Keloids are considered benign fibroproliferative tumors. Overabundance of extracellular matrix resulting from hyperproliferation of keloid fibroblasts (KFs), and dysregulation of apoptosis represent the main pathophysiological mechanisms underlying the formation of keloids. High-mobility group box 1 (HMGB1) is considered relevant in various diseases as it plays important roles in inflammation, mitogenesis, and the regulation of cell death. Suppression of intracellular HMGB1 expression inhibits autophagy, which predominantly serves as a cell survival mechanism, whilst increasing apoptotic cell death. Suppression of HMGB1 with glycyrrhizin has therapeutic benefits in fibrotic diseases. In this study, we explore the possible involvement of autophagic activity and HMGB1 as a cell death regulator in keloid pathogenesis. We highlight the potential utility of glycyrrhizin as an antifibrotic agent via the regulation of the aberrant balance between autophagy and apoptosis in keloids. The expression of HMGB1 in keloid and normal dermal tissue was investigated by immunohistochemistry (IHC). Transmission electron microscopic examination was performed to detect autophagosomes in both KFs and HDFs. Further, IHC for Beclin 1 and light chain protein 3 (LC3), which are markers of autophagy, was performed in tissue samples. Using an autophagy defect kit, the basal autophagy levels in KFs, HDFs, transforming growth factor-β (TGF-β)-treated HDFs, and HMGB1-treated HDFs were investigated. After treatment of KFs and human dermal fibroblasts (HDFs) with glycyrrhizin (0, 100, 200, and 500 μM), cell proliferation was assessed. Apoptosis as well as autophagic activity were determined in keloids treated with glycyrrhizin. Expression of major ECM components and factors associated with fibrosis were investigated in keloid spheroids treated with glycyrrhizin. Levels of autophagy and collagen accumulation in the TGB-β-treated fibrotic condition were examined following the application of the autophagy inhibitor 3-methyladenine (3-MA). Higher HMGB1 expression was observed in keloid than in normal tissue. Additionally, high autophagic activity was found in KFs as well as in keloid tissue. In TGF-β- or HMGB1-treated fibrotic condition, an enhanced autophagy level was detected in HDFs. Decreased HMGB1 expression was confirmed in keloid spheroids treated with glycyrrhizin. The proliferation of KFs was decreased following glycyrrhizin treatment. While the level of autophagy was decreased, the rate of apoptosis was increased in KFs following glycyrrhizin application. The expression levels of profibrogenic molecules, namely ERK1/2, Akt, and NF-κB, were enhanced in HMGB1-teated HDFs but significantly decreased following glycyrrhizin treatment. The expression levels of types I and III collagen, fibronectin, and elastin were reduced in glycyrrhizin-treated keloid spheroids. TGF-β, Smad2/3, ERK1/2, and HMGB1 were decreased significantly in keloid spheroids following the application of glycyrrhizin. Enhanced autophagy was detected in KFs and keloid tissue. While enhanced apoptosis was detected in keloids after glycyrrhizin treatment, autophagy markers were significantly reduced. Treatment with the autophagy inhibitor 3-MA resulted in a marked decrease in the levels of autophagy markers and collagen expression in the TGF-β-induced fibrotic condition. The results indicate that autophagy plays an important role in the pathogenesis of keloids. Because the HMGB1 blocker, glycyrrhizin, appears to degrade ECM and downregulate autophagic cell death in keloids, its potential use for treatment of keloids is indicated.
Keloid는 발생기전 및 치료방법이 명확히 밝혀지지 않은 섬유성 피부 병변으로 비정상적으로 세포의 증식이 증가되고 자멸사가 감소하며 과도하게 세포외기질이 축적되는 것을 특징으로 한다. High mobility group box 1 (HMGB1)은 대부분의 진핵 세포에 존재하는 핵 단백질로 염증, 면역, 세포의 증식 및 사멸 등을 조절하여 다양한 섬유성 병변과 관련되어 있는 것으로 알려져 있다. 자가포식은 에너지 결핍 등의 스트레스 조건에서 세포가 생존할 수 있도록 돕는 세포 사멸의 한 방법으로, HMGB1은 세포자멸과 자가포식간 균형을 조절하는 것으로 알려져 있다. 이에 우리는 자가포식이 keloid의 병인과 관계되어 있지 않은지 확인해보고자 하였다. 또한, HMGB1의 세포외 작용을 억제하는 것으로 알려진 glycyrrhizin을 이용하여 keloid에서 HMGB1과 glycyrrhizin이 세포 사멸의 균형에 미치는 영향과, 이를 통한 항섬유 효과를 확인해 보고자 하였다. 면역화학염색을 통해 정상조직에 비해 keloid 조직에서 HMGB1의 발현이 증가함을 확인하였고, 투과전자현미경을 이용해 자가포식소체가 켈로이드에서 증가되어있으며, 켈로이드 섬유모세포 및 조직에서 자가포식 표지인자인 Beclin 1과 LC3이 증가되어 있음을 확인하였다. 유세포분석을 통해 대표적 섬유성 인자인 TGF-β와 HMGB1을 처리한 섬유모세포에서 자가포식이 증가함을 확인하였다. 켈로이드 세포구에 glycyrrhizin을 처리하여 HMGB1의 발현이 감소함을 면역화학염색을 통해 확인하였으며, MTT 분석을 통해 켈로이드 섬유모세포와 정상 섬유모세포에서 glycyrrhizin (0, 100, 200, 500 μM) 처리후 세포의 증식이 유의하게 감소함을 확인하였다. 자가포식 표지인자의 면역화학염색을 통해 glycyrrhizin의 처리에 따라 자가포식이 유의하게 감소하나, 유세포분석 및 TUNEL 검사에서 세포자멸은 유의하게 증가하는 결과를 확인하였다. Western blot을 통해 HMGB1을 처리한 섬유모세포에서 ERK1/2, Akt, NF-κB가 유의하게 증가하나 glycyrrhizin을 처리 후 다시 감소함을 확인하였다. 또한 켈로이드 세포구에서 type 1, 3 collagen, fibronectin, elastin과 TGF-β, Smad2/3, ERK1/2의 발현이 glycyrrhizin 처리에 따라 유의하게 감소함을 western blot을 통해 확인하였다. TGF-β를 처리한 정상 섬유모세포에 자가포식 억제제인 3-methyladenine을 처리해 자가포식이 감소하고, 교원질의 발현도 감소함을 PCR검사를 통해 확인하였다. 이 결과들을 토대로, 켈로이드에서 자가포식이 증가되어 있고, HMGB1의 억제제인 glycyrrhizin의 처리에 따라 자가포식이 억제되고, 세포자멸이 증가하며, 섬유화가 감소하는 결과를 확인하였다. 이를 통해 keloid의 억제 및 치료방법 개발에 응용할 수 있을 것으로 사료된다.