摘要
目的 探讨南蛇藤素(Cel)对微小RNA(miR)-223-3p/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)轴对脂多糖(LPS)诱导的人牙周膜干细胞(hPDLSCs)增殖、凋亡和炎症反应的影响。方法 分别给予不同浓度的Cel(1、2、4μmol/L)培养经1μg/ml LPS诱导的hPDLSCs,以未处理的hPDLSCs作为对照组。采用MTT法检测细胞存活率,选取合适的Cel浓度用于后续实验研究。将对数生长期hPDLSCs分为对照组、LPS组、Cel组(2μmol/L),采用LipofectamineTM2000转染试剂盒在Cel组的基础上转染细胞miR-223-3p inhibitor及阴性对照(inhibitor NC),并命名为Cel+inhibitor NC组、Cel+miR-223-3p inhibitor组。分别检测以上各组细胞凋亡、增殖以及炎症因水平;q RT-PCR检测各组细胞中miR-223-3p表达水平,Western blot检测细胞中NLRP3蛋白及凋亡蛋白-天冬氨酸蛋白水解酶-1(Caspase-1)表达;双荧光素酶报告基因检测实验验证miR-223-3p、NLRP3靶向关系。结果 2μmol/L Cel作用细胞后其活力最高。与对照组相比,LPS组细胞存活率、mi R-223-3p表达显著下降,肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6含量、细胞凋亡率、NLRP3、Caspase-1表达显著升高(P<0.05);与LPS组相比,Cel组细胞存活率、miR-223-3p表达显著升高,TNF-α、IL-1β、IL-6含量、细胞凋亡率、NLRP3、Caspase-1表达显著降低(P<0.05);与Cel+inhibitor NC组相比,Cel+miR-223-3p inhibitor组LPS组细胞存活率、miR-223-3p表达显著下降,TNF-α、IL-1β、IL-6含量、细胞凋亡率、NLRP3、Caspase-1表达显著升高(P<0.05)。结论 Cel可以促进LPS诱导的hPDLSCs增殖,抑制细胞凋亡及炎症反应,可能与上调miR-223-3p、抑制NLRP3表达有关。
Objective To investigate the effect of celastrol(Cel)on lipopolysaccharide(LPS)-induced proliferation,apoptosis and inflammatory response of human periodontal ligament stem cells(hPDLSCs).Methods The hPDLSCs were cultured with different concentrations of Cel,and the untreated hPDLSCs were used as the control group.The cell viability was detected by MTT method.hPDLSCs in logarithmic growth phase were divided into control group,LPS group and Cel group(2μmol/L).The cells of Cel group were transfected with miR-223-3p inhibitor and negative control(inhibitor NC)using lipofectamineTM2000 transfection kit,and named as Cel+inhibitor NC group and Cel+miR-223-3p inhibitor group.The apoptosis,proliferation and the levels of inflammatory factors in the above groups were detected respectively.The expression levels of miR-223-3p in the cells of each group were detected by qRT-PCR.Western blot was performed to detect the expression of NLRP3 protein and apoptosis protein-aspartate proteolytic enzyme-1(Caspase-1)in cells,dual-luciferase reporter gene detection experiment was performed to verify the targeting relationship between miR-223-3p and NLRP3.Results The cell viability was the highest after 2μmol/L Cel treatment.Compared with the control group,the cell viability and the expression of miR-223-3p in the LPS group were obviously decreased,the contents of tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-6,the apoptosis rate,and the expression of NLRP3 and Caspase-1 were obviously increased(P<0.05).Compared with the LPS group,the cell viability and the expression of miR-223-3p in the Cel group were obviously increased,the contents of TNF-α,IL-1βand IL-6,the apoptosis rate,and the expression of NLRP3 and Caspase-1 were obviously decreased(P<0.05).Compared with the Cel+inhibitor NC group,the cell viability and the expression of miR-223-3p in the Cel+miR-223-3p inhibitor group were obviously decreased,the contents of TNF-α,IL-1βand IL-6,the apoptosis rate,and the expression of NLRP3 and Caspase-1 were obviously inc
作者
刘洁
王文洁
常颖
LIU Jie;WANG Wen-jie;CHANG Ying(Department of Stomatology,Aerospace Center Hospital,Beijing 100049,China)
出处
《北京口腔医学》
CAS
2024年第1期11-15,共5页
Beijing Journal of Stomatology
基金
航天医疗健康科技集团有限公司科研项目(2020YK17)。
