研究背景

瘢痕疙瘩是一种皮肤结缔组织异常增生性疾病，以细胞外基质大量沉积和成纤维细胞过度增殖为特点。患者除外貌受损之外，还会发生瘙痒、疼痛等不适症状，严重影响身心健康。因其发病机制尚未研究清楚，瘢痕疙瘩的治疗一直是整形外科及皮肤科的难题。Warburg效应是指在有氧条件下糖酵解作用增强的代谢异常现象，最先在肿瘤中发现，在肿瘤的发生、发展过程中起着重要作用。有研究发现，瘢痕疙瘩来源的成纤维细胞（KFs）出现了糖酵解作用增强的现象，可能存在类似肿瘤细胞的Warburg效应。本研究旨在明确KFs中是否存在Warburg效应，并阐明Warburg效应在KFs中所发挥的作用及相关机制，从能量代谢的角度探索瘢痕疙瘩的发病机制及新的治疗手段。

研究目的

研究KFs中是否存在Warburg效应，并明确Warburg效应是瘢痕疙瘩的特殊能量代谢方式还是其他瘢痕共有的代谢特征。

阐明Warburg效应与KFs的生物学活性及功能的关系，探究抑制Warburg效应能否为瘢痕疙瘩的治疗提供新的可能。

研究方法

从临床获取废弃的瘢痕疙瘩、增生性瘢痕、萎缩性瘢痕、增生期瘢痕及正常皮肤组织标本，分离培养成纤维细胞。检测瘢痕疙瘩成纤维细胞（KFs）、增生性瘢痕成纤维细胞（HSFs）、萎缩性瘢痕成纤维细胞（ASFs）及增生期瘢痕成纤维细胞（PSSFs）中是否存在Warburg效应：

各组取相同数量的细胞进行培养，用葡萄糖检测试剂盒及乳酸检测试剂盒检测对比各组细胞的葡萄糖消耗量及乳酸产生量；

提取细胞总RNA及总蛋白，应用qPCR技术及Western-blot技术检测各组细胞中糖酵解关键酶mRNA的转录情况及蛋白的表达情况；

取相同数量的KFs及正常皮肤成纤维细胞（NFs），用糖酵解抑制剂2-脱氧-D-葡萄糖(2-DG)处理48h后，应用CCK8试剂盒检测KFs与NFs增殖活性变化。

分离培养KFs，用乳酸脱氢酶抑制剂草氨酸钠 (0, 20, 40, 80 mmol/l)进行处理。检测抑制Warburg效应对KFs细胞生物学活性及功能的影响，并探究相关机制：

检测草氨酸钠处理后KFs糖酵解作用的产物—乳酸的产生量变化，以此来反应草氨酸钠对KFs中Warburg效应的抑制作用；

抑制Warburg效应后，应用划痕实验检测各组KFs细胞迁移力的变化；提取细胞内总RNA，应用qPCR技术检测各组细胞中I型胶原、III胶原、TGF-β1、VEGF及α-SMA基因mRNA的转录情况；应用流式细胞仪及Annexin V PE凋亡试剂盒检测细胞凋亡情况；应用CCK8试剂盒检测细胞增殖活性的变化。

应用流式细胞仪及细胞周期检测试剂盒检测各组KFs的细胞周期分布情况；提取细胞内总蛋白，应用Western-blot技术检测各组KFs细胞周期相关蛋白表达情况；

研究结果

KFs、HSFs、ASFs及PSSFs中是否存在Warburg效应的检测：

KFs组的葡萄糖消耗量及乳酸产生量较NFs组显著增高；而HSFs组、ASFs组、PSSFs组与NFs组相比，无显著统计学差异。

KFs组糖酵解关键酶己糖激酶（HK2）、丙酮酸激酶（PKM2）、乳酸脱氢酶（LDHA）mRNA及蛋白的相对表达量较NFs组显著增高。而HSFs组、ASFs组、PSSFs组与NFs组相比，无显著统计学差异。

在2-DG的作用下，KFs组的细胞增殖活性较NFs组降低更加显著，差异有统计学意义。

抑制Warburg效应对KFs细胞生物学活性、功能的影响及其相关机制研究：

随着草氨酸钠浓度的升高，KFs所产生的乳酸量逐渐降低，差异有统计学意义。草氨酸钠可抑制KFs中的Warburg效应，抑制作用呈浓度依赖性。

抑制Warburg效应对KFs生物学活性及功能的影响：

在草氨酸钠作用24h及48h后，各组KFs细胞迁移率均随草氨酸钠浓度提高而逐渐降低。草氨酸钠可呈浓度依赖性地抑制KFs细胞迁移力。

与无草氨酸钠的对照组相比，20mmol/l及40 mmol/l草氨酸钠组I型胶原mRNA转录水平显著降低，40 mmol/l草氨酸钠组α-SMA的mRNA转录水平显著降低。

与对照相比，草氨酸钠作用组KFs细胞凋亡比率显著增高。

在草氨酸钠作用24h及48h后，随草氨酸钠浓度提高，各组KFs细胞增殖活性均逐渐降低，且48h后降低幅度更大。草氨酸钠可呈浓度及时间依赖性抑制KFs细胞增殖活性。

抑制Warburg效应后引起KFs增殖活性降低的分子机制研究

随草氨酸钠浓度的提高，各组KFs中G0/G1期细胞占比逐渐增高，S期及G2/M期细胞占比相应降低。草氨酸钠可呈浓度依赖性诱导KFs发生G0/G1周期阻滞。

在草氨酸钠的作用下，周期相关蛋白Cyclin D1蛋白表达量显著降低。其上游调控因子Akt蛋白表达量及GSK3β (phospho S9)蛋白量也显著降低。

结论

KFs中既有Warburg效应又有氧化磷酸化反应，而其他类型瘢痕及增生期瘢痕无类似现象发生。Warburg效应是瘢痕疙瘩的特殊能量代谢方式而非其他瘢痕共有的代谢特征。

抑制KFs的Warburg效应可引起细胞增殖活性降低、迁移力降低、胶原分泌减少，并可通过Akt-GSK3β-Cyclin D1信号通路诱导KFs发生G0/G1周期阻滞。KFs的生物学活性及功能与Warburg效应关系密切，抑制Warburg效应可为瘢痕疙瘩的治疗提供新的可能。

Background

Keloid is a dermal fibroproliferative disease characterized by abnormal proliferation of fibroblasts and overproduction of extracellular matrix (ECM). It can not only affect the appearance, but also cause pain, itching, and other symptoms, which would become a cosmetic and psychological burden for patients. However, there is no satisfactory treatment for keloid as the key factor responsible for the disease has not been identified. Warburg effect is first found in tumors and refers to enhanced glycolysis under aerobic conditions, which takes an important part in the occurrence and development of tumors. It was found that the glycolysis in keloid fibroblasts (KFs) was enhanced and there might be a Warburg effect like tumors in KFs. This study aims to demonstrate whether there is a Warburg effect in KFs, to study the effects of the Warburg effect in KFs and related mechanisms, and to explore the pathogenesis and new treatment of keloids in terms of energy metabolism.

Objective

The aim of this study is to demonstrate whether there is a Warburg effect in KFs and find out whether Warburg effect is a special way of energy metabolism in keloid or a common metabolic feature of other scars.

This study aims to investigate the relationship between the Warburg effect and the biological activity, the function of KFs, and to explore whether inhibition of Warburg effect can provide new hope for the treatment of keloid.

Methods

Fibroblasts were isolated from keloid samples, hypertrophic scar samples, atrophic scar samples, proliferative stage scar samples, and normal skin samples. Study whether there is a Warburg effect in keloid fibroblasts (KFs), hypertrophic scars fibroblasts (HSFs), atrophic scar fibroblasts (ASFs), and proliferative stage scar fibroblasts (PSSFs).

The same amounts of cells in each group were cultured under the same conditions. Then, the glucose consumption and the lactate production were detected using a glucose assay kit and a lactic acid assay kit.

Cells in each group were cultured in the same condition, and total RNA and total protein were extracted. The mRNA and protein expression levels of glycolysis key enzyme genes in each group were detected by qPCR and Western-blot.

The same amounts of KFs and NFs were cultured and treated with 2-DG at different concentrations. After 48h, the cell proliferation of KFs and NFs was detected with a Cell Counting Kit-8 (CCK-8).

KFs were isolated and cultured with different concentrations of oxamate (0,20,40,80 mmol/l) in vitro. After the Warburg effect was inhibited, we detected the biological activity and function of KFs, and explored the related mechanism.

The product of glycolysis (the production of lactate) in KFs was detected after the treatment of oxamate to reflect the inhibition effects of oxamate on the Warburg effect in KFs.

After the inhibition of the Warburg effect, a scratch wound assay was used to detect the cell migration ability of KFs. Then, total RNA was extracted and the mRNA expression levels of type I collagen, type III collagen, TGF-β1, VEGF, and α-SMA were detected using qPCR. Besides, the apoptosis rate in KFs was detected using flow cytometry and the Annexin V PE apoptosis kit. What’s more, a CCK-8 was used to detect the cell proliferation in KFs.

The cell cycle distribution in KFs was detected using flow cytometry and the Cell Cycle Analysis Kit. Then, total protein was extracted and the protein expression levels of proteins related to cell cycles were detected using Western-blot.

Results

The detection of Warburg effect in KFs, HSFs, ASFs, and PSSFs:

Glucose consumption and lactate production in KFs were significantly higher than that in NFs. Glucose consumption and lactate production in HSFs, ASFs, and PSSFs were not significantly different from those in NFs.

The mRNA and protein expression levels of hexokinase 2 (HK2), pyruvate kinase isoform M2 (PKM2), and lactate dehydrogenase A (LDHA) in KFs were significantly higher than those in NFs. The mRNA and protein expression levels of glycolysis key enzyme genes in HSFs, ASFs, and PSSFs were not significantly different from those in NFs.

After the treatment of 2-DG, the cell proliferation of KFs decreased more pronounced than that of NFs.

The effect of inhibition of Warburg effect on the biological activity, function of KFs and related mechanism:

The lactate production in KFs decreased gradually when treated with increasing concentrations of oxamate. Oxamate could inhibit Warburg effect in KFs in a dose-dependent manner.

Effect of inhibition of Warburg effect on biological activity and function of KFs:

The migration ability of KFs decreased gradually when treated with increasing concentrations of oxamate for 24h and 48h. Oxamate could inhibit the migration ability of KFs in a dose-dependent manner.

Compared with the control group, the mRNA expression levels of type I collagen in KFs treated with 20 mmol/l and 40 mmol/l oxamate decreased significantly. And the mRNA expression levels of α-SMA in KFs treated with 40 mmol/l oxamate also decreased significantly.

The apoptosis rate in KFs treated with oxamate increased significantly compared with the control group.

The cell proliferation of KFs decreased gradually when treated with increasing concentrations of oxamate for 24h and 48h. Cell proliferation decreased more pronounced when treated with oxamate for 48h. Oxamate could inhibit the cell proliferation of KFs in a dose- and time-dependent manner.

Study on the Molecular Mechanism of the decrease of the cell proliferation of KFs after inhibition of Warburg effect:

When treated with increasing concentrations of oxamate, cells in G0/G1 phase increased significantly, and cells in S phase and G2/M phase decreased correspondingly. Oxamate can induce G0/G1 arrest in KFs in a dose-dependent manner.

When treated with oxamate, the protein expression levels of cell cycle related protein – Cyclin D1 decreased significantly and so does its upstream regulators – Akt and GSK3β (phospho S9).

Conclusion

1. There is both aerobic glycolysis and oxidative phosphorylation in KFs, and there is no similar phenomenon in HSFs, ASFs, or PSSFs. The Warburg effect in KFs is a special feature, which not exists in other types of scars or in the proliferative stage of scars.

2. Inhibition of the Warburg effect in KFs could significantly suppress cell proliferation, migration ability, and collagen production, and could induce G0/G1 arrest through Akt-GSK3β-Cyclin D1 pathway in KFs. The biological activity and function of KFs were closely associated with the Warburg effect. And inhibition of the Warburg effect in keloid can provide new hope for the treatment of keloid.