金合欢素调节HMGB1/TLR4信号通路对脂多糖诱导牙髓细胞凋亡的影响  

Effect of acacetin on lipopolysaccharide induced apoptosis of dental pulp cells by regulating the HMGB1/TLR4 signaling pathway

作　　者：林瑶 刘从娜 王世霞 张志勇[2] LIN Yao;LIU Congna;WANG Shixia;ZHANG Zhiyong(Department of Stomatology,Tangshan Maternal and Child Health Hospital,Tangshan 063000,China;Department of Stomatology,Second Hospital of Hebei Medical University)

机构地区：[1]唐山市妇幼保健院口腔科,063000 [2]河北医科大学第二医院口腔内科

出　　处：《天津医药》2024年第12期1238-1243,共6页Tianjin Medical Journal

基　　金：河北省医学科学研究重点课题计划项目(20231762)。

摘　　要：目的探究金合欢素(ACE)通过调节高迁移率族蛋白1(HMGB1)/Toll样受体4(TLR4)信号通路对脂多糖(LPS)诱导牙髓细胞凋亡的影响。方法从5例正畸、阻生患者拔除的健康牙齿的牙髓中分离、克隆、纯化第三代牙髓细胞,观察细胞形态并通过免疫荧光鉴定基质细胞表面抗原1(STRO1);流式细胞术鉴定表皮抗原CD90、CD105、CD34、CD45;MTT鉴定ACE对原代牙髓细胞(DPSCs)活性影响。将DPSCs分为Control组(0μmol/L ACE培养)、LPS组(1 ng/L LPS处理)、L-ACE组(LPS组基础上加20μmol/L ACE)、H-ACE组(LPS组基础上加40μmol/L ACE)、pcDNA-NC组(H-ACE组基础上转染pcDNA-NC质粒),HMGB组(H-ACE组基础上转染pcDNA-HMGB)。CCK-8检测细胞增殖;克隆形成实验检测克隆形成数;Western blot实验检测HMGB1/TLR4通路、凋亡、炎症相关蛋白的表达;酶联免疫吸附试验检测细胞上清液中白细胞介素(IL)-1β、IL-4、肿瘤坏死因子α(TNF-α)的含量。结果从细胞形态、STRO1阳性表达以及CD90、CD105呈阳性,CD34、CD45呈阴性鉴定成功分离DPSCs。与Control组比较,LPS组细胞增殖活力、克隆细胞数、B细胞淋巴瘤-2(Bcl-2)、IL-4水平减少,细胞凋亡率、通路相关蛋白HMGB1、TLR4,凋亡相关蛋白胱天蛋白酶(Caspase)-3、Caspase-7、Bcl-2相关X蛋白(Bax)以及炎症因子IL-1β、TNF-α水平升高(P<0.05);与LPS组比较,L-ACE组、H-ACE组细胞增殖活力、Bcl-2、IL-4依次升高,细胞凋亡率、HMGB1、TLR4、Caspase-3、Caspase-7、Bax以及IL-1β、TNF-α水平依次降低(P<0.05);过表达HMGB逆转了H-ACE对细胞增殖、凋亡、相关蛋白及炎症因子表达的影响(P<0.05)。结论ACE通过影响HMGB1/TLR4信号通路活化,实现抑制LPS诱导的DPSCs凋亡和炎症反应的作用。Objective To investigate the effect of acacetin(ACE)on the apoptosis of dental pulp cells induced by lipopolysaccharide(LPS)by regulating the high mobility histone 1(HMGB1)/Toll-like receptor 4(TLR4)signaling pathway.Methods Third generation dental pulp cells were isolated,cloned and purified from pulp of five patients with healthy teeth extracted from orthodontics and impaction.Cell morphology was observed and identification of stromal cell surface antigen1(STRO1)by immunofluorescence,and epidermal antigens CD90,CD105,CD34 and CD45 were identified by flow cytometry.MTT was applied to identify the effect of ACE on the activity of primary dental pulp cells(DPSCs).DPSCs were divided into the control group(0μmol/L ACE culture),the LPS group(1 ng/L LPS treatment),the L-ACE group(added 20μmol/L ACE on the basis of LPS group),the H-ACE group(added 40μmol/L ACE on the basis of LPS group),the pcDNA-NC group(transfected pcDNA-NC plasmid on the basis of H-ACE group)and the HMGB group(transfected pcDNA-HMGB on the basis of H-ACE group).Cell proliferation was detected by CCK-8.Clone formation number was detected by clone formation assay.Western blot experiments were applied to detect the expression of HMGB1/TLR4 pathway,apoptosis and inflammation related proteins.ELISA assay was applied to detect levels of interleukin(IL-)-1β,IL-4 and tumor necrosis factor-α(TNFα)in cell supernatant.Results DPSCs were successfully isolated and identified based on cell morphology,positive expression of STRO1,positive expression of CD90 and CD105,and negative identification of CD34 and CD45.Compared with the control group,cell proliferation activity,clonal cell count,B-cell lymphoma-2(Bcl-2)and IL-4 levels were decreased in the LPS group,while apoptosis rate,the pathway related proteins HMGB1,TLR4,apoptosis related proteins aspartate-specific cysteine protease(Caspase)-3,Caspase-7,Bcl-2 associated X protein(Bax),and inflammatory factors IL-1β,TNF-αwere increased in the LPS group(P<0.05).Compared with the LPS group,cell proliferation ac

关 键 词：HMGB1蛋白质 Toll样受体4 金合欢属 脂多糖类 牙髓炎 细胞凋亡 细胞增殖 牙髓细胞 

分 类 号：R781.31[医药卫生—口腔医学] R34[医药卫生—临床医学]