探討檜木醇於糖尿病相關牙周炎抑制發炎老化及鐵死亡效用與分子機制

糖尿病是全球公共衛生的重大議題，亦為牙周炎的主要危險因子。高血糖促進糖化終產物（advanced glycation end products）的累積，引發由氧化壓力、細胞衰老及衰老相關分泌表型（senescence-associated secretory phenotype）所驅動的慢性低度發炎狀態，此現象又被稱之為發炎老化（inflammaging）。此外，糖化終產物與鐵凋亡（ferroptosis）密切相關，鐵凋亡為鐵依賴性細胞死亡，參與糖尿病相關併發症（包括牙周炎）之病理機制。現今治療糖尿病相關牙周炎（diabetes-associated periodontitis）的策略主要著重於抑制細菌生長，卻未能有效解決其免疫失衡的根本問題。檜木醇（hinokitiol）是一種源自於柏科（Cupressaceae）植物的單萜類化合物，其具有抗發炎老化、抗細胞焦亡及抗鐵死亡等特性。然而，檜木醇在糖尿病相關牙周炎中的治療潛力尚未被充分研究，因此，本研究旨在探討檜木醇在糖尿病相關牙周炎中對發炎老化及鐵死亡的影響。為了模擬糖尿病相關牙周炎的環境，本研究使用糖化終產物刺激人類牙齦纖維母細胞（human gingival fibroblasts）。本實驗以細胞存活率分析以及傷口癒合試驗評估檜木醇對細胞活性及傷口癒合的影響，並透過流式細胞儀、衰老相關β-半乳糖苷酶（senescence-associated β-galactosidase）染色及p16蛋白表現量來分析檜木醇對氧化壓力及細胞衰老的影響。衰老相關分泌表型細胞激素介白素-6（interleukin-6）和介白素-8，則使用酶聯免疫吸附試驗（ELISA）進行測定。此外，利用即時定量聚合酶連鎖反應（real-time quantitative polymerase chain reaction）測定鐵死亡相關基因SLC7A11、GPX4與TFR1表現變化。研究結果顯示，檜木醇顯著促進傷口癒合，並以劑量依賴性方式減少糖化終產物誘導的活性氧產生、細胞衰老及衰老相關分泌表型細胞激素分泌。此外，檜木醇可顯著上調SLC7A11和GPX4表達，並降低TFR1表達，顯示其可透過此機制有效抑制鐵死亡。總結來說，檜木醇能夠透過減輕發炎老化和抑制鐵死亡進而促進人類牙齦纖維母細胞傷口癒合，顯示其作為糖尿病相關牙周炎輔助治療劑的潛力。

Diabetes mellitus (DM) is a major public health concern and a key risk factor for periodontitis. Hyperglycaemia accelerates the accumulation of advanced glycation end products (AGEs), which drive inflammaging—a chronic, low-grade inflammatory state characterized by oxidative stress, cellular senescence, and the senescence-associated secretory phenotype (SASP). Additionally, AGEs contribute to ferroptosis, an iron-dependent form of regulated cell death implicated in diabetes-related complications, including periodontitis. Current therapeutic strategies for diabetes-associated periodontitis (DP) primarily focus on bacterial reduction but fail to address the underlying dysregulated immune response central to disease progression. Hinokitiol (β-thujaplicin), a monoterpenoid from the Cupressaceae family, has demonstrated potent anti-inflammaging and anti-ferroptotic properties. However, its potential therapeutic role in DP remains largely unexplored. To address this gap, this study investigated the effects of hinokitiol on inflammaging and ferroptosis in an in vitro DP model. To mimic DP conditions, human gingival fibroblasts (HGFs) were stimulated with AGEs. Hinokitiol’s effects on cell viability and wound healing were assessed using MTT and wound healing assays. Additionally, key aspects of inflammaging, including oxidative stress and cellular senescence, were analysed through flow cytometry, senescence-associated β-galactosidase (SA-β-Gal) staining, and p16 protein expression via Western blot. Senescence-associated secretory phenotype (SASP) cytokines, interleukin-6 (IL-6) and interleukin-8 (IL-8), were assessed utilizing enzyme-linked immunosorbent assay (ELISA), while ferroptotic markers, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and transferrin receptor 1 (TFR1), were assessed via reverse transcription-polymerase chain reaction (RT-PCR). It was discovered that hinokitiol significantly enhanced wound healing while dose-dependently reducing AGE-induced ROS production, senescence activity and SASP secretion. Furthermore, hinokitiol suppressed ferroptosis by upregulating SLC7A11, GPX4 while suppressing TFR1. In conclusion, hinokitiol effectively promotes wound healing in HGFs through the mitigation of inflammaging and ferroptosis. These findings highlight its potential as a novel adjunct therapeutic model for DP, offering a targeted approach to addressing immune dysregulation in addition to bacterial control.