Mechanism Of Orientin On Inhibiting Periodontal Inflammation And Osteoclast Differentiation Via Autophagy

Periodontitis,a chronic inflammatory illness characterised by gum inflammation and loss of periodontal attachment,is one of the most common diseases of the oral cavity,caused by complex biofilm colonisation of subgingival plaque,which causes resorption of the alveolar bone by destroying the supporting tissues of the teeth and eventually leads to tooth loss.Osteoclasts are the main cells involved in alveolar bone resorption and are a key target for the treatment of periodontitis.The current treatment of periodontitis focuses on systemic or local medication after basic periodontal treatment,however,although commonly used drugs have some therapeutic effect on controlling periodontitis,they have significant systemic side effects and are not recommended for long-term use,therefore,it is necessary to find a drug with less toxic side effects for periodontitis.Moringa leaf extract has been found to delay the development of chronic inflammatory diseases like osteoarthritis and ulcerative colitis,while recent studies have proposed that the Moringa oleifera leaf monomer orientin has good anti-inflammatory effects,however,the effect of orientin on osteoclast differentiation and on periodontal inflammation and alveolar bone resorption in vivo has not been reported.Autophagy is a highly conserved catabolic process,and its activity is closely related to the m TOR signaling pathway.Autophagy has recently been found to promote osteoclast formation and differenti ation,resulting in alveolar bone resorption.Thus,in the current study,we examined for the first time the effect of orientin on osteoclast differentiation as well as periodontal inflammation and alveolar bone resorption in vitro,and further explored the role of autophagy in this process.Objective:To investigate experimentally whether orientin inhibits osteoclast differentiation through autophagy and has an effect on periodontal inflammation and alveolar bone resorption in vivo.Methods:1.BMMs were treated with 0μM-80μM concentrations of Orientin for 24 h,72h and 120 h and cytotoxicity was detected by CCK8;During the differentiation of osteoclasts,appropriate concentrations of orientin were selected to intervene with rapamycin to promote autophagic activity,TRAP staining to detect the degree of osteoclast differentiation,phalloidin staining to assess osteoclast resorption function,Osteoclast associated gene expression was detected by q RT-PCR,the autophagyspecific response was detected by MDC staining,and the expression of proteins associated with autophagy and the AKT/m TOR signaling pathway was dete cted by Western blot.2.The animal model of periodontitis was established and split into blank control group,periodontitis group and orientin group,and executed after 1 month.HE staining was performed to detect the toxic effects of drugs on each maj or organ group and the expression of inflammatory cells.q RT-PCR was performed to detect the expression of inflammation-related factors,Micro-CT was performed to scan the alveolar bone and measure the proximal and distal middle bone resorption,TRAP staining was performed to detect osteoclast differentiation,and immunohistochemistry was performed to assess the expression of autophagy-related protein LC3.Results:1.Results of in vitro experiments:(1)In the concentration range of 0μM-20μM,orientin had little effect on the cell activity of BMMs,and the cell survival rate was greater than 80%;(2)TRAP staining and ghost pen cyclic peptide staining showed a reduction in the volume and number of TRAP+ multinucleated osteoblasts and a reduction in the circumference of fibrillar actin rings after treatment with orientin;(3)q RT-PCR results showed that orientin inhibited the expression of the osteoclastassociated genes c-Fos,TRAP,DC-STAMP and CTSK in vitro;(4)WB and MDC staining results showed that orientin could inhibit the expression of autophagy-related proteins LC3-II and Beclin-1,and the expression of acidic autophagic vesicles in the cells;(5)Rapamycin-promoted autophagy reverses the inhibitory effect of orientin on osteoblasts;(6)Orientin promoted the expression of AKT/m TOR signaling pathway,while the addition of rapamycin decreased the expression of AKT/m TOR.2.Results of in vivo experiments:(1)HE staining of important organ tissues from all groups of rats showed no organic lesions and no toxic effects of orientin on experimental rats;(2)q RT-PCR results showed that orientin inhibited the expression of pro-inflammatory factors TNF-α and IL-1β,while promoting the expression of anti-inflammatory factor IL-10;(3)HE staining showed that there were a lot of inflammatory cells in the periodontal tissues of periodontitis group,however,the number of inflammatory cells in orientin group was significantly decreased;(4)Micro-CT results showed that orientin could reduce the absorption of alveolar bone in experimental rats;(5)TRAP staining showed that orientin reduced the differentiation of osteoclasts;(6)immunohistochemical results showed that orientin inhibited the expression of the autophagy protein LC3-II.Conclusion:1.Orientin has good biosafety and low toxicity in a certain concentration range;2.In vitro experiments found that orientin can inhibit osteoclast differentiation by affecting AKT/m TOR signaling pathway through autophagy;3.In vivo experiments revealed that orientin can inhibit periodontal inflammation and alveolar bone resorption through autophagy.