二氧化硫体内衍生物通过ROS/JNK/AP-1通路诱导大鼠切牙成釉器细胞AE2蛋白表达上调
Internal Derivatives of Sulfur Dioxide Induced Up-regulation of AE2 Protein Expression of Rat Enamel Organ through ROS/JNK/AP-1 Pathway
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摘要: 探究二氧化硫体内衍生物混合液(亚硫酸钠与亚硫酸氢钠物质的量比为3∶1)对雄性大鼠切牙组织抗氧化防御系统及切牙成釉器细胞丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)信号转导通路关键蛋白与阴离子交换蛋白2(anion exchanger 2, AE2)表达的影响。30只雄性Wistar大鼠按体质量随机分为高(100 mg·kg-1·d-1)、中(50 mg·kg-1·d-1)、低(25 mg·kg-1·d-1)二氧化硫衍生物混合液染毒组、高剂量二氧化硫衍生物混合液+N-乙酰半胱氨酸(N-acetylcysteine, NAC)染毒组及生理盐水对照组(0.9%氯化钠注射液)。各组均以2 mL·kg-1每日行腹腔注射一次，高剂量+NAC组每次腹腔注射前30 min予200 mg·kg-1 NAC水溶液灌胃再施予高剂量组相同染毒操作，连续4周。取大鼠下颌骨切牙组织进行超氧化物歧化酶(superoxide dismutase, SOD)活性、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)活性、还原型谷胱甘肽(reduced glutathione, GSH)及丙二醛(malondialdehyde, MDA)含量、活性氧簇(reactive oxygen species, ROS)水平测定。剥离包绕切牙根部的下颌骨分离成釉器细胞，以Western blot法检测成釉器细胞MAPKs信号通路关键蛋白及AE2蛋白表达情况。结果显示，低剂量染毒组与对照组比较，大鼠切牙组织多类抗氧化指标及过氧化产物含量的改变均不具统计学意义。中、高剂量染毒组较对照组大鼠切牙组织SOD活性、GSH-Px活性及GSH含量下降、MDA、ROS水平升高，差异有统计学意义(P＜0.05)。Western blot结果显示，随二氧化硫衍生物剂量升高，各组大鼠成釉器细胞p-p38 MAPK/p38 MAPK及p-ERK/ERK水平较对照组未见明显变化，而中、高剂量组p-JNK/JNK水平明显升高，AP-1蛋白发生核转位分布，AE2蛋白表达明显升高，差异均有统计学意义(P＜0.05)；而ROS抑制剂NAC能使高剂量下AP-1蛋白核转位情况得到改善，并能在一定程度上逆转高剂量二氧化硫衍生物暴露下引起的p-JNK/JNK水平升高及AE2蛋白表达上调。以上结果提示，二氧化硫体内衍生物混合液可造成大鼠切牙组织氧化应激水平升高并通过ROS/JNK/AP-1通路诱导切牙成釉细胞AE2表达上调，具有干扰成釉器细胞成釉功能的潜在毒性。Abstract: To investigate the effects of sulfur dioxide derivative mixture (molar ratio of sodium sulfite to sodium bisulfite is 3∶1) on antioxidant defense system and expression of key proteins in mitogen-activated protein kinase signal transduction pathway and anion exchanger protein 2 (AE2) in enamel organ of incisors of male rats. Thirty male Wistar rats were randomly divided into high (100 mg·kg-1·d-1), medium (50 mg·kg-1·d-1), low (25 mg·kg-1·d-1) sulfur dioxide derivative mixture group, high-dose sulfur dioxide derivative mixture+N-acetylcysteine (NAC) group and normal saline control group (0.9% sodium chloride injection). Each group received intraperitoneal injection every day with a volume of 2 mL·kg-1 for four weeks. The high-dose +NAC group was given 200 mg·kg-1 NAC solution 30 min before each intraperitoneal injection. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), contents of reduced glutathione (GSH) and malondialdehyde (MDA), and levels of reactive oxygen species (ROS) of incisors tissue were determined by assay kit. The enamel organ was separated from the root of the incisor, and the expression of key proteins in mitogen-activated protein kinase signal transduction pathway and AE2 of enamel organ was determined by Western blot. The results showed that compared with the control group, there was no statistical significance in the changes of antioxidant indexes and content of peroxide products in the incisor tissue of rats in the low-dose group, while SOD activity, GSH-Px activity and GSH content in the incisor tissue of rats in the medium-dose group and the high-dose group were decreased and level of MDA and ROS were increased. The difference was statistically significant (P<0.05). As for the protein expression, Western blot results showed that with the increase of sulfur dioxide derivative dose, the levels of p-p38 MAPK/p38 MAPK and p-ERK/ERK did not change significantly in each group compared with the control group, while the levels of p-JNK/JNK in medium and high-dose groups increased significantly and expression of AE2 protein increased with nuclear translocation distribution of AP-1 protein occurred (P<0.05). NAC, a ROS inhibitor can improve the nuclear translocation of AP-1 protein and partly reverse the elevated p-JNK/JNK level and the up-regulation of AE2 protein expression induced by exposure to high dose of sulfur dioxide derivatives. These results suggest that mixture of sulfur dioxide derivatives can increase the oxidative stress level and induce the up-regulation of AE2 expression of enamel organ of rat incisors through ROS/JNK/AP-1 pathway, which has the potential toxicity of interfering with the amelogenesis function of ameloblasts.
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