Experimental Study Of Nesfatin-1’s Anti-inflammatory, Antioxidant, And Anti-apoptotic Properties In The Treatment Of Periodontitis

ObjectiveIn this study,we will explore the anti-inflammatory effect of nesfatin-1 on mouse macrophages RAW264.7 and the antioxidant and anti-apoptotic effects of nesfatin-1 on mouse precursor osteoblast MC3T3-E1,so as to explore the application prospect of nesfatin-1 in enhancing the anti-inflammatory ability of immune cells and enhancing the antioxidant and anti-apoptotic ability of osteoblasts for periodontitis（PD）treatment.Methods1.In this experiment,10 cases of periodontitis gums and healthy gums were collected clinically,and after hematoxylin and eosin（HE）staining determined the histological inflammatory state of the gums,the relative expression of nesfatin-1 genes in different groups was detected by real-time real-time polymerase chain reaction（qRT-PCR）technology.The relationship between the onset of periodontitis and the relative gene expression of nesfatin-1 was analyzed,and the potential relationship between periodontitis and nesfatin-1 was explored.2.In this experiment,lipopolysaccharide（LPS）was used to stimulate RAW264.7 cells to establish an immune cell inflammatory model.The cell counting kit-8（CCK-8）was used to detect the effect of different concentrations of nesfsatin-1（20,40,80 ng/m L）on RAW264.7 cells for 12 h and 24 h,and the biocompatibility of nesfatin-1 on RAW264.7 cells was detected;qRT-PCR and enzyme linked immunosorbent assay（ELISA）technology detected the relative gene expression and extracellular release content of tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）,which are related inflammatory mediators,to determine the successful establishment of the inflammatory model.Subsequently,RAW264.7 cells were pretreated with different concentrations of nesfatin-1（20,40,80 ng/m L）,and the relative gene expression and release of inflammatory factors such as TNF-αand IL-6 were detected by qRT-PCR and ELISA technology to study the effect of nesfatin-1 concentration on its anti-inflammatory activity,and the concentration with better anti-inflammatory effect was selected to study the effect of intervention time on its anti-inflammatory effect.Therefore,the application prospect of the anti-inflammatory properties of nesfatin-1 in the treatment of periodontitis was explored.3.In this experiment,H₂O₂ was used to stimulate MC3T3-E1 cells to establish a model of oxidative damage of osteoblasts.The biocompatibility of nesfatin-1 on MC3T3-E1 cells and the effects of different concentrations of H₂O₂（50,100,200,400μmol/L）on the survival rate of MC3T3-E1 cells were detected by CCK-8,and the oxidative damage model was optimized.Cells were pretreated with different concentrations of nesfatin-1（20,40,80 ng/m L）for 24 h,and the commonly used antioxidant N-acetylcysteine（NAC）was used as a control to detect the cell survival rate of the oxidative damage model.Different concentrations of nesfatin-1（20,40,80 ng/m L）were pretreated and the cell state changed under microscopy.The fluorescence intensity of ROS after pretreatment with different concentrations of nesfatin-1（20,40,80 ng/m L）was observed by fluorescence microscopy,and the content of reactive oxygen species（ROS）was quantitatively detected by flow cytometer.After pretreatment with different concentrations of nesfatin-1（20,40,80 ng/m L）,the contents of superoxide dismutase（SOD）and malondialdehyde（MDA）were detected in cells with related kits.After pretreatment with different concentrations of nesfatin-1（20,40,80 ng/m L）,apoptosis staining was observed by fluorescence microscopy,and the apoptosis ratio was detected by flow cytometry dual channel.Results1.HE staining results showed that the connective tissue of the inflammatory group was dissolved and destroyed,and the inflammatory cells were extensively infiltrate;The connective tissue of the normal group is arranged neatly,rarely destroyed,and the infiltration of inflammatory cells is limited;The qRT-PCR results showed that the relative gene expression of nesfatin-1 in the inflammation group was higher than that in the normal group,suggesting that the change of nesfatin-1 content in periodontal tissue may be potentially related to the onset of periodontitis.2.The results of CCK-8 showed that nesfatin-1 had good biocompatibility with RAW264.7 cells;microscopic observation showed that RAW264.7 cells extended pseudopodia and increased atypia after LPS stimulation;qRT-PCR showed that the m RNA expression of its inflammation-related genes TNF-αand IL-6 was significantly increased,and the release of TNF-αand IL-6 was also significantly increased by ELISA detection,and 1μg/m L LPS was selected to stimulate 12 h to build an inflammatory model;After pretreatment with different concentrations of nesfatin-1,m RNA expression of inflammation-related genes TNF-αand IL-6 was detected by qRT-PCR to be reduced to varying degrees,and the release of TNF-αand IL-6 was also reduced by ELISA detection,indicating that nesfatin-1 pretreatment had the effect of weakening the inflammatory level of RAW264.7 cells,and 80 ng/m L concentration of nesfatin-1 found that its anti-inflammatory effect was relatively continuous,and pretreatment of RAW264.7 cells had good anti-inflammatory effect for 12 h and 24 h.3.The results of CCK-8 showed that nesfatin-1 had good biocompatibility with MC3T3-E1 cells.CCK-8 detected the survival rate of MC3T3-E1 cells after different concentrations of H₂O₂stimulation at different times,and determined that 100μmol/L concentration of H₂O₂ stimulated MC3T3-E1 cells for 0.5 h to establish a model of oxidative damage.Compared with the H₂O₂ group,the cell state pretreated by nesfatin-1 was better under microscopy,with high brightness,full morphology,less atypia,and stronger adhesion.The results of CCK-8 showed that nesfatin-1pretreatment increased the survival rate of MC3T3-E1 cells after oxidative stimulation.The results of flow cytometry showed that nesfatin-1 pretreatment significantly reduced the content of intracellular ROS compared with the H₂O₂ group.Fluorescence microscopy showed that the green fluorescence intensity of ROS was weakened compared with the H₂O₂ group after nesfatin-1pretreatment.The results of SOD detection showed that nesfatin-1 pretreatment significantly increased the content of intracellular SOD compared with the H₂O₂ group.The results of MDA detection showed that nesfatin-1 pretreatment significantly reduced the content of intracellular MDA compared with the H₂O₂ group.The results of flow cytometry showed that nesfatin-1pretreatment reduced the apoptosis rate of oxidative damage cells compared with the H₂O₂ group.Fluorescence microscopy showed that the proportion of orange-yellow apoptotic bodies after nesfatin-1 pretreatment was significantly reduced compared with that in the H₂O₂ group,indicating that nesfatin-1 had antioxidant and anti-apoptotic effects on MC3T3-E1.ConclusionThe expression of Nesfatin-1 is potentially related to the onset of periodontitis:the relative gene expression of Nesfatin-1 in the gum tissue of periodontitis people is higher than that in healthy people.Nesfatin-1 acts on RAW264.7 cell inflammation model can effectively reduce the gene expression and extracellular release of TNF-αand IL-6,its inflammatory mediators;Nesfatin-1 acts on MC3T3-E1 cell oxidative damage model can enhance its antioxidant and anti-apoptosis ability by reducing ROS and MDA production and increasing SOD content;Nesfatin-1 can enhance the anti-inflammatory ability of immune cells and at the same time enhance the antioxidant and anti-apoptosis ability of osteoblasts,and has good application prospects in the treatment of periodontitis through the synergy of anti-inflammatory and osteogenesis.