Treatment Of Periodontitis With Bempedoic Acid(ETC-1002) Via Activation Of AMPK Pathway

Periodontitis is a chronic inflammatory disease characterized by bleeding gums,periodontal pocket formation and alveolar bone resorption,which ultimately leads to tooth loss.Periodontitis not only harms the oral function,but is also closely related to systemic diseases.The goal of periodontitis treatment is to achieve periodontal tissue regeneration.At present,none of the traditional treatment methods in the clinic can obtain complete periodontal regeneration.Periodontal regeneration efforts have shown limited success due to the inability to control the inflammation that negatively affects stem cell function.Resolution of inflammation may be a possible key early event in periodontal regeneration,by kickstarting the resolution of the inflammation process and creating a pro-regenerative environment for periodontal tissue regeneration that is otherwise inhibited by local inflammation.Strategies other than resolution of inflammation that could promote stem cell regeneration are also important.Therefore,it is urgent to develop effective treatments which is able to both reduce the infections and inflammation in injured sites and improve the regeneration of damaged tissues.AMP-activated protein kinase(AMPK)plays a regulatory role in inflammation and bone metabolic processes.Bempedoic acid(ETC-1002),a pharmacological activator of AMPK,showed a promising beneficial effect in in vivo models of inflammation,indicating this drug as an alternative tool to manage periodontitis.Given that,we visualize that ETC-1002 may promote periodontal tissue regeneration by modulating macrophage mediated inflammatory responses and promoting the osteogenic differentiation of r BMSCs,through AMPK pathway.Subsequently,specific research work was elaborated from the following three aspects:(1)The role and mechanism of ETC-1002 on inflammation in RAW264.7 induced by Pg-LPS.First,we examined the effects of ETC-1002 on the expression of inflammatory cytokines(IL-6,IL-1β and TNF-α)in macrophage by Real-time PCR and ELISA.The results suggest that ETC-1002 inhibits expression of inflammatory cytokines and alleviates inflammatory responses in macrophage.Then,the effect of ETC-1002 on phosphorylation of AMPK under inflammatory and normal conditions was examined by Western blot.The results indicate that ETC-1002 promotes phosphorylation of AMPK under both inflammatory and normal conditions.Next,to verify the role of AMPK signaling pathway in ETC-1002-inhibited inflammatory responses in macrophages,AMPKαsi RNA was applied to block the AMPK signaling pathway,and the expression of inflammatory cytokines were detected in macrophages by Real-time PCR and Western blot.The results show that the expression of inflammatory cytokines in macrophages was significantly decreased after the AMPK signaling pathway blocked,suggesting that ETC-1002 inhibits inflammatory responses in macrophages through AMPK signaling pathway.NF-κB signaling pathway is a classical signaling pathway in periodontal inflammatory response.In order to clarify the relationship between AMPK and NF-κB signaling pathways in the process of inflammation inhibited by ETC-1002,the phosphorylation levels of IκBα and p65 were examined by Western blot,when Pg-LPS,ETC-1002 or AMPK inhibitors were added.The results show that Pg-LPS promotes the phosphorylation of IκBα and p65,and activates NF-κB signaling pathway;ETC-1002 reduces the phosphorylation of IκBαand p65,and suppresses NF-κB signaling pathways in inflammatory conditions;however,the phosphorylation of IκBα and p65 is elevated again after blocking the AMPK signaling pathway,and the inhibition of NF-κB signaling pathway by ETC-1002 is disappeared.These results suggest that ETC-1002 exerts anti-inflammatory effects by promoting AMPKα phosphorylation and negatively regulating NF-κB signaling pathway.(2)Effects and mechanisms of osteogenic differentiation in r BMSCs promoted by ETC-1002 under inflammatory and normal conditions.First,the expression of osteogenic genes(RUNX2,OSX,ALP and COL1)by Real-time PCR were performed to determine the effective concentration of ETC-1002 on the osteogenic differentiation of r BMSCs.The results show that 100 μM is the most effective concentration of ETC-1002.Then,in order to determine the effects of ETC-1002 on the osteogenic differentiation of r BMSCs under inflammatory and normal conditions,the Real-time PCR was performed to detect the expression of early osteogenic differentiation genes(RUNX2,OSX,ALP and COL1).It is found that ETC-1002 can promote gene expression of RUNX2,OSX,ALP and COL1 in r BMSCs under both inflammatory and normal conditions.In addition,the expression of ALP was detected by ALP staining.The results indicate that ETC-1002 can promote the expression of ALP in r BMSCs under inflammatory and normal conditions.We further detected the formation of calcium nodules by ARS staining and evaluated the ability of r BMSCs osteogenic differentiation in later stage.The results show that ETC-1002 can promote the formation of calcium nodules in r BMSCs under inflammatory and normal conditions.Finally,we explored the mechanism of ETC-1002 promoting osteogenic differentiation of r BMSCs under inflammatory and normal conditions.The AMPK inhibitor Compound C was used to block AMPK signaling pathway,and Real-time PCR,ALP staining,and ARS were performed to evaluate the effects of ETC-1002 on the osteogenic differentiation of r BMSCs under inflammatory and normal conditions.The results show that the expression of osteogenic differentiation genes and ALP and formation of calcium nodule in r BMSCs induced by ETC-1002 were inhibited after AMPK signaling pathway blocked,under inflammatory and normal conditions.It is well documented that AMPK signaling pathway is involved in the process of ETC-1002-induced osteogenic differentiation of r BMSCs under inflammation and normal conditions.Therefore,in this part,it is found that ETC-1002 can promote the osteogenic differentiation of r BMSCs by through AMPK signaling pathway both under inflammation and normal conditions.(3)The effects of ETC-1002 on experimental periodontitis in mice were evaluated.C57 BL / 6 mice were used to establish the periodontitis model,which were divided into three groups: Periodontitis group,ETC-1002 group and Compound C group.After 10 days,it is found that ETC-1002 reduces sulcus bleeding index and tooth mobility index and improves periodontitis symptoms in mice.The samples were scanned by micro-CT and the distance from the enamel cementum boundary(CEJ)to the top of the alveolar ridge(ABC)in the maxillary first molars were measured as the index of alveolar bone loss.The relevant bone and bone trabecular parameters were also measured between the maxillary first and second molars.It is found that the alveolar bone resorption is severe in the periodontitis group.Moreover,the bone mass and bone mineralization density are also decreased.To our delight,the height of alveolar bone,bone mass,and bone mineralization density are almost restored to the normal level in the ETC-1002 group.But,the alveolar bone resorption is severe,and the bone mass and bone mineralization density are also decreased again,after the AMPK signaling pathway inhibited.We illustrate that ETC-1002 promotes alveolar bone regeneration through the AMPK signaling pathway.To further verify the effect of ETC-1002 on the osteogenic differentiation,the expression of osteogenesis genes(RUNX2,OSX,COL1,ALP,OCN and OPN)in periodontium was examined by Real-time PCR.The results show that the expression of osteogenesis genes(RUNX2,OSX,COL1,ALP,OCN and OPN)were decreased in the periodontitis group,and increased in the ETC-1002 group.But,the expression of osteogenesis genes was decreased again,after AMPK signaling pathway inhibited.We demonstrate that ETC-1002 exerts promoting osteogenic effects through AMPK signaling pathway.To clarify the effect of ETC-1002 on inflammation of periodontium,HE staining was performed to observe the distribution of inflammatory cells in the periodontium between the first and second maxillary molars.The results show that a large number of inflammatory cells are observed in the periodontium of the periodontitis group,with loss of connective tissue attachment,disarrangement of periodontal fibers and resorption of alveolar bone.Scattered inflammatory cells and regular arrangement of collagen fibers are observed in the ETC-1002 group.But,massive lymphocyte and plasma cells infiltrate again in the periodontium,after AMPK signaling pathway inhibited.We demonstrate that ETC-1002 exerts anti-inflammatory effects through AMPK signaling pathways.To verify the anti-inflammatory effect of ETC-1002 furtherly,the expression of inflammatory cytokines(IL-6,IL-1 β and TNF-α)was detected by Real-time PCR.It is found that the expression of inflammatory cytokines was increased in the periodontitis group,descended in the ETC-1002 group,and increased again in the Compound C group.It proves again that ETC-1002 exerts anti-inflammatory effects through AMPK signaling pathway.In addition,the biosafety of ETC-1002 in vivo was evaluated by HE staining based on vital organs,including thymus,livers,spleens,and kidneys,as well as detecting the contents of AST,ALT and creatinine in blood.The results show that here is no significant difference in the cell morphology and contents of AST,ALT and Creatinine in blood between the control and the ETC-1002 group.Therefore,this part of the experiment demonstrates that ETC-1002 exerts anti-inflammatory and promoting osteogenic effects in vivo,and are safe for applications.In summary,we demonstrate that ETC-1002 alleviates inflammation in macrophages induced by Pg-LPS through AMPK/NF-κB signaling pathway and promotes osteogenic differentiation of r BMSCs through AMPK signaling pathway under both inflammatory and normal conditions.Importantly,ETC-1002 can inhibit inflammation of periodontium and enhance alveolar bone regeneration through AMPK pathway in mice with periodontitis vivo.It follows that ETC-1002 plays a therapeutic role in periodontitis via AMPK signaling pathway and it may serve as a candidate drug for periodontitis treatment.