高糖炎性环境下KLF4调控人牙周膜干细胞能量代谢的机制初探
Preliminary study on the mechanism of KLF4 regulation on energy metabolism in human periodontal ligament stem cells un-der high glucose inflammatory environment
唐婧淇 1徐萱雯 1周逸 1苟惠清 1李璐 1徐艳1
作者信息
- 1. 南京医科大学附属口腔医院牙周病科,江苏省口腔疾病研究重点实验室,江苏省口腔转化医学工程研究中心,江苏 南京 210029
- 折叠
摘要
目的:探究高糖炎性环境下Krüppel样因子 4(KLF4)对人牙周膜干细胞(hPDLSCs)线粒体能量代谢和成骨分化的影响.方法:利用免疫荧光和Western blot实验研究高糖炎性环境下KLF4 在hPDLSCs的定位和表达情况.通过碱性磷酸酶(ALP)和茜素红染色等检测高糖炎性环境下hPDLSCs成骨能力.通过慢病毒敲低和过表达技术,结合实时荧光定量PCR与Western blot实验检测病毒感染后 KLF4 的表达,检测 KLF4 对 hPDLSCs 成骨分化的影响.采用核酸酶靶向切割和释放(CUT&RUN)技术,分析高糖炎性环境下hPDLSCs中KLF4 结合的DNA及相关信号通路变化.使用Seahorse能量代谢仪探索KLF4 对hPDLSCs氧化呼吸能力的影响.结果:高糖炎性环境下,hPDLSCs中KLF4 表达下调、ALP活性降低、钙结节形成减少(均P＜0.001),Runt相关转录因子2(RUNX2)、骨钙素(OCN)表达降低(均P＜0.001).敲低KLF4,导致hPDLSCs的ALP活性降低(P＜0.001),钙结节形成减少(P＜0.001);过表达KLF4 促进hPDLSCs的成骨分化.高糖炎性环境下,KLF4 下游调控基因富集于能量代谢、成骨等相关通路.敲低KLF4 导致hPDLSCs线粒体耗氧率(OCR)水平下调,而细胞外酸化率(ECAR)水平上调;过表达KLF4 使OCR水平上调,而ECAR水平下调.结论:在高糖炎性环境下,hPDLSCs的成骨分化能力受损,KLF4 可能通过上调线粒体氧化磷酸化能力来恢复hPDLSCs的成骨分化能力.
Abstract
Objective:To investigate the effects of Krüppel-like factor 4(KLF4)in a high glucose and inflammatory environment on mitochondrial energy metabolism and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs).Methods:The localization and expression of KLF4 in hPDLSCs was investigated under high glucose inflammatory environment using immunofluo-rescence and Western blot.ALP staining and alizarin red staining were used to study osteogenic differentiation of hPDLSCs in hypergly-cemic inflammatory environment.Lentivirus knockdown and overexpression techniques were used to detect the expression of KLF4 after virus infection by qRT-PCR and Western blot.CUT&RUN technique was used to analyze the changes of KLF4 binding DNA and related signaling pathways in hPDLSCs under high glucose inflammatory environment.The impact of KLF4 on oxidative respiratory capacity of hPDLSCs was explored using a Seahorse energy metabolism analyzer.Results:KLF4 expression was downregulated under high glucose inflammatory conditions,accompanied by reduced ALP activity,calcium nodule formation(all P＜0.001),and expression of RUNX2 and OCN(P＜0.001).Knockdown of KLF4 resulted in a significant decrease in ALP activity(P＜0.001)and reduced calcium nodule formation(P＜0.001)in hPDLSCs,while overexpression of KLF4 promote osteogenic differentiation.Under high glucose inflammatory environment,the downstream regulatory genes of KLF4 were enriched in energy metabolism,osteogenesis and other related pathways.KLF4 knockdown reduced OCR levels and increased ECAR levels in hPDLSCs,while KLF4 overexpression increased OCR levels and decreased ECAR levels.Conclusions:In a high glucose inflammatory environment,the osteogenic differentiation ability of hPDLSCs was compromised,and KLF4 may enhance the mitochondrial oxidative phosphorylation capacity to restore this ability.
关键词
人牙周膜干细胞/Krüppel样因子4/牙周炎/糖尿病/线粒体能量代谢Key words
human periodontal ligament stem cells/Krüppel-like factor 4/periodontitis/diabetes mellitus/mitochondrial energy metabolism引用本文复制引用
基金项目
国家自然科学基金(82170962)
国家自然科学基金(82370961)
江苏省科教能力提升工程江苏省研究型医院项目(YJXYYJSDW4)
江苏省医学创新中心项目(CXZX202227)
出版年
2024
NETL
NSTL
万方数据