Effects Of Armillaria Mellea Polysaccharide On Inflammation And Alveolar Bone Resorption In Experimental Periodontitis Mice

Background: Periodontitis is a chronic progressive disease caused by bacterial infection,which is mainly manifested by the destruction of periodontal supporting tissue.Pathogenic microorganisms invade periodontal tissue,activate local defense cells to secrete inflammatory mediators,and cause periodontal tissue damage.At present,the effective method of periodontal treatment is to mechanically remove local irritants,but some special tooth structures such as root bifurcation area will limit the enter of instruments,and the accumulated plaque cannot be effectively removed.Drugs are often used as adjunctive therapy.Natural products derived from plants have attracted attention in the research on the treatment of periodontitis in recent years because of their various biological activities and low toxicity.It has been confirmed that a variety of plant polysaccharides can participate in the treatment of periodontitis through anti-inflammatory effect,antibacterial effect and regulating bone tissue metabolism.Armillaria mellea polysaccharide（AMP）is the main active substance of Armillaria mellea,a traditional drug and food fungus in China.It has many biological activities,such as immune regulation,antioxidant effect,anti-inflammatory effect and hypoglycemic.Among them,the anti-inflammatory effect is more significant in AMPs’ biological activity.Studies have shown that AMP inhibits the inflammatory response in inflammatory diseases such as colitis,pneumonia,etc.,by regulating the inflammatory cytokines interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,interleukin-10（IL-10）.It is considered to be one of the important resources for the development of new anti-inflammatory drugs.Although the effect of Armillaria polysaccharides on periodontal inflammation has not been reported so far,it is well known that the above-mentioned inflammatory cytokines IL-1β,TNF-α,and IL-10 play an important role in the occurrence and development of periodontal inflammation.In addition,studies have confirmed that Armillaria polysaccharides have positive improvement effects on certain diseases closely related to periodontitis,such as cardiovascular disease,diabetes,respiratory disease,Alzheimer’s disease.In view of this,we speculate that AMP may improve periodontitis inflammation by regulating the above inflammatory cytokines.In order to confirm this hypothesis,AMP was extracted in this study and its physical and chemical properties were analyzed.It was applied to the experimental model of periodontitis in mice.The effects of AMP on periodontitis inflammation and alveolar bone resorption in mice were evaluated by quantitative real-time PCR（q RT-PCR）,micro-CT,H＆E staining and other experimental techniques,aiming to explore the therapeutic effect of AMP on periodontitis,and provide a theoretical basis for the application of AMP in the treatment of periodontitis.Methods: AMP was extracted by the water extraction-ethanol precipitation technology.The physico-chemical properties of AMP were analyzed by gel permeation chromatography-refractive index-multi-angle laser light scattering detection（GPC-RIMALLS）techniques,high-performance anion exchange chromatography（HPAEC）and Fourier transform infrared（FT-IR）spectroscopy.Sixty male C57BL/6 mice were randomly divided into five groups: blank control group（CON）,mouse periodontitis model control group（MOL）,and periodontitis mice given different working concentrations of AMP.The experimental group（APL group 100 mg/kg,APM group 200 mg/kg,APH group 500 mg/kg）were given daily intragastric administration,and the CON group and MOL group were given the same amount of normal saline.The gene expression changes of periodontal tissue inflammatory factors IL-1β,TNF-α and IL-10 were detected by real-time fluorescence quantitative PCR（q RT-PCR）at 1 w and 2 w of administration to evaluate the effect of AMP on local inflammation in periodontal tissue.Then,Micro-CT scanning and H＆E staining were performed on the right maxilla tissue of mice in each group to detect the effect of AMP on alveolar bone resorption.The important organs of mice were isolated,and the biosafety of AMP was preliminarily evaluated by observing the general body and calculating the organ index.Results: AMP in brown powder was successfully extracted,with a weight-average molecular weight of 269.448 k Da.FT-IR analysis of AMP had characteristic absorption peaks of polysaccharides.AMP was a heteropolysaccharide with glucose as the main component.The results of Micro-CT and H＆E showed that compared with the MOL group,different concentrations of AMP could inhibit the alveolar bone resorption in periodontitis mice.The inhibitory effect was enhanced with the increase of the working concentration of Armillaria polysaccharide at 2 weeks.The results of q RT-PCR showed that AMP at different concentrations had regulatory effects on the gene expression of inflammatory factors IL-1β,TNF-α and IL-10 in the periodontal tissue of mice with periodontitis at 1 week and 2 weeks.AMP of different concentrations had no obvious effect on the organs of periodontitis model mice,and the biological safety was good.Conclusion: 1.In this study,AMP was successfully extracted.The analysis of physical and chemical properties confirmed that the extracted Armillaria polysaccharide had a weight-average molecular weight of 269.448 k Da and was a heteropolysaccharide with glucose as the main component.2.AMP at different working concentrations inhibited alveolar bone resorption in periodontitis mice at different time points（1 w,2 w）.3.AMP can reduce the inflammatory response of periodontitis in mice by regulating the gene expression of pro-inflammatory factor IL-1β,TNF-α and antiinflammatory factor IL-10 in periodontal tissue.