吐纳麝香(6-乙酰基-1,1,2,4,4,7-六甲基四氢萘)对人永生化角质形成细胞HaCaT间隙连接通讯的影响
Effects of 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (AHTN) on Gap Junctional Intercellular Communication in Human Immortalized Keratinocytes Cells HaCaT
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摘要: 吐纳麝香（tonalide，AHTN）广泛存在于各种生物和环境介质，易生物富集，具有内分泌干扰效应，皮肤接触是其进入人体的主要途径。细胞间隙连接通讯（gap junction intercellular communication，GJIC）是普遍存在于相邻细胞间的直接通讯通道，其功能的抑制与癌症的发生发展密切相关。目前AHTN对人体皮肤细胞GJIC的影响尚未见报道。本研究选用人永生化角质形成细胞HaCaT为体外（in vitro）暴露实验的材料，MTT法检测环境相关浓度AHTN对HaCaT细胞活性的影响，细胞划痕标记染料示踪技术（scrape-loading and dye transfer，SLDT）检测AHTN对GJIC功能的影响，通过对GJIC生物标志物及MAPK/ERK和PI3K/AKT信号通路的定量分析，探究AHTN调控HaCaT细胞GJIC功能的分子机制。细胞活性检测发现，10-8 mol·L-1的AHTN暴露24 h和10-9 mol·L-1的AHTN暴露48 h均可显著促进HaCaT细胞增殖（P≤0.01）；10-6 mol·L-1的AHTN暴露48 h可显著抑制HaCaT细胞增殖（P≤0.01）。SLDT检测发现，10-8 mol·L-1的AHTN暴露24 h，GJIC功能增强；10-8 mol·L-1和10-9 mol·L-1的AHTN暴露48 h，GJIC功能减弱。对不同暴露浓度和时间下雌激素受体α（estrogen receptor，ERα）的定量分析及抑制实验证明，AHTN可通过上调ERα的表达，促进或抑制下游Cx26和Cx43的表达，进而影响HaCaT细胞的GJIC功能。对信号通路的定量分析发现，10-8 mol·L-1和10-9 mol·L-1的AHTN暴露可上调级联分子MEK、ERK、PI3K和AKT的磷酸化水平，激活MAPK/ERK和PI3K/AKT信号通路，进而上调Cx43磷酸化水平，抑制GJIC功能。本研究首次通过体外实验证明了环境相关浓度AHTN对人体皮肤细胞GJIC的影响及可能的分子机制，为客观评价AHTN的生物毒性和致癌风险提供了科学依据。
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关键词:
- 吐纳麝香 /
- 细胞间隙连接通讯 /
- 雌激素受体 /
- MAPK/ERK通路 /
- PI3K/AKT通路
Abstract: 6-acetyl-1,1,2,4,4,7-hexamethyltetralin (AHTN), a widely used synthetic musk, exists in various biota and environmental media. It is easy to be accumulated in organisms and has been proven to have endocrine disrupting effects. The main way of AHTN entering human body is skin exposure. Gap junction intercellular communication (GJIC) is a common means of communication between adjacent cells and the inhibition of GJIC function is closely related to the occurrence and development of cancer. However, there is almost no effect study of AHTN on GJIC function in skin cells till now. In this study, human immortalized keratinocytes cell line (HaCaT) was exposed to different environmental relevant concentrations of AHTN in vitro, to explore the effects of AHTN on cell viability and GJIC function in skin cells. The MTT method was used to detect cell proliferation, while the scrape-loading and dye transfer (SLDT) method was used to demonstrate GJIC function. Biomarkers related to GJIC and its regulation pathways (MAPK/ERK and PI3K/AKT) were quantitative analyzed by RT-qPCR and Western blot, to reveal the possible molecular mechanism of AHTN on GJIC function in HaCaT cells. MTT assay showed that low concentration of AHTN exposure (10-8 mol·L-1 for 24 h and 10-9 mol·L-1 for 48 h) significantly promoted HaCaT cell proliferation, while high concentration of AHTN exposure (10-6 mol·L-1 for 48 h) significantly inhibited HaCaT cell proliferation. SLDT test demonstrated that GJIC function was increased after 24 h exposure to 10-8 mol·L-1 AHTN, but decreased after 48 h exposure to 10-8 mol·L-1 and 10-9 mol·L-1 AHTN. Quantitative analysis of estrogen receptor α (ERα) and connexins proved that different concentrations of AHTN exposure could up-regulate the expression of ERα, promote or inhibit the expression of downstream Cx26 and Cx43, and thereby affect the GJIC function in HaCaT cells. The ERα inhibitor experiments further identified the important role of ERα in AHTN-induced GJIC function alteration. Quantitative analysis of related signaling pathways proved that exposure to 10-8 mol·L-1 and 10-9 mol·L-1 AHTN could activate MAPK/ERK and PI3K/AKT signaling pathways by up-regulating the phosphorylation levels of cascade molecules MEK, ERK, PI3K and AKT, and inhibit GJIC function by up-regulating the phosphorylation level of Cx43. Taken together, the influence and possible molecular mechanisms of environmental relevant concentrations of AHTN on GJIC function in human skin cells was first explored in this study. Our results will provide scientific bases for objectively evaluating on the biological toxicity and carcinogenic risk of AHTN. -
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