Abstract：
BACKGROUND: Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis.
METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis.
RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium.
CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.
METHODS: C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis.
RESULTS: In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium.
CONCLUSIONS: The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.
摘要：
背景：氟斑牙是由于过量消耗氟化物而引起的牙齿变色。它代表了牙齿组织中慢性氟中毒的明显表现，对人体产生不良影响，特别是在牙齿上。跨膜蛋白16a(TMEM16A)在内质网和质膜的连接处表达。其通道活性的改变可以破坏内质网钙稳态和细胞内钙离子浓度，从而诱导内质网应激（ERS）。本研究旨在探讨钙补充剂和TMEM16A对氟斑牙ERS的影响。
方法：对出现氟斑牙的C57BL/6小鼠进行为期八周的不同钙浓度治疗：低（0.071％），中等（0.79%），和高（6.61%）。各种化验，包括苏木精和伊红（HE）染色，免疫组织化学，实时荧光定量聚合酶链反应(qPCR),和蛋白质印迹,用于评估钙补充剂对氟化物含量的影响，成釉细胞形态学，TMEM16A表达，和内质网应激相关蛋白(钙网蛋白(CRT)，葡萄糖调节蛋白78（GRP78），需要肌醇的激酶1α（IRE1α），PKR样ER激酶(PERK),激活转录因子6（ATF6））在受氟斑牙影响的小鼠切牙中。此外，用TMEM16A抑制剂T16Ainh-A01和中等剂量钙治疗氟斑牙小鼠，以研究TMEM16A对氟化物含量的影响，成釉细胞形态学，和内质网应激相关蛋白在小鼠切牙氟中毒的背景下。
结果：与模型小鼠相比，补钙后，门牙中的氟化物含量显着降低（p<0.01）。此外，TMEM16A的表达，CRT,GRP78,IRE1α,PERK,ATF6也表现出显著降低(p<0.01)，在中剂量钙组中观察到最明显的效果。此外,氟含量（p<0.05）和CRT的表达，GRP78,IRE1α,PERK,在用TMEM16A抑制剂T16Ainh-A01和中等剂量的钙同时治疗后,ATF6(p<0.01)进一步减少。
结论：补充钙或抑制TMEM16A表达似乎可以通过抑制内质网应激来减轻氟中毒的有害影响。这些发现对确定解决氟斑牙的潜在治疗目标具有重要意义。
方法：对出现氟斑牙的C57BL/6小鼠进行为期八周的不同钙浓度治疗：低（0.071％），中等（0.79%），和高（6.61%）。各种化验，包括苏木精和伊红（HE）染色，免疫组织化学，实时荧光定量聚合酶链反应(qPCR),和蛋白质印迹,用于评估钙补充剂对氟化物含量的影响，成釉细胞形态学，TMEM16A表达，和内质网应激相关蛋白(钙网蛋白(CRT)，葡萄糖调节蛋白78（GRP78），需要肌醇的激酶1α（IRE1α），PKR样ER激酶(PERK),激活转录因子6（ATF6））在受氟斑牙影响的小鼠切牙中。此外，用TMEM16A抑制剂T16Ainh-A01和中等剂量钙治疗氟斑牙小鼠，以研究TMEM16A对氟化物含量的影响，成釉细胞形态学，和内质网应激相关蛋白在小鼠切牙氟中毒的背景下。
结果：与模型小鼠相比，补钙后，门牙中的氟化物含量显着降低（p<0.01）。此外，TMEM16A的表达，CRT,GRP78,IRE1α,PERK,ATF6也表现出显著降低(p<0.01)，在中剂量钙组中观察到最明显的效果。此外,氟含量（p<0.05）和CRT的表达，GRP78,IRE1α,PERK,在用TMEM16A抑制剂T16Ainh-A01和中等剂量的钙同时治疗后,ATF6(p<0.01)进一步减少。
结论：补充钙或抑制TMEM16A表达似乎可以通过抑制内质网应激来减轻氟中毒的有害影响。这些发现对确定解决氟斑牙的潜在治疗目标具有重要意义。
