不同根管封填劑之氧化鋅顆粒對MG-63類骨細胞誘發內質網壓力及缺氧誘發因子/熱休克蛋白訊息路徑之關係與機制

Zinc oxide particles of root canal sealers stimulate ER stress and HIF/HSP signaling pathways in MG-63 osteoblastic cells

林晏如(Yan-Ru Lin)

指導教授 ： 鄭景暉

國立臺灣大學/醫學院/臨床牙醫學研究所/碩士(2020年)

https://doi.org/10.6342/NTU202002744

摘要

實驗目的：氧化鋅是根管封填劑的主要成分之一。本篇研究的目的為比較不同劑量之市售氧化鋅丁香油酚基底的根管充填劑粉末在MG-63類骨細胞中對細胞活性氧化物質、內質網壓力和缺氧誘發因子/熱休克蛋白路徑之間的關係及機制。
實驗方法：本實驗使用不同濃度的氧化鋅顆粒刺激MG-63類骨細胞，將MG-63類骨細胞暴露在不同濃度（0-200 μg/ ml）的市售氧化鋅丁香油酚基底根管充填劑 (Canals®, Roth’s 801®) 24小時，部分組別加入NAC (ROS抑制劑) (5 mM, 10mM)做預處理。以即時聚合酶連鎖反應（Real-time PCR）及西方墨點法（Western Blot）探討氧化鋅顆粒對MG-63類骨細胞活性氧化物產生、內質網壓力、熱休克蛋白及缺氧誘發因子之間傳訊路徑的關係。
實驗結果: MG-63類骨細胞加入市售氧化鋅丁香油酚基底根管治療充填劑(Canals®, Roth’s 801®)暴露24小時後，我們發現會刺激活性氧化物反應產生，並啟動MAPK蛋白質p-ERK、p-p38、細胞調節氧化反應的p-Nr2及HO-1表現量增加，加入NAC的組別則抑制HO-1的表現量。內質網壓力的傳導因子ERO1L、GRP78、GRP94、PERK、IRE1α、eIF2α、XBP1s、CHOP等隨著氧化鋅顆粒濃度的上升表現量均會提高，其中PERK、IRE1α、eIF2α、XBP1s、CHOP在氧化鋅顆粒濃度上升超過50 μg/ ml，表現量卻明顯下降，推測與氧化鋅顆粒在高濃度環境造成的細胞毒性有關。熱休克蛋白Hsp27、Hsp70及缺氧誘發因子HIF-1α也都有類似反應，隨著氧化鋅顆粒濃度上升表現量提高，當濃度上升超過50μg/ ml，表現量下降的趨勢。熱休克蛋白Hsp60及Hsp90相對不同的濃度則沒有顯著的變化。
結論：這是首次檢測市售氧化鋅丁香油酚基底根管充填劑(Canals®, Roth’s 801®)對於細胞受到損傷產生活性氧化物反應、內質網壓力及熱休克蛋白的研究，我們發現氧化鋅顆粒會促使細胞產生過氧化物反應、內質網壓力反應發生、熱休克蛋白及缺氧誘發因子的活化，且彼此之間有密切的相關性訊號能夠彼此影響傳導，當毒性超過細胞可以負荷的範圍，細胞基因亦可能因損傷而無法表現。

關鍵字

氧化鋅 ； 氧化鋅丁香油酚根管充填劑 ； MG-63類骨細胞 ； 活性氧化物 ； 內質網壓力 ； 熱休克蛋白 ； 缺氧誘發因子

並列摘要

Aim: Zinc oxide is a main component of zinc oxide-eugenol based root canal sealers. The purpose of this study is to evaluate and compare the effects of different doses of commercially available root canal filling powder. The aim is to find a link between MG-63 osteoblast-like cells and its interactions with reactive oxygen species, endoplasmic reticulum stress, hypoxia inducible factors, and heat shock proteins all while figuring out the relationship and mechanism of action between these protein pathways.
Materials and methods: This experiment stimulated MG-63 osteoblast-like cells by exposing the cells to different concentrations (0-200 μg/ml) of zinc oxide particles from commercially available zinc oxide-eugenol based root canal sealer (Canals®, Roth's 801®) for 24 hours. Several groups were pretreated by adding NAC (ROS scavenger) (5 mM, 10 mM). After exposure, Real-time PCR and Western Blot techniques were used to examine the effects of zinc oxide particles on MG-63 osteoblast-like cells and on reactive oxygen species, endoplasmic reticulum stress, hypoxia inducible factor-1α, and heat shock proteins which can help us to understand their relationships and signaling pathways.
Results: After 24 hours of exposure to MG-63 osteoblast-like cells with commercially available zinc oxide-eugenol based root canal fillers (Canals®, Roth's 801®), it was found to stimulate the production of reactive oxygen species and increased the expression of the MAPK protein, p-ERK, p-p38, cell regulated oxidation reacted p-Nr2, and HO-1. The group with NAC added inhibited HO-1 expression. Endoplasmic reticulum stress conduction factors such as ERO1L, GRP78, GRP94, PERK, IRE1α, eIF2α, XBP1s, and CHOP increased in accordance with an increase in zinc oxide concentration. Of the conduction factors tested, PERK, IRE1α, eIF2α, XBP1s, and CHOP expression decreased after being exposed to concentrations of 50 μg/ ml or higher of zinc oxide due to the cytotoxic nature of zinc oxide. Heat shock proteins Hsp27 and Hsp70 along with hypoxia induced factor HIF-1α also experienced the same type of reaction, increasing in expression as zinc oxide concentrations increased until a concentration of 50 μg/ ml was achieved which then reversed it. Heat shock proteins Hsp60 and Hsp90 expression did not change when exposed to different concentrations of zinc oxide.
Conclusion: This is the first time a commercially available zinc oxide-eugenol based root canal sealer (Canals®, Roth’s 801®) was tested for reactive oxygen species, endoplasmic reticulum stress, and heat shock proteins against injured cells. It was found that zinc oxide particles would produce a peroxidation response from cells. It would additionally increase endoplasmic reticulum stress, heat shock protein, hypoxia inducible factor activation and are intrinsically related, mutually affecting each other’s conduction process. When cells are exposed to toxicity levels which they cannot handle, the genetic code of the cells will be damaged and cannot be further expressed correctly.

並列關鍵字

zinc oxide ； zinc oxide-eugenol based root canal sealer ； MG-63 osteoblast-like cell ； reactive oxygen species ； endoplasmic reticulum stress ； heat shock protein ； hypoxia inducible factor

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