Asiatic Acid Inhibits LPS-induced Inflammatory And Apoptotic Response In Human Gingival Fibroblasts

BackgroundPeriodontal disease is a disease that occurs in the supporting tissues of the teeth and is one of the major causes of tooth loss in adults.It is also a major oral disease that endangers the health of human teeth and body.Lipopolysaccharides(LPS)is a recognized pathogenic factor of periodontal disease,which can induce the cells of the host to express and secrete a series of inflammatory cytokines,mediate the pathophysiological response of the body,and to the periodontal tissue.With antigenicity and high toxicity,regulate and destroy periodontal tissue.Human gingival fibroblasts(HGFs)are the main components of the gingival connective tissue and play an important role in the repair of periodontal tissues.Under the infection of LPS,HGFs can effectively increase the level of leukocytes.The production of inflammatory factors such as prostaglandins and prostaglandins causes the occurrence of inflammatory effects in the body.Asiatic acid(AA)is a pentacyclic triterpene compound extracted from Centella asiatica,and has the pharmacological effects of promoting wound healing,anti-inflammatory action,anti-microbial,anti-oxidation and anti-tumor effects.However,there is no report about the therapeutic effect and mechanism of Asiatic acid on periodontal disease.ObjectiveThe aim of this study is to investigate the inducing effect of LPS on inflammatory and apoptotic responses to HGFs,to analyze the mechanism of inflammatory and apoptotic responses of HGFs to asiatic acid in the presence of LPS,and to explore the role of NF-κB signaling pathway in the inflammatory expression of HGFs.The new approach to periodontal disease provides experimental and theoretical evidence.Methods(1)During the period from 2014.1 to 2014.9,we selected healthy patients with complete impacted teeth of impacted teeth of the third molar.A total of 59 patients were collected and the gums were collected during the extraction of wisdom teeth.After primary culture of gingival fibroblasts,the morphological changes of LPS before and after LPS were observed under light microscope and transmission electron microscope;the degree of intracellular damage was determined qualitatively by membrane potential assay;flow cytometry and MTT analysis Cell proliferation inhibition.(2)The inflammatory and apoptotic responses of gingival fibroblasts induced by LPS were studied in vitro.The expression and contents of related inflammatory cytokines IL-1,IL-6,IL-8 and TNF-α were detected by immunocytochemistry and ELISA.Changes;EXpression of inflammatory protein MIP-2 and autophagy proteins Atg5 and LC3-Ⅱ by Western blot;Apoptosis detected by flow cytometry;Changes in Bax mRNA,Bc1-2 mRNA content detected by RT-PCR.(3)Analysis of inflammatory and apoptotic responses of ginseng fibroblasts to asiatic acid under LPS treatment Cell viability in-each group was detected by MTT assay;PGE2,IL-6 and IL-8 expressions were detected by ELISA;NO was detected by Griess method.Apoptosis and cell cycle changes were detected by flow cytometry.Western blot was used to detect the expression of cell-associated proteins NF-κB and PPAR-γ.Results(1)Under different concentrations of LPS,fibroblasts were structurally altered,mitochondria and Golgi were enlarged,and the membrane potential crossed the barrier and increased cell proliferation activity.(2)LPS significantly increased the expression of inflammation-related factors IL-1,IL-6,IL-8,TNF-α and inflammatory related protein MIP-2,autophagy proteins Atg5 and LC3-Ⅱ in HGFs.The increase of LPS concentration increased significantly(P<0.05).(3)With the increase of LPS concentration,the expression of Bax in HGFs increased,the expression of Bcl-2 gradually decreased,and the degree of apoptosis increased(P<0.05).(4)Asiatic acid significantly inhibited the expression of inflammatory cytokines PGE2,IL-6,IL-8 and NO in LPS-induced HGFs(P<0.05).(5)The effect of asiatic acid on the cell cycle arrest was significantly decreased after HGFs were fully used.With the increase of asiatic acid concentration,the apoptosis rate was significantly decreased(P<0.05).(6)Asiatic acid inhibits the activation and occurrence of NF-κB signaling pathway through activation of PPAR-γ.Conclusion:1.LPS caused a substantial destruction of the structure of HGFs,increased the inflammatory response of HGFs,and promoted the apoptosis of HGFs;2.Asiatic acid significantly inhibited the apoptosis and necrosis of HGFs induced by LPS.As the concentration of asiatic acid increased,the apoptosis rate decreased gradually.3.Asiatic acid can effectively activate PPAR-γ,and then regulate the activation and occurrence of NF-κB nucleic acid signal pathway under LPS,weaken the related inflammatory factor expression,inhibit inflammation.