三七总皂苷通过抑制槟榔碱诱导的HaCaT细胞铁死亡减轻口腔黏膜下纤维化  

Panax notoginseng saponins attenuate oral submucous fibrosis by pre⁃venting arecaline-induced ferroptosis of HaCaT cells

作　　者：邹红 孙宇婕 赵晨曦 张馨月 胡亮 谢璐 文礼湘[1] 唐群[1] 胡立娟 ZOU Hong;SUN Yujie;ZHAO Chenxi;ZHANG Xinyue;HU Liang;XIE Lu;WEN Lixiang;TANG Qun;HU Lijuan(School of Medicine,Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学医学院,湖南长沙410208

出　　处：《中国病理生理杂志》2023年第9期1563-1569,共7页Chinese Journal of Pathophysiology

基　　金：湖南省自然科学基金项目(No.2021JJ30506);湖南省教育厅重点项目(No.21A0242);湖南省大学生创新创业训练项目(No.S202210541130)。

摘　　要：目的:探讨三七总皂苷(PNS)是否通过调控铁死亡减轻槟榔碱(ANE)诱导的口腔黏膜下纤维化(OSF),并初步研究其机制。方法:采用ANE作用于人永生化角质形成细胞系HaCaT构建OSF模型,设置空白组,模型组(50 mg/L ANE),低、中、高剂量(12.5、25和50 mg/L)PNS组,以及liproxstatin-1(铁死亡抑制剂)组,共计6组。光镜观察HaCaT细胞形态学变化;CCK-8法检测细胞活力;Western blot检测细胞中Ⅰ型胶原(Col-Ⅰ)、溶质载体家族7成员11(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)的蛋白表达水平;透射电镜观察细胞线粒体形态学变化;Ferro-Orange荧光探针检测细胞Fe^(2+)水平;DCFH-DA荧光探针检测细胞内活性氧(ROS)含量;比色法检测细胞内谷胱甘肽(GSH)含量。结果:与空白组比较,模型组HaCaT细胞多数呈长梭形,纤维化样外观,贴壁不牢靠,形态学改变显著,电镜下线粒体双层膜密度增加,嵴显著减少,呈典型的铁死亡表现,Col-Ⅰ蛋白表达增加,GPX4和SLC7A11蛋白表达降低,Fe^(2+)和ROS水平升高,GSH含量降低(均P<0.05);与模型组相比,低、中、高剂量PNS组细胞多数恢复圆润,呈上皮细胞样,线粒体膜密度减小,嵴数目增加,形态趋于正常,Col-Ⅰ蛋白表达减少,ROS水平下降,GSH含量升高(均P<0.05);与模型组相比,中、高剂量PNS组GPX4和SLC7A11蛋白表达显著上升,Fe^(2+)含量显著降低(均P<0.05);liproxstatin-1组细胞和线粒体形态及各指标水平与高剂量PNS组相比无显著差异(均P>0.05)。结论:PNS可通过抑制ANE诱导的HaCaT细胞铁死亡减轻OSF。AIM:To investigate whether Panax notoginseng saponins(PNS)attenuate arecoline(ANE)-in-duced human oral submucous fibrosis(OSF)by regulating ferroptosis,and to study its mechanism.METHODS:The OSF model was established by treating human immortalized keratinocyte cell line HaCaT with ANE.There were 6 groups created:blank group,model group(50 mg/L ANE),low-,medium-and high-dose(12.5,25 and 50 mg/L)PNS groups,and liproxstatin-1(ferroptosis inhibitor)group.The morphological changes of the cells were observed under a light micro-scope.Cell viabilty was detected by CCK-8 assay.Collagen type I(Col-I),solute carrier family 7 member 11(SLC7A11)and glutathione peroxidase 4(GPX4)protein expression levels were detected by Western blot.The morphological changes of mitochondria were observed by transmission electron microscopy.The Fe^(2+)level was detected by FerroOrange fluores-cent probe.The intracellular reactive oxygen species(ROS)content was detected by DCFH-DA fluorescent probe.The content of glutathione(GSH)was determined by colorimetry.RESULTS:Compared with blank group,the majority of the cells in model group displayed long spindle shape,fibrotic appearance,unstable adhesion,and obvious morphological changes.Under electron microscope,mitochondrial double membrane density increased,and cristae decreased signifi-cantly,presenting typical ferroptosis.The expression of Col-I increased,GPX4 and SLC7A11 protein expression de-creased,Fe^(2+)and ROS levels increased,and GSH content decreased(all P<0.05).Compared with model group,most of the cells in low-,medium-and high-dose PNS groups were round and epitheloid,with decreased mitochondrial membrane density,increased ridge number and normal morphology,decreased Col-I expression and ROS level,and increased GSH content(all P<0.05).Compared with model group,the protein expression levels of GPX4 and SLC7A11 in medium-and high-dose PNS groups were significantly increased,while the content of Fe^(2+)was significantly decreased(all P<0.05).There were no significant differences in cell a

关 键 词：口腔黏膜下纤维化 铁死亡 三七总皂苷 

分 类 号：R781.5[医药卫生—口腔医学] R363.2[医药卫生—临床医学]