重组贻贝黏蛋白Mfp151的皮肤屏障修复功能和抗炎活性评价

Evaluation of the skin barrier repair function and anti-inflammatory activity of recombinant mussel mucin Mfp151

摘要:

炎症是导致皮肤衰老的主要诱因，贻贝黏蛋白是一种天然来源的抗炎蛋白，但其天然提取效率低下。为突破天然来源的产量限制，并验证重组蛋白的生物活性，本研究通过合成生物学技术高效制备并分离纯化得到重组贻贝黏蛋白Mfp151，并对其抗炎和皮肤屏障修复功能进行评价。以人永生化角质形成细胞(human immortalized keratinocytes, HaCaT)、人包皮成纤维细胞(human foreskin fibroblasts, HFF-1)及人单核细胞白血病细胞(human monocytic leukemia cells, THP-1)为模型，采用细胞计数试剂盒-8 (cell counting kit-8, CCK-8)、反转录实时荧光定量聚合酶链式反应(reverse transcription quantitative real-time polymerase chain reaction, RT-qPCR)、跨膜电阻测量、划痕实验、ELISA及自由基清除等方法，评估Mfp151在修复、抗氧化及抗炎方面的功效，同时利用斑马鱼晒伤模型进行体内验证。结果表明，Mfp151可显著促进细胞增殖(80 μg/mL处理使HaCaT细胞增殖率提高约32%)与迁移，并上调皮肤屏障关键基因IVL、OVOL1和AQP3的表达(相比正常对照组，上调2.14-3.88倍)。Mfp151能有效清除1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)和2,2′-联氮-双(3-乙基苯并噻唑啉-6-磺酸)[2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS]自由基，并通过激活Nrf2通路增强细胞抗氧化能力。在抗炎方面，Mfp151能有效抑制脂多糖(lipopolysaccharide, LPS)诱导的THP-1细胞产生促炎因子白细胞介素-6 (interleukin-6, IL-6)、肿瘤坏死因子-α (tumor necrosis factor-alpha, TNF-α)和白细胞介素-1β (interleukin-1β, IL-1β) (80 μg/mL处理使其表达水平降低至模型组的59.87%-79.12%)，并显著下调关键炎症介质环氧合酶-2 (cyclooxygenase-2, COX-2)与诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的编码基因表达及其下游产物前列腺素E2 (prostaglandin E2, PGE2)的水平(80 μg/mL处理使COX-2和iNOS表达及PGE2的分泌降低至模型组的48.91%-67.30%)。斑马鱼实验证实其可显著促进晒伤后的尾鳍修复。本研究结果表明，通过合成生物学技术获得的重组Mfp151蛋白具有促进皮肤修复的功能和抗氧化抗炎活性，在皮肤抗衰化妆品原料及创面敷料等领域展现出良好的应用前景。

Abstract:

Inflammation is a primary driver of skin ageing. Mussel mucin, a naturally occurring anti-inflammatory protein, suffers from inefficient extraction from its natural source. To overcome the yield limitation of natural sources and to verify the biological activity of the recombinant protein, this study employed synthetic biology techniques to efficiently produce and purify the recombinant mussel mucin Mfp151 and subsequently evaluated its anti-inflammatory and skin barrier repair functions. Using human immortalized keratinocytes (HaCaT), human foreskin fibroblasts (HFF-1), and human monocyte leukemia cells (THP-1) as models, we assessed the skin-repairing, antioxidant, and anti-inflammatory effects of Mfp151 via the CCK-8 assay, qPCR, transmembrane resistance measurement, scratch assay, ELISA, and free radical scavenging assays. In vivo validation was conducted with a zebrafish sunburn model. The results demonstrated that Mfp151 significantly promoted cell proliferation (80 μg/mL treatment increased HaCaT cell proliferation by approximately 32%) and migration, while upregulating the expression of key skin barrier genes IVL, OVOL1, and AQP3 by 2.14-3.88 folds. Mfp151 effectively scavenged DPPH and ABTS free radicals while enhancing cellular antioxidant capacity by activating the Nrf2 pathway. Regarding anti-inflammatory effects, Mfp151 significantly suppressed lipopolysaccharide-induced production of pro-inflammatory factors IL-6, TNF-α, and IL-1β (80 μg/mL treatment reduced expression levels to 59.87%-79.12% of that in the model group), while significantly downregulating the expression of key inflammatory mediators COX-2 and iNOS and their downstream product PGE2 (80 μg/mL treatment reduced COX-2, iNOS expression and PGE2 secretion to 48.91%-67.30% those in the model group.) Zebrafish experiments confirmed the significant promotion of Mfp151 on caudal fin repair following sunburn. The data indicate that the recombinant Mfp151 obtained through synthetic biology techniques possesses skin-repairing functions alongside antioxidant and anti-inflammatory activities, thus demonstrating promising application prospects in fields such as anti-ageing cosmetic ingredients and wound dressings.