活性氧响应的纳米颗粒对炎性环境下牙龈成纤维细胞功能的影响及机制

Effect and mechanism of reactive oxygen species⁃responsive nanoparticles on the regulation of human gingival fibroblast function and inflammation induced by lipopolysaccharide

目的 探讨在牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis-lipopolysaccharide,P.g-LPS)诱导的牙龈成纤维细胞(human gingival fibroblasts,HGFs)炎症环境下,活性氧(reactive oxygen species,ROS)响应型纳米颗粒PssL-NAC对HGFs内ROS、炎症因子、胶原蛋白生成、细胞迁移功能以及Toll样受体4(Toll-like receptor 4,TLR4)-核因子κB(nuclear factor-κB,NF-κB)通路蛋白表达的影响。方法 本研究已通过单位伦理委员会审查批准。通过响应ROS的硫缩酮键(thioketal,TK)连接聚己内酯(polycaprolactone,PCL)的疏水端和聚乙二醇(polyethylene glycol,PEG)的亲水端,水相油相自主装的方式得到内部包裹油溶性的抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine-NAC)的PssL-NAC微球,制备NAC水溶液,并使用透射电镜观察验证纳米粒子合成成功。在提取的HGFs中分别加入P.g-LPS(0、5、10μg/mL),P.g-LPS(0、5、10μg/mL)+NAC,P.g-LPS(0、5、10μg/mL)+PssL-NAC,共聚焦显微镜观察不同组别细胞内ROS水平,确定后续实验使用的P.g-LPS浓度。将HGFs随机分为空白对照组(对照组,不做处理),P.g-LPS组(P.g-LPS处理细胞),NAC组(P.g-LPS+NAC处理细胞),PssL-NAC组(P.g-LPS+PssL-NAC处理细胞)。通过细胞增殖与毒性实验验证PssL-NAC的生物安全性。使用2',7'-二氯荧光黄双乙酸盐探针与细胞共孵育通过荧光实时定量检测细胞内炎症因子白细胞介素6(interleukin-6,IL-6)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和Ⅰ型胶原蛋白(collagen 1,COL1)、Ⅲ型胶原蛋白(collagen 3,COL3)的基因水平;通过划痕实验观察PssL-NAC对牙龈成纤维细胞的迁移功能的影响;通过免疫印迹法检测TLR4-NFκB信号通路相关蛋白的表达。结果 PssL-NAC具有良好生物相容性,对HGFs的细胞增殖能力无显著影响(P>0.05);在P.g-LPS浓度升高的条件下,PssL-NAC能维持细胞内大约两倍对照组的ROS水平(P<0.001);PssL-NAC能显著降低P.g-LPS诱导升高的IL-6(P<0.001)、TNF-α基因水平(P<0.001),上调COL1基因水平(P<0.001);P.g-LPS刺激后,PssL-NAC能将细胞迁移能力恢复至对照组水平(P>0.05),并降低TLR4-NF-κB通路中TLR4(P<0.001)、p65(P=0.006)、p-p65(P=0.017)蛋白的表达。结论PssL-NAC维持细胞内适宜浓度ROS,通过TLR4-NF-κB通路减轻P.g-LPS诱导的细胞炎症,并恢复炎症条件下的细胞胶原生成和细胞迁移功能。

Objective To investigate the effects of PssL-NAC reactive oxygen species(ROS)-responsive nanoparti-cles on intracellular ROS production,inflammatory factor levels,collagen production,cell function and Toll-like recep-tor 4(TLR4),NF-κB nuclear factor-κB(p65)pathway protein expression in human gingival fibroblasts(HGFs)in-duced by Porphyromonas gingivalis-lipopolysaccharide(P.g-LPS).Methods This study was reviewed and approved by the ethics committee.PssL-NAC microspheres containing oil soluble antioxidant N-acetylcysteine(NAC)were obtained by connecting the hydrophobic end of polycaprolactone(PCL)and the hydrophilic end of polyethylene glycol(PEG)via thioketal(TK)bonds in response to ROS,and self loading in the aqueous and oil phases.After preparation of the PssL-NAC microspheres and aqueous NAC solution,successful synthesis of the nanoparticles was verified by transmission electron microscopy.Then,HGFs were exposed to P.g-LPS(0,5,or 10μg/mL),P.g-LPS(0,5,or 10μg/mL)+NAC,and P.g-LPS(0,5,or 10μg/mL)+PssL-NAC,and the ROS levels in the different groups were observed under confocal micros-copy to determine the concentration of P.g-LPS for use in subsequent experiments.The groups were as follows:control group(no treatment),P.g-LPS group(HGFs treated with P.g-LPS),NAC group(HGFs treated with P.g-LPS and NAC),and PssL-NAC group(HGFs treated with P.g-LPS and PssL-NAC).Cell counting kit-8(CCK-8)assays verified the bio-safety of PssL-NAC.The ROS levels in the different groups were detected by DCFH-DA probes and observed via confo-cal microscopy.Real-time qPCR(RT-qPCR)was used to monitor the gene expression levels of the intracellular inflam-matory cytokines interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),collagen 1(COL1)and collagen 3(COL3).The effect of PssL-NAC on the migration of HGFs was observed via the scratch test.The protein expression of TLR4-NF-κB,and phosphorylated p65(p-p65)in the TLR4-NF-κB pathway was evaluated by Western blot.Results PssL-NAC had no significant effect on HGF proliferation(P>0.05).At elevated P.g-LPS concentrations,PssL-NAC maintained intra-cellular ROS levels approximately twice those in the control group(P<0.001).PssL-NAC significantly decreased P.g-LPS-induced IL-6(P<0.001)and TNF-α(P<0.001)gene expression and increased COL1 gene expression(P<0.001).After P.g-LPS stimulation,PssL-NAC restored cell migration to the control level(P>0.05)and decreased the protein expression of TLR4(P<0.001),p65(P=0.006),and p-p65(P=0.017)in the TLR4-NF-κB pathway.Conclusion PssL-NAC maintains the appropriate intracellular ROS concentration,alleviates P.g-LPS-induced inflam-mation in HGFs through the TLR4-NF-κB pathway,and restores the cell functions of collagen production and migration in an inflammatory environment.