岩黄连总碱下调miR-223表达调控人牙龈成纤维细胞增殖与凋亡的分子机制研究

Molecular mechanism of corydalis saxicola bunting total alkaloids regulating roliferation and apoptosis of human gingival fibroblasts by down-regulating expression of miR-223

目的:探讨岩黄连总碱对牙龈卟啉单胞菌脂多糖(Pg-LPS)诱导的人牙龈成纤维细胞增殖与凋亡的影响及其可能作用机制。方法:体外培养人牙龈成纤维细胞,分为对照组(NC组)、Pg-LPS组、5μg/ml岩黄连总碱+Pg-LPS组、10μg/ml岩黄连总碱+Pg-LPS组、20μg/ml岩黄连总碱+Pg-LPS组、miR-NC+Pg-LPS组、miR-223+Pg-LPS组、anti-miR-NC+Pg-LPS组、anti-miR-223+Pg-LPS组、20μg/ml岩黄连总碱+Pg-LPS+miR-NC组、20μg/ml岩黄连总碱+Pg-LPS+miR-223组。采用EDU、流式细胞术分别检测细胞增殖与凋亡;RT-qPCR法检测miR-223的表达量;ELISA法检测白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)水平;Western blot检测剪切的半胱天冬氨酸蛋白酶3(Cleaved-caspase-3)、pro-caspase-3、磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(AKT)蛋白表达。结果:岩黄连总碱(5、10、20μg/ml)和下调miR-223表达可显著升高Pg-LPS诱导的人牙龈成纤维细胞增殖活力和pro-caspase-3蛋白表达水平,降低细胞凋亡率、IL-1β和TNF-α水平以及Cleaved-caspase-3、p-PI3K/PI3K、p-AKT/AKT蛋白表达水平(均P<0.05)。过表达miR-223可逆转岩黄连总碱对Pg-LPS诱导的人牙龈成纤维细胞增殖、凋亡及PI3K/AKT通路的作用。结论:岩黄连总碱可通过下调miR-223表达而抑制PI3K/AKT信号通路从而促进Pg-LPS诱导的人牙龈成纤维细胞增殖,并抑制细胞凋亡和炎性反应。

Objective:To explore the effect of corydalis saxicola bunting total alkaloids on the proliferation and apoptosis of human gingival fibroblasts induced by porphyromonas gingivalis lipopolysaccharide(Pg-LPS)and its possible mechanism.Methods:Human gingival fibroblasts were cultured in vitro and divided into control group(NC group),Pg-LPS group,5μg/ml corydalis saxicola bunting total alkaloids+Pg-LPS group,10μg/ml corydalis saxicola bunting total alkaloids+Pg-LPS group,20μg/ml corydalis saxicola bunting total alkaloids+Pg-LPS group,miR-NC+Pg-LPS group,miR-223+Pg-LPS group,anti miR-NC+Pg-LPS group,anti miR-223+Pg-LPS group,20μg/ml corydalis saxicola bunting total alkaloids+Pg-LPS+miR-NC group,20μg/ml corydalis saxicola bunting total alkaloids+Pg-LPS+miR-223 group.Cell proliferation and apoptosis were detected by EDU and flow cytometry;the expression of miR-223 was detected by qRT-PCR;the levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were determined by ELISA.Western blot was used to detect the expression of Cleaved caspase-3,pro-caspase-3,phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)proteins.Results:The corydalis saxicola bunting total alkaloids(5,10,20μg/ml)and down-regulation of miR-223 expression significantly increased the proliferation activity and pro-caspase-3 protein level of Pg-LPS-induced human gingival fibroblasts,decreased apoptosis rate,IL-1βand TNF-αlevels,and Cleaved caspase-3,p-PI3K/PI3K,p-AKT/AKT protein levels(all P<0.05).Overexpression of miR-223 reversed the effects of corydalis saxicola bunting total alkaloids on Pg-LPS-induced proliferation,apoptosis and PI3K/AKT pathway of human gingival fibroblasts.Conclusion:Corydalis saxicola bunting total alkaloids can inhibit the PI3K/AKT signaling pathway by downregulating the expression of miR-223,thereby promoting the proliferation of human gingival fibroblasts induced by Pg-LPS,and inhibiting cell apoptosis and inflammatory reactions.