Effects of in situ cross-linked graphene oxide-containing gelatin methacrylate anhydride hydrogel on wound vascularization of full-thickness skin defect in mice
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摘要:
目的 制备含氧化石墨烯（GO）的甲基丙烯酸酐化明胶（GelMA）水凝胶并探讨原位光聚合GO-GelMA复合水凝胶对小鼠全层皮肤缺损创面血管化的影响。 方法 采用实验研究方法。将0.2 mg/mL的GO溶液50 μL均匀涂抹于导电胶上，烘干后于场发射扫描电子显微镜下观察GO的结构和大小。将人皮肤成纤维细胞（HSF）分为采用相应终质量浓度GO处理的0 μg/mL GO（不加GO溶液，下同）组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组、10.0 μg/mL GO组，用酶标仪检测细胞培养48 h的吸光度值，以此表示细胞增殖活性（样本数为6）。将HSF和人脐静脉血管内皮细胞（HUVEC）分别分为采用相应终质量浓度GO处理的0 μg/mL GO组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组，采用划痕试验检测划痕后24、36 h HSF的迁移率（样本数为5）及划痕后12 h HUVEC的迁移率（样本数为3），采用酶联免疫吸附测定法检测培养4、6、8 h后HSF分泌的血管内皮生长因子（VEGF）水平（样本数为3）。将配制的含相应终质量浓度GO的GO-GelMA复合水凝胶设为0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，观察其交联前后的性状，检测用磷酸盐缓冲液浸泡3、7 d后GO的释放情况（样本数为3）。在16只6周龄雌性C57BL/6小鼠背部制作全层皮肤缺损创面，将采用原位交联的含相应终质量浓度GO的GO-GelMA复合水凝胶处理的小鼠按随机数字表法分为0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，每组4只，观察治疗3、7、14 d创面大体情况并计算创面愈合率，采用激光多普勒血流仪检测治疗3、7、14 d创面血流灌注并计算平均灌注单位（MPU）比值，采用苏木精-伊红染色观察治疗7 d创面血管新生情况并计算血管密度（样本数均为3）。取0 μg/mL GO复合水凝胶组和0.1 μg/mL GO复合水凝胶组治疗7 d的创面组织，采用苏木精-伊红染色观察GO分布与血管新生的关系（样本数为3），行免疫组织化学染色后观察VEGF的表达。对数据行重复测量方差分析、单因素方差分析、Tukey法。 结果 GO为多层片状结构，宽度约为20 μm、长度约为50 μm。培养48 h，10.0 μg/mL GO组HSF的吸光度值明显低于0 μg/mL GO组（q=7.64，P<0.01）。划痕后24 h，4组HSF迁移率相近（P>0.05）；划痕后36 h，0.1 μg/mL GO组HSF迁移率明显高于0 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组（q值分别为7.48、10.81、10.20，P值均<0.01）。划痕后12 h，0.1 μg/mL GO组HUVEC迁移率明显高于0 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组（q值分别为7.11、8.99、14.92，P值均<0.01），5.0 μg/mL GO组HUVEC迁移率明显低于0 μg/mL GO组和1.0 μg/mL GO组（q值分别为7.81、5.33，P<0.05或P<0.01）。培养4、6 h，4组HSF的VEGF表达均相近（P>0.05）；培养8 h，0.1 μg/mL GO组HSF的VEGF表达明显高于0 μg/mL GO组和5.0 μg/mL GO组（q值分别为4.75、4.48，P值均<0.05）。4组GO-GelMA复合水凝胶在交联前均呈红色液体状，交联后呈微黄色凝胶状且流动性无明显差异。0 μg/mL GO复合水凝胶组复合水凝胶各时间点均无GO释放，其余3组GO-GelMA复合水凝胶中的GO于浸泡3 d部分释放，至浸泡7 d全部释放。治疗3~14 d，4组小鼠创面可见水凝胶敷料覆盖在位并保持湿润，创面逐渐愈合。治疗3、7、14 d，4组小鼠创面愈合率均相近（P>0.05）。治疗3 d，0.1 μg/mL GO复合水凝胶组小鼠创面MPU比值明显高于0 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组（q值分别为10.70、11.83、10.65，P<0.05或P<0.01）。治疗7、14 d，4组小鼠创面MPU比值均相近（P>0.05）。0.1 μg/mL GO复合水凝胶组小鼠创面治疗7 d的MPU比值明显低于治疗3 d（q=14.38，P<0.05），治疗14 d的MPU比值明显低于治疗7 d（q=27.78，P<0.01）。治疗7 d，0.1 μg/mL GO复合水凝胶组小鼠创面新生血管密度为每200倍视野下（120.7±4.1）根，明显高于0 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组的每200倍视野下（61.7±1.3）、（77.7±10.2）、（99.0±7.9）根（q值分别为12.88、7.79、6.70，P值均<0.01）；1.0 μg/mL GO复合水凝胶组和5.0 μg/mL GO复合水凝胶组小鼠创面新生血管密度均明显高于0 μg/mL GO复合水凝胶组（q值分别为5.10、6.19，P<0.05）。治疗7 d，相较于0 μg/mL GO复合水凝胶组，0.1 μg/mL GO复合水凝胶组小鼠创面中成簇新生血管更多，且聚集于GO附近；0.1 μg/mL GO复合水凝胶组小鼠创面中GO和新生血管分布区域有大量VEGF表达。 结论 GO质量浓度低于10.0 μg/mL对HSF增殖活性无明显影响，0.1 μg/mL的GO能够促进HSF和HUVEC迁移，能促进HSF分泌VEGF。原位光聚合GO-GelMA复合水凝胶敷料能够通过促进小鼠全层皮肤缺损创面血管新生，增加创面早期血流灌注，且GO对新生血管有富集作用，其机制可能与GO促进创面细胞分泌VEGF相关。 Abstract:Objective To prepare graphene oxide (GO)-containing gelatin methacrylate anhydride (GelMA) hydrogel and to investigate the effects of in situ photopolymerized GO-GelMA composite hydrogel in wound vascularization of full-thickness skin defect in mice. Methods The experimental study method was used. The 50 μL of 0.2 mg/mL GO solution was evenly applied onto the conductive gel, and the structure and size of GO were observed under field emission scanning electron microscope after drying. Human skin fibroblasts (HSFs) were divided into 0 μg/mL GO (without GO solution, the same as below) group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, 5.0 μg/mL GO group, and 10.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the absorbance value was detected using a microplate analyzer after 48 h of culture to reflect the proliferation activity of cells (n=6). HSFs and human umbilical vein vascular endothelial cells (HUVECs) were divided into 0 μg/mL GO group, 0.1 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group treated with GO of the corresponding final mass concentration, and the migration rates of HSFs at 24 and 36 h after scratching (n=5) and HUVECs at 12 h after scratching (n=3) were detected by scratch test, and the level of vascular endothelial growth factor (VEGF) secreted by HSFs after 4, 6, and 8 h of culture was detected by enzyme-linked immunosorbent assay method (n=3). The prepared GO-GelMA composite hydrogels containing GO of the corresponding final mass concentration were set as 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group to observe their properties before and after cross-linking, and to detect the release of GO after soaking with phosphate buffer solution for 3 and 7 d (n=3). The full-thickness skin defect wounds were made on the back of 16 6-week-old female C57BL/6 mice. The mice treated with in situ cross-linked GO-GelMA composite hydrogel containing GO of the corresponding final mass concentration were divided into 0 μg/mL GO composite hydrogel group, 0.1 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group according to the random number table, with 4 mice in each group. The general condition of wound was observed and the wound healing rate was calculated on 3, 7, and 14 d of treatment, the wound blood perfusion was detected by laser Doppler flowmetry on 3, 7, and 14 d of treatment and the mean perfusion unit (MPU) ratio was calculated, and the wound vascularization on 7 d of treatment was observed after hematoxylin-eosin staining and the vascular density was calculated (n=3). The wound tissue of mice in 0 μg/mL GO composite hydrogel group and 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was collected to observe the relationship between the distribution of GO and neovascularization by hematoxylin-eosin staining (n=3) and the expression of VEGF by immunohistochemical staining. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and Tukey's method. Results GO had a multilayered lamellar structure with the width of about 20 μm and the length of about 50 μm. The absorbance value of HSFs in 10.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group after 48 h of culture (q=7.64, P<0.01). At 24 h after scratching, the migration rates of HSFs were similar in the four groups (P>0.05); at 36 h after scratching, the migration rate of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.48, 10.81, and 10.20, respectively, P<0.01). At 12 h after scratching, the migration rate of HUVECs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group, 1.0 μg/mL GO group, and 5.0 μg/mL GO group (with q values of 7.11, 8.99, and 14.92, respectively, P<0.01), and the migration rate of HUVECs in 5.0 μg/mL GO group was significantly lower than that in 0 μg/mL GO group and 1.0 μg/mL GO group (with q values of 7.81 and 5.33, respectively, P<0.05 or P<0.01 ). At 4 and 6 h of culture, the VEGF expressions of HSFs in the four groups were similar (P>0.05); at 8 h of culture, the VEGF expression of HSFs in 0.1 μg/mL GO group was significantly higher than that in 0 μg/mL GO group and 5.0 μg/mL GO group (with q values of 4.75 and 4.48, respectively, P<0.05). The GO-GelMA composite hydrogels in the four groups were all red liquid before cross-linking, which turned to light yellow gel after cross-linking, with no significant difference in fluidity. The GO in the GO-GelMA composite hydrogel of 0 μg/mL GO composite hydrogel group had no release of GO at all time points; the GO in the GO-GelMA composite hydrogels of the other 3 groups was partially released on 3 d of soaking, and all the GO was released on 7 d of soaking. From 3 to 14 d of treatment, the wounds of mice in the 4 groups were covered with hydrogel dressings, kept moist, and gradually healed. On 3, 7, and 14 d of treatment, the wound healing rates of mice in the four groups were similar (P>0.05). On 3 d of treatment, the MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 10.70, 11.83, and 10.65, respectively, P<0.05 or P<0.01). On 7 and 14 d of treatment, the MPU ratios of wound of mice in the four groups were similar (P>0.05). The MPU ratio of wound of mice in 0.1 μg/mL GO composite hydrogel group on 7 d of treatment was significantly lower than that on 3 d of treatment (q=14.38, P<0.05), and that on 14 d of treatment was significantly lower than that on 7 d of treatment (q=27.78, P<0.01). On 7 d of treatment, the neovascular density of wound of mice on 7 d of treatment was 120.7±4.1 per 200 times of visual field, which was significantly higher than 61.7±1.3, 77.7±10.2, and 99.0±7.9 per 200 times of visual field in 0 μg/mL GO composite hydrogel group, 1.0 μg/mL GO composite hydrogel group, and 5.0 μg/mL GO composite hydrogel group (with q values of 12.88, 7.79, and 6.70, respectively, P<0.01), and the neovascular density of wound of mice in 1.0 μg/mL GO composite hydrogel group and 5.0 μg/mL GO composite hydrogel group was significantly higher than that in 0 μg/mL GO composite hydrogel group (with q values of 5.10 and 6.19, respectively, P<0.05). On 7 d of treatment, cluster of new blood vessels in wound of mice in 0.1 μg/mL GO composite hydrogel group was significantly more than that in 0 μg/mL GO composite hydrogel group, and the new blood vessels were clustered near the GO; a large amount of VEGF was expressed in wound of mice in 0.1 μg/mL GO composite hydrogel group in the distribution area of GO and new blood vessels. Conclusions GO with mass concentration lower than 10.0 μg/mL had no adverse effect on proliferation activity of HSFs, and GO of 0.1 μg/mL can promote the migration of HSFs and HUVECs, and can promote the secretion of VEGF in HSFs. In situ photopolymerized of GO-GelMA composite hydrogel dressing can promote the wound neovascularization of full-thickness skin defect in mice and increase wound blood perfusion in the early stage, with GO showing an enrichment effect on angiogenesis, and the mechanism may be related to the role of GO in promoting the secretion of VEGF by wound cells. -
皮肤纤维化疾病是一类严重影响患者身心健康的疾病，可由烧创伤、手术或疾病引起 ［ 1］ ，主要包括增生性瘢痕、瘢痕疙瘩及系统性硬化病等 ［ 2, 3, 4］ 。皮肤纤维化疾病由于治疗周期长、个体差异大及复发率高等原因，造成了沉重的社会卫生经济负担。目前，临床仍然缺乏能有效缓解或治疗皮肤纤维化疾病的理想药物。
皮肤纤维化疾病的主要病理特点是以胶原蛋白为主的ECM异常沉积，导致瘢痕过度增生或皮肤增厚。Fb在调节ECM稳态中发挥主要作用，其在多条信号通路（包括TGF-β、Wnt和血小板衍生生长因子信号通路）的调控下实现活化和增殖；其次，促炎性细胞因子如TNF-α、IL-1或IL-6等也可作用于Fb促进其活化和增殖 ［ 5, 6］ 。此外，内皮细胞、巨噬细胞等其他细胞也参与皮肤纤维化疾病的发生与发展 ［ 7］ 。近年来的相关机制研究为该疾病的治疗带来了新希望，特别是细胞代谢调节领域研究的快速进展，为研究者们提供了新的思考及探索途径。本文就有氧糖酵解相关酶和产物在皮肤纤维化疾病发生与发展中的作用及靶向有氧糖酵解治疗皮肤纤维化疾病的药物进行综述。
1. 皮肤纤维化疾病中异常的有氧糖酵解
糖酵解是指细胞质内葡萄糖在无氧或缺氧条件下分解为乳酸，并产生ATP的过程。在氧气充足的正常细胞中，葡萄糖分解为丙酮酸后易位至线粒体，通过三羧酸循环和氧化呼吸链产生大量的ATP ［ 8］ 。有氧糖酵解是指即使在氧气充足的情况下，细胞仍然倾向于糖酵解，丙酮酸经乳酸脱氢酶（lactate dehydrogenase，LDH）催化产生大量乳酸，同时抑制线粒体氧化磷酸化（oxidative phosphorylation，OXPHOS），这种现象最初在1927年由Otto Warburg于肿瘤细胞中观察到，并被命名为Warburg效应 ［ 9］ 。
近年来研究表明，有氧糖酵解与皮肤纤维化疾病的发生与发展密切相关，有氧糖酵解不仅与Fb的增殖、活化相关，也参与ECM的过量积累 ［ 10, 11, 12］ 。研究显示，在皮肤伤口愈合早期，有氧糖酵解可能作为一种机体自我保护机制，有利于细胞快速供能并促进伤口愈合，但是后期持续增强的有氧糖酵解会影响组织重塑，导致增生性瘢痕形成 ［ 13］ 。研究者运用氟脱氧葡萄糖正电子发射断层扫描对5例瘢痕疙瘩患者病损处进行扫描，结果显示瘢痕疙瘩较周围正常皮肤葡萄糖聚集增加，提示瘢痕疙瘩中糖代谢发生了改变 ［ 14］ 。后续研究显示患者瘢痕疙瘩中糖酵解标志物含量明显升高 ［ 15］ ，瘢痕疙瘩来源的Fb中葡萄糖消耗、乳酸生成、丙酮酸激酶M2（pyruvate kinase M2，PKM2）及Ⅰ型胶原水平均高于正常皮肤Fb ［ 16, 17, 18］ 。系统性硬化病患者Fb中的糖酵解相关酶储量增加，表明这些细胞在OXPHOS受损的条件下糖酵解水平升高，并产生更多的乳酸 ［ 19, 20］ 。针对小鼠ECM致密处的足垫皮肤和ECM菲薄的腹部皮肤基因测序结果显示，ECM致密处糖酵解相关分子基因表达水平上调 ［ 13］ 。这些研究显示，有氧糖酵解在皮肤损伤发生时启动，在整个损伤修复过程中发挥重要作用，但组织重塑期持续增强的有氧糖酵解导致了皮肤纤维化疾病的发生。
2. 有氧糖酵解在皮肤纤维化疾病中的作用
有氧糖酵解在皮肤纤维化疾病中通过促进乳酸生成，调节缺氧诱导因子-1α（hypoxia-inducible factor-1α，HIF-1α）的表达，增强PKM2、葡萄糖转运蛋白1（glucose transporter 1，GLUT1）及磷酸甘油酸激酶1（phosphoglycerate kinase 1，PGK1）的表达，刺激Fb活化和增殖、内皮-间质转化及胶原蛋白合成，从而在皮肤纤维化疾病的发生与发展过程中发挥重要作用。见 图1。
2.1 促进乳酸生成
乳酸由丙酮酸经LDH催化产生，通过单羧酸转运蛋白转运。在纤维化组织中，糖酵解水平的升高导致乳酸大量堆积，促进Fb活化、增殖及内皮-间质转化，增加ECM分泌。有研究表明，人瘢痕疙瘩中央的Fb中LDH、单羧酸转运蛋白4表达水平升高 ［ 15］ ，这些Fb较正常真皮Fb消耗更多的葡萄糖，产生更多乳酸 ［ 21］ 。系统性硬化病患者真皮中的Fb有氧糖酵解增强导致乳酸释放增加，随后细胞外酸化促进内皮-间质转化，加重了系统性硬化病中的组织纤维化 ［ 20］ 。研究者在急性肾损伤小鼠模型中观察到，增强的有氧糖酵解产生的乳酸被肾Fb摄取，诱导Fb活化为肌Fb，引起ECM堆积 ［ 22］ 。TGF-β 1在Fb转分化为肌Fb中起重要作用，是主要的促纤维化因子之一 ［ 23］ ，其活化依赖于细胞微环境中的蛋白酶、pH或活性氧 ［ 24］ 。大量堆积的乳酸可以通过改变人肺Fb微环境pH激活TGF-β 1 ［ 25］ 。同时，乳酸可加快多种癌细胞系增殖期细胞中有丝分裂的完成，缩短细胞倍增时间，提高增殖效率 ［ 26］ 。有关肾纤维化的研究表明，乳酸可加快小鼠肾间质Fb的增殖 ［ 22］ 。进一步的研究揭示了多种癌细胞系和人胚胎肾细胞内乳酸浓度随有丝分裂的进行而增加，乳酸通过在小泛素样修饰物特异性蛋白酶1活性位点与锌形成复合物，结合并抑制小泛素样修饰物特异性蛋白酶1，这一作用稳定了后期促进复合物/细胞周期体亚基4上2个残基的小泛素化修饰，驱动了泛素结合酶E2与后期促进复合物/细胞周期体的结合，刺激细胞周期蛋白B1和分离酶抑制蛋白的定时降解，加快了细胞增殖 ［ 26］ 。二氯乙酸钠是线粒体丙酮酸脱氢酶激酶（pyruvate dehydrogenase kinase，PDK）的抑制剂，可优先驱动线粒体氧化丙酮酸拮抗乳酸的产生，从而维持细胞周期蛋白B1和分离酶抑制蛋白的稳定，延长有丝分裂的时间，减缓细胞增殖 ［ 26］ 。
2.2 调节HIF-1α的表达
HIF-1α是调节HIF-1活性的主要亚单位，其可显著提高多种细胞包括Fb的糖酵解水平 ［ 27］ 。研究者在小鼠胚胎Fb中观察到，HIF-1会上调 PDK1基因表达，抑制丙酮酸脱氢酶，进一步抑制三羧酸循环，使葡萄糖代谢物（丙酮酸）由OXPHOS反应转变为糖酵解 ［ 28］ 。GLUT对葡萄糖的吸收和转运至关重要，其中GLUT1是分布最为广泛的GLUT。HIF-1作用于多种癌细胞 GLUT1基因的增强子，上调其表达，升高糖酵解水平 ［ 29］ 。此外，HIF-1α可直接促进Fb活化。关于人肺纤维化的研究表明，肌Fb中6-磷酸果糖-2-激酶/果糖-2，6-二磷酸酶3（6-phosphofructo-2-kinase/fructose-2，6-bisphosphatase 3，PFKFB3）表达增加使得糖酵解水平升高，导致三羧酸循环中间产物琥珀酸水平上调，琥珀酸可稳定HIF-1α的结构，染色质免疫沉淀试验证实HIF-1α在经TGF-β 1处理后直接结合于α平滑肌肌动蛋白基因的启动子区域，促进人肺Fb转分化为肌Fb ［ 10］ 。
2.3 增强PKM2的表达
PKM2是糖酵解中催化葡萄糖生成丙酮酸的主要限速酶之一。人瘢痕疙瘩Fb中的PKM2在低氧刺激下较在正常Fb中的表达升高 ［ 30］ 。人增生性瘢痕来源的内源性多肽-增生性瘢痕失调多肽1通过与黏着斑激酶和PKM2结合，下调两者活性，并抑制Smad2磷酸化，达到抑制增生性瘢痕Fb增殖，促进Fb凋亡，降低S期Fb比例及减少胶原合成的作用 ［ 31］ 。
PKM2以4种不同的酶促状态存在：无活性单体、几乎无活性的二聚体、无活性T态四聚体和活性R态四聚体，四者之间的比例决定PKM2最终的功能 ［ 32］ 。目前，关于增大PKM2四聚体占比在不同组织纤维化中的作用机制有不同的研究结果。有研究显示，四氯化碳诱导的小鼠肝纤维化模型中肝Fb的PKM2表达上调，敲除 PKM2基因或使PKM2四聚体化会显著抑制体外小鼠肝Fb的活化和增殖 ［ 33］ 。在选择性PKM2四聚体化激活剂焦磷酸四乙酯-46作用下，小鼠主动脉内皮细胞产生较低浓度的乳酸，抑制了体内外的内皮-间质转化 ［ 34］ 。然而，PKM2四聚体在人纤维化肺组织中的Fb内表达上调，PKM2四聚体可与Smad7形成络合物，阻断Smad7与TGF-β 1Ⅰ型受体结合，抑制TGF-β 1Ⅰ型受体泛素化，使其保持稳定，从而增强TGF-β 1信号通路转导，促进纤维化进程 ［ 35］ 。综合以上研究，考虑PKM2在纤维化中发挥不同作用的原因可能基于以下2点：（1）上述研究均未对PKM2四聚体的活性状态进行区分，因此产生了不同的研究结果；（2）PKM2不同构象调控不同的纤维化信号通路，这些信号通路同时受纤维化发展阶段中其他分子的调控。因此，PKM2不同构象在纤维化中的具体作用机制仍需进一步研究明确。
2.4 增强GLUT1的表达
研究显示，烧伤患者皮肤形成的瘢痕疙瘩组织中糖酵解和GLUT1表达增强 ［ 36］ 。有研究表明，TGF-β 1在小鼠原代肝Fb中可通过经典和非经典通路上调GLUT1的表达，从而促进肝纤维化，通过根皮素抑制GLUT1则可逆转TGF-β 1对肝Fb迁移和增殖的影响，并延迟肝Fb转分化为肌Fb的过程；动物实验结果显示，腹腔注射根皮素缓解了四氯化碳诱导的小鼠肝纤维化 ［ 37］ 。
2.5 促进PGK1的表达
PGK1在糖酵解过程中催化1，3-二磷酸甘油酸转变成3-磷酸甘油酸，并产生ATP。研究者观察到PGK1在人瘢痕疙瘩组织和瘢痕疙瘩Fb中均高表达，敲除 PGK1基因后，瘢痕疙瘩Fb的增殖、迁移、侵袭和Ⅰ型胶原表达均受到抑制，并且磷脂酰肌醇-3-激酶（phosphoinositide 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）通路的磷酸化被显著抑制，在PI3K抑制剂LY294002作用于瘢痕疙瘩Fb后，GLUT1、LDHA和Ⅰ型胶原的表达均下调，研究者推测PGK1通过PI3K/Akt途径介导上述基因在瘢痕疙瘩Fb中的表达 ［ 38］ 。
3. 抑制有氧糖酵解的药物
在皮肤纤维化疾病的发生与发展过程中，有氧糖酵解参与并发挥了重要作用，以抑制有氧糖酵解为治疗靶点的药物为皮肤纤维化疾病提供了新的治疗思路。抑制有氧糖酵解的药物通过靶向有氧糖酵解相关酶和综合抑制有氧糖酵解发挥作用。
3.1 靶向有氧糖酵解相关酶的药物
研究显示，紫草素通过抑制PKM2下调糖酵解水平并抑制核苷酸结合寡聚化结构域样受体热蛋白结构域相关蛋白3介导的炎症反应，改善了小鼠烧伤创面的异常愈合，防止了瘢痕疙瘩的形成 ［ 36］ 。另有研究表明，紫草素可降低机械通气诱导的肺纤维化小鼠肺泡灌洗液中的乳酸和Ⅰ型原胶原羟基端延长肽，减轻肺组织中的胶原沉积，抑制小鼠肺纤维化 ［ 39］ 。2-脱氧-D-葡萄糖（2-deoxy-D-glucose，2-DG）是己糖激酶-2的抑制剂 ［ 11］ ，研究者通过构建人真皮原代Fb的Smad3驱动荧光素酶报告基因，使用2-DG抑制糖酵解可下调由TGF-β 1诱导的 Smad3依赖性转录，表明2-DG可下调ECM水平，从而改善皮肤纤维化 ［ 13］ 。Li等 ［ 40］ 关于瘢痕疙瘩的研究表明，2-DG可以剂量和时间依赖的方式抑制人瘢痕疙瘩Fb的增殖。3-（3-吡啶基）-1-（4-吡啶基）-2-丙烯-1-酮是PFKFB3的抑制剂 ［ 10］ ，可缓解由TGF-β 1刺激人真皮Fb发生的胶原沉积 ［ 19］ 。草氨酸盐可通过竞争性结合LDH而后抑制LDH ［ 22］ ，继而减少小鼠肾小管上皮细胞的乳酸生成，抑制叶酸诱导损伤后的小鼠肾Fb活化和增殖 ［ 22］ 。化合物408可抑制LDH5并逆转TGF-β 1介导的人原代肺Fb中代谢方式向有氧糖酵解的转变 ［ 41］ 。木蝴蝶素A通过抑制LDHA下调人肝Fb内的糖酵解，抑制肝Fb收缩，从而缓解肝纤维化 ［ 42］ 。
3.2 综合抑制有氧糖酵解的药物
目前研究表明，二甲双胍可下调Warburg效应的关键因子HIF-1α、GLUT1、PDK1、己糖激酶和LDH，在长时程快速心房起搏诱导的犬慢性房颤模型中，通过口服给予二甲双胍，缓解了犬心房纤维化 ［ 43］ 。另有研究表明，二甲双胍通过调节人肺Fb腺苷酸激活蛋白激酶/哺乳动物雷帕霉素靶蛋白途径抑制人肺Fb胶原合成 ［ 44］ 。3-溴丙酮酸是一种小分子烷化剂，有研究者在单侧输尿管结扎诱导的小鼠肾纤维化模型中，通过腹腔注射3-溴丙酮酸，使得小鼠肾Fb中有氧糖酵解相关酶己糖激酶-2、LDHA和PKM2的表达水平下调，小鼠肾Fb的活化、增殖和ECM的合成均受到抑制，从而缓解了小鼠肾纤维化 ［ 45］ 。博来霉素诱导的小鼠肺纤维化研究表明，血管活性肽可通过血管活性肽-MAS相关G蛋白偶联受体D轴下调小鼠肺Fb中己糖激酶-2、PFKFB3表达水平，抑制糖酵解，并缓解博来霉素诱导的小鼠肺纤维化 ［ 46］ 。雷公藤红素可下调糖酵解酶（如GLUT1、己糖激酶-2、LDHA、PKM2）和相关信号蛋白（如Akt、HIF-1α、哺乳动物雷帕霉素靶蛋白）的表达水平，抑制Warburg效应，减轻小鼠非酒精性脂肪性肝病肝脏纤维化 ［ 47］ 。
4. 总结与展望
有氧糖酵解在皮肤损伤发生时同步启动，持续存在于整个损伤修复过程中，后期不断增强的有氧糖酵解打破了组织重塑期的能量代谢平衡，通过多种复杂机制促使纤维化发生。使用有氧糖酵解相关酶的抑制剂或其他小分子化合物及目前已经研制出的药物抑制有氧糖酵解，可达到抑制皮肤纤维化的目的，说明有氧糖酵解参与皮肤纤维化的过程并在其中发挥重要作用，抑制有氧糖酵解可以为皮肤纤维化疾病的防治提供一个新方向。但是，尽管目前已有许多关于有氧糖酵解促纤维化相关机制的研究报道，但其大多着眼于内脏器官纤维化，关于皮肤纤维化的研究仍非常有限，缺乏精准的信号通路转导、蛋白相互作用关系及皮肤纤维化疾病发生、发展和稳定过程中的持续监测数据。因此，有氧糖酵解促进皮肤纤维化疾病发生与发展的具体及关键机制的揭秘和整个疾病发展过程中的变化情况亟待探索。相关机制的深入研究会有助于筛选更好的靶向有氧糖酵解的治疗药物，从而为皮肤纤维化疾病提供更精准、更有效的临床治疗方法。
梁莉婷：设计实验、实施研究、采集数据、分析/解释数据、文章撰写；宋薇、李曌、姚斌：对文章的知识性内容作批评性审阅、技术和材料支持；张超、张孟德、袁星宇、恩和吉日嘎拉：实施研究、采集数据、解释数据、统计分析；付小兵、黄沙、朱平：酝酿和设计实验、研究过程指导、研究经费支持、对文章的知识性内容作批评性审阅所有作者均声明不存在利益冲突糖尿病引起的慢性难愈合创面会导致患者生活质量下降、感染乃至截肢，而这类创面的治疗方式的选择有限。该研究团队曾利用胎盘生长因子-2123-144（PIGF-2123-144）的肝素结合域设计生长因子，使得其与创面环境中暴露的ECM结合。在1型糖尿病小鼠模型中，工程生长因子（eGF）可促进创面再上皮化和肉芽组织形成。联合使用eGF则更为有效，如联合血管内皮生长因子A（VEGF-A）与PIGF-2123-144形成的VEGF-A-PIGF-2123-144、血小板衍生生长因子BB（PDGF-BB）与PIGF-2123-144形成的PDGF-BB-PIGF-2123-144和肝素结合EGF（HB-EGF）与PIGF-2123-144形成的HB-EGF-PIGF-2123-144的“三联疗法”可显著促进创面愈合，其在给药部位停留的时间明显长于野生型生长因子。此外，该研究团队还观察到，在1型糖尿病小鼠模型中创面的细胞环境的变化，包括M1型巨噬细胞、M2型巨噬细胞和效应T细胞的数量变化，对创面愈合情况有显著预测价值。这些结果提示，VEGF-A-PIGF-2123-144与PDGF-BB-PIGF-2123-144和HB-EGF-PIGF-2123-144的三联疗法可能是促进糖尿病慢性难愈合溃疡愈合的一种有效治疗手段。张凡，编译自《NPJ Regen Med》，2021,6(1):76；肖健，审校 -
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2 细胞计数试剂盒8法检测5组人皮肤成纤维细胞培养48 h后的吸光度值 (样本数为6，
注：以吸光度值表示细胞增殖活性；GO为氧化石墨烯；横坐标下方1、2、3、4、5分别为0 μg/mL GO组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组、10.0 μg/mL GO组；5组细胞吸光度值总体比较，F=17.16，P<0.001；与0 μg/mL GO组比较，aP<0.01
3 划痕试验观察4组人皮肤成纤维细胞划痕后36 h的迁移情况 倒置荧光显微镜×50，图中标尺为500 μm。3A、3B、3C、3D.分别为0 μg/mL GO组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组细胞迁移情况，图3B剩余划痕面积最小，图3A、3C、3D剩余划痕面积相近且均明显大于图3B
注：GO为氧化石墨烯
4 划痕试验观察4组人脐静脉血管内皮细胞划痕后12 h的迁移情况 倒置荧光显微镜×50，图中标尺为500 μm。4A、4B、4C、4D.分别为0 μg/mL GO组、0.1 μg/mL GO组、1.0 μg/mL GO组、5.0 μg/mL GO组细胞迁移情况，图4B剩余划痕面积最小，图4A、4C、4D剩余划痕面积相似且均大于图4B
注：GO为氧化石墨烯
5 4组GO-GelMA复合水凝胶交联前后性状。5A、5B、5C、5D.分别为交联前0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，均为红色液体状；5E、5F、5G、5H.分别为交联后0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，均为微黄色凝胶状，且5.0 μg/mL GO复合水凝胶组颜色最深，4组流动性无明显差别
注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶
6 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗各时间点愈合情况。6A、6B、6C、6D.分别为治疗3 d时0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，创面均不同程度缩小；6E、6F、6G、6H.分别为治疗14 d 时0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，创面均基本愈合，且均未见水凝胶敷料残留
注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶
7 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗各时间点血流灌注情况。7A、7B、7C.分别为治疗3 d时0 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组创面血流灌注情况；7D、7E、7F.分别为0.1 μg/mL GO复合水凝胶组治疗3、7、14 d 创面血流灌注情况，随着治疗时间延长，血流灌注逐渐减少，但图7D血流灌注明显高于图7A、7B、7C
注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶；黑色代表无血流灌注，绿色代表血流灌注少，红色代表血流灌注多
8 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗7 d血管新生情况。8A、8B、8C、8D.分别为0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组、1.0 μg/mL GO复合水凝胶组、5.0 μg/mL GO复合水凝胶组，均有血管生成 苏木精-伊红×200，图中标尺为100 μm；8E、8F.分别为0 μg/mL GO复合水凝胶组和0.1 μg/mL GO复合水凝胶组，0.1 μg/mL GO复合水凝胶组有成簇新生血管且集中分布于GO附近 苏木精-伊红×400，图中标尺为50 μm
注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶；黑色箭头指示GO
9 2组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗7 d时VEGF的表达 二氨基联苯胺-苏木精×400，图中标尺为50 μm。9A、9B.分别为0 μg/mL GO复合水凝胶组、0.1 μg/mL GO复合水凝胶组VEGF表达，0.1 μg/mL GO复合水凝胶组VEGF表达明显高于0 μg/mL GO复合水凝胶组
注：VEGF阳性染色为深褐色；GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶，VEGF为血管内皮生长因子；白色箭头指示的是GO
表1 4组人皮肤成纤维细胞划痕后各时间点细胞迁移率比较（%，
组别 样本数 24 h 36 h 0 μg/mL GO组 5 23.5±7.7 55.0±7.2a 0.1 μg/mL GO组 5 30.1±1.1 82.2±13.9 1.0 μg/mL GO组 5 19.1±6.9 42.8±2.6a 5.0 μg/mL GO组 5 19.1±8.9 45.1±9.5a F值 2.85 19.08 P值 0.070 <0.001 注：GO为氧化石墨烯；处理因素主效应，F=1.97，P=0.159；时间因素主效应，F=441.00，P<0.001；两者交互作用，F=0.71，P=0.641；F值、P值为组间各时间点总体比较所得；与0.1 μg/mL GO组比较， aP<0.01 
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表2 4组人皮肤成纤维细胞培养各时间点血管内皮生长因子的表达比较（
组别 样本数 4 h 6 h 8 h 0 μg/mL GO组 3 17.90±0.89 17.14±0.92 17.11±1.28a 0.1 μg/mL GO组 3 17.72±0.45 19.62±1.64 19.56±0.51 1.0 μg/mL GO组 3 16.98±0.53 17.31±1.21 17.76±0.86 5.0 μg/mL GO组 3 16.78±0.59 17.07±0.08 17.25±0.60a F值 2.25 3.61 5.11 P值 0.159 0.065 0.029 注：GO为氧化石墨烯；处理因素主效应，F=8.52，P<0.001；时间因素主效应，F=1.35，P=0.279；两者交互作用，F=1.52，P=0.215；F值、P值为组间各时间点总体比较所得；与0.1 μg/mL GO组比较，aP<0.05
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表3 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗各时间点创面愈合率比较（%，
组别 样本数 3 d 7 d 14 d 0 μg/mL GO复合水凝胶组 3 22.1±5.5 66.5±8.8 97.1±2.0 0.1 μg/mL GO复合水凝胶组 3 30.8±7.9 66.5±4.9 94.5±1.4 1.0 μg/mL GO复合水凝胶组 3 30.8±9.6 72.4±5.7 97.3±1.0 5.0 μg/mL GO复合水凝胶组 3 33.3±6.4 70.3±4.5 97.8±1.0 F值 1.32 0.89 5.77 P值 0.316 0.434 0.062 注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶；处理因素主效应，F=2.89，P=0.068；时间因素主效应，F=656.20，P<0.001；两者交互作用，F=1.06，P=0.405；F值、P值为组间各时间点总体比较所得
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表4 4组小鼠全层皮肤缺损创面经GO-GelMA复合水凝胶治疗各时间点MPU比值比较 （
组别 样本数 3 d 7 d 14 d 0 μg/mL GO复合水凝胶组 3 1.77±0.11a 1.29±0.16 1.85±0.22 0.1 μg/mL GO复合水凝胶组 3 2.68±0.16b 2.13±0.24 1.46±0.24d 1.0 μg/mL GO复合水凝胶组 3 1.80±0.08c 1.74±0.17 2.45±0.56 5.0 μg/mL GO复合水凝胶组 3 1.82±0.11a 1.87±0.30 1.75±0.36 F值 30.22 7.83 3.07 P值 0.011 0.093 0.201 注：GO为氧化石墨烯，GelMA为甲基丙烯酸酐化明胶，MPU为平均灌注单位；处理因素主效应，F=2.86，P=0.104；时间因素主效应，F=2.55，P=0.104；两者交互作用，F=8.81，P<0.001；F值、P值为组间各时间点总体比较所得；与0.1 μg/mL GO复合水凝胶组比较，aP<0.01，cP<0.05;与0.1 μg/mL GO复合水凝胶组治疗7 d比较，bP<0.05,dP<0.01
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