牙髓成纤维细胞乳酸堆积对牙髓炎的影响

Effect of dental pulp fibroblasts lactate accumulation on pulpitis

目的:探究牙髓炎中牙髓成纤维细胞(DPF)乳酸堆积与炎症之间的关系。方法:构建大鼠牙髓炎模型,苏木素-伊红(HE)染色观察牙髓炎症浸润情况;免疫组织化学染色观察炎症因子白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α),以及乳酸脱氢酶A(LDHA)的表达情况;免疫荧光观察波形蛋白(Vimentin)与LDHA共定位情况。收集人健康及龋坏第三磨牙,提取正常及炎症牙髓组织检测乳酸含量。体外培养人DPF,加入LDHA抑制剂GSK2837808A,分为对照组、脂多糖(LPS)组、抑制剂组,检测乳酸产生情况;实时荧光定量PCR检测DPF中炎症因子IL-1β、TNF-α及LDHA mRNA表达水平。结果:与大鼠正常牙髓相比,大鼠牙髓炎模型建模1、3 d时炎症浸润逐渐增加,炎症因子IL-1β、TNF-α及LDHA表达随之增高,3 d时最为显著(P<0.05);7 d炎症减轻,炎症因子IL-1β、TNF-α及LDHA表达降低,与对照组无明显差异(P>0.05)。大鼠炎症牙髓组织中DPF的Vimentin与LDHA可见共定位。人炎症牙髓组织乳酸含量显著高于健康牙髓(P<0.05)。体外使用GSK2837808A处理人DPF,乳酸分泌减少,LDHA及炎症因子表达降低(P<0.05)。结论:在牙髓炎发展中,DPF的乳酸生成与炎症程度相关,乳酸的堆积可促进炎症因子的表达;通过抑制LDHA可致人DPF的乳酸产生及炎症因子表达降低。

Objective:To explore the relationship between dental pulp fibroblast(DPF)lactate accumulation and inflammation in pulpitis.Methods:SD rats were used to construct the pulpitis model.Hematoxylin-eosin was used to observe the inflammatory infiltration of pulpitis.The expression of inflammatory cytokines,IL-1βand TNF-α,as well as the key enzyme that catalyzes lactate production,lactate dehydrogenase A(LDHA),were visualized by immunohistochemical staining.Immunofluorescence staining was used to evaluate the colocalization of LDHA and the marker of dental pulp fibroblasts Vimentin.Human healthy and carious third molars were collected,then extracting normal and inflammatory pulp tissues to measure lactate content.DPF was cultured in vitro and LDHA inhibitor GSK2837808A was added.The cells were divided into control group,lipopolysaccharide(LPS)group,and LDHA inhibitor group.Lactate test kit was used to measure cellular lactate content.qRT-PCR was used to detect the mRNA expression of inflammatory cytokines IL-1βand TNF-α,and LDHA.Results:Compared with the normal pulp of rats,the inflammatory infiltration increased during the modeling of pulpitis for 1 d,3 d,meanwhile the expression of LDHA and inflammatory cytokines IL-1β,TNF-αincreased,especially 3 d(P<0.05),while decreased at 7 days,which has no significant difference as compared with that of the control group(P>0.05).Vimentin and LDHAof DPF in inflammatory pulp tissue in rats could be seen co-localization.The content of lactate in human inflammatory pulp tissue was higher than that of healthy pulp(P<0.05).By treating dental pulp fibroblasts with lactate dehydrogenase inhibitor GSK2837808A,lactate secretion decreased,and the expression of LDHA and inflammatory cytokines decreased(P<0.05).Conclusions:In the development of pulpitis,the production of lactate of DPF is related to the degree of inflammation,and the accumulation of lactate promoted the expression of inflammatory cytokines;and by inhibiting LDHA,human DPF lactate production and expression of inflammatory factors were reduced.