skin wounds in mice
3 讨论 3.1 EPO对创面愈合的影响

表 1可知:造模后前10天,低、高剂量组创伤愈合速率均高于2个对照组,可有效加快创伤愈合。创伤愈合后期,各实验组创伤愈合速率逐渐趋于平衡,阳性对照组和低、高剂量组促愈效果明显。据报道,生物活性肽具有促进消化吸收、增强免疫力、调节营养代谢等功能活性[24],且已经成为药物开发领域的天然药物资源库[25]。肉芽组织中胶原蛋白含量的增加有助于胶原蛋白的沉积和细胞与细胞的相互作用,有利于创伤愈合过程中的再上皮化[26]。从愈合率结果看,阳性对照组在造模后第10~14天(组织重塑期)作用效果明显,这时期胶原蛋白起主要作用[27],而阳性对照组(初元Ⅰ型产品)配方中海洋鱼皮胶原低聚肽、大豆低聚肽、酪蛋白磷酸肽均为低聚肽混合物,具有独特愈合功效,与研究结果相吻合,这表明生物活性肽有利于胶原蛋白的沉积和细胞增殖,从而加快皮肤创伤愈合。EPO肽含量较高,且低剂量组在整个创伤愈合过程起作用,更具有愈合优势。

3.2 EPO对炎症期的影响

炎症是创伤愈合的重要阶段,已经受损或死亡的细胞将被清除,还有助于去除细菌和其他感染性病原体[28]。白细胞介素(IL-6和IL-10)作为炎症细胞因子,在炎症的调节中起主要作用。IL-6是一种促炎因子,在急性炎症中,IL-6将会增加;IL-10具有抗炎活性,抑制IL-6的表达和炎性细胞在损害部位处迁移[29]。IL-10抑制中性粒细胞和巨噬细胞向损伤部位的浸润以及促炎细胞因子(IL-1β,IL-6,肿瘤坏死因子-α)的体内表达[30]。本研究IL-10含量在第3天达到高峰期,之后开始下降的结果与文献[31]报道一致。有研究[32]表明,通过减少促炎M1巨噬细胞极化和促进抗炎M2极化可减轻炎症。本研究结果表明,EPO对IL-6有一定的抑制效果,这可能会促进M2型巨噬细胞分泌IL-10等抗炎因子,从而起到抑制炎症反应的作用。阳性对照组配方中海洋鱼皮胶原低聚肽、酪蛋白磷酸肽[33]具有抑制炎症功效,且由EPO基本性质结果可知,EPO有较高含量的蛋白及肽,推测有利于EPO具有促进IL-10分泌从而起到抑制炎症反应的功能活性因子。在皮肤中,IL-10可通过抑制炎症反应,减轻瘢痕增生,实现优质创伤修复[34], 且在创伤愈合的炎症阶段结束时,巨噬细胞发展成抗炎,促进基质沉积,血管生成和再上皮化过程[35],这与照片呈现的直观创伤修复效果一致,推测EPO还可通过促进IL-10分泌,预防瘢痕增生,实现优质修复。

3.3 EPO对增殖期的影响

生长因子在创伤愈合和组织再生的复杂过程中起着促进趋化性(吸引细胞进入伤口),诱导细胞迁移和增殖,刺激细胞上调蛋白质的产生,且重塑细胞外基质并促进血管生成,形成有利于皮肤伤口愈合过程的理想环境的重要作用[36]。成纤维细胞作为胶原蛋白和其他细胞外基质成分的主要来源,在正常的伤口愈合和增生性瘢痕形成中起着重要作用,FGF-2对多种细胞有促进基因合成和细胞分裂作用[37]。TGF-β是在组织重塑和瘢痕形成期间表现出重要功能的生长因子,作用于多种细胞类型,并提供过多的生物活性,包括刺激炎症、促进血管生成和增强成纤维细胞增殖[38]。CCND1主要功能是促进细胞增殖[39]。EGF促进创面愈合最主要的作用是促进创面组织的增殖和分化,增殖和分化形成的新细胞代替衰老和已经死亡的细胞[40]。由此可推测EPO促进创面愈合的过程中通过加速细胞因子TGF-β、EGF的分泌促进成纤维细胞分泌胶原,增加胞外基质沉积,新血管形成,促进表皮细胞生长,同时也通过加速细胞周期调控蛋白CCND1的表达促进成纤维细胞等细胞周期向分裂期转化,从而加速细胞的有丝分裂[41]以实现同愈合率结果一致的创伤愈合。

3.4 EPO对重塑期的影响

创伤愈合过程的最后阶段称为重塑,由于迁移和凋亡,观察到受伤区域的细胞含量减少。重塑是一个动态阶段,胶原蛋白与其他胶原蛋白和其他蛋白质分子形成紧密的交联[42],可通过测定羟脯氨酸含量来反映胶原蛋白含量的变化情况。由图 4可知,低剂量给药组能有效提高瘢痕缩小率(P < 0.05),这与炎症抑制及TGF-β含量分泌结果一致,表明EPO对瘢痕增生具有一定的抑制作用。

综上,通过灌胃给药EPO进行动物实验,研究表明其具有一定抑制炎症作用并促进生长因子分泌以及胶原蛋白的沉积与交联,从而加快小鼠软组织开放性创伤愈合,对浅表瘢痕增生具有一定的抑制作用。这为进一步研究EPO促进创伤愈合实验提供理论依据。

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Effect of enzymolysis products of oyster on wound healing of skin
YANG Faming1, LIN Haisheng1,2,3,4,5, QIN Xiaoming1,2,3,4,5, ZHANG Chaohua1,2,3,4,5, CAO Wenhong1,2,3,4,5, GAO Jialong1,2,3,4,5     
1. College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, Guangdong, China;
2. Guangdong Provincial Key Laboratory of Aquatic Products Processing and Safety, Zhanjiang 524088, Guangdong, China;
3. Key Laboratory of Advanced Processing ofAquatic Products of Guangdong Higher Education Institution, Zhanjiang 524088, Guangdong, China;
4. National Research and Development Branch Center for Shellfish Processing, Zhanjiang 524088, Guangdong, China;
5. South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Zhanjiang 524088, Guangdong, China
Abstract: We compared photos, wound healing rate, scar reduction rate, IL-6, IL-10, TGF-β, etc. in the skin wound healing process of the groups of IG low, medium and high dose enzymolysis products of oyster by animal experiment, and the results are shown as follows:The wound healing rate of the low and high dose groups was higher than that of the two control groups at 6 and 10 days and on the 14th day the healing rate of the low and high dose groups was significantly higher than that of the negative control group(P < 0.05); Enzymolysis products of oyster can inhibit the production of inflammatory factor IL-6 and promote IL-10 secretion(P < 0.05); The FGF-2 content in the low dose group was higher than the other groups but there was no significant difference (P>0.05), compared with the TGF-β content in the negative control group(P < 0.05), the levels of CCND1 in the three dose groups were significantly higher than the two control groups (P>0.05), and the EGF content in the low and high dose groups was significantly higher than that in the other groups(P>0.05); On the 7th day the hydroxyproline content in the low dose group was significantly higher than that in the negative control group (P < 0.05), and on the 14th day the hydroxyproline content in the skin tissue was significantly increased in each administration group and low dose group improves scar contraction rate (P < 0.05).Enzymolysis products of oyster has accelerated wound healing of soft tissue in mice and has a certain inhibitory effect on superficial scar hyperplasia.
Key words: oyster     enzymolysis products     skin     wound healing