Effect And Mechanism Of Antler Polypeptide Inhibites Alveolar Bone Resorption Via NF-κB Signaling Pathway

Background:Periodontitis is a chronic inflammatory disease of the periodontal tissues caused by microbial infection from dental plaque,which can lead to irreversible damage to the periodontium,ultimately resulting in tooth looseness and even loss.China is a high-risk country for periodontal disease.According to the data from the fourth national oral health epidemiological survey,only 9.1% of middle-aged and 5.0% of elderly people have healthy periodontal tissue.Respectively,with China entering an aging population,the disease burden caused by periodontal disease and its social and economic burden will become increasingly severe.Currently,the treatment of periodontitis mainly relies on mechanical therapy with adjunctive drug therapy.Although mechanical therapy can effectively scrape away the inflammatory substances,instruments cannot reach deep areas within complex structures,making it difficult to eliminate the infection factors.Therefore,local pharmacotherapy remains an indispensable part of periodontal treatment.However,due to the limitations of commonly used antibiotics,such as resistance and adverse reactions,it is of profound significance to continuously optimize or search for drugs that are more beneficial to clinical application in periodontics.Multiple active ingredients exist in traditional Chinese medicine,which have demonstrated significant therapeutic effects in various diseases.Antler polypeptide(AP),the main active ingredient in traditional Chinese medicine antler glue,has been proven to possess antibacterial,anti-inflammatory,immune-regulatory,antioxidant,hypoglycemic,bone remodeling,and anticancer effects.Among them,AP has more significant effects in anti-inflammatory and bone remodeling.Studies have shown that AP can regulate the expression of inflammatory factors by modulating the nuclear factor kappa-B(NF-κB)signaling pathway in inflammatory diseases such as pneumonia,osteoarthritis,and intestinal inflammation.and inhibit bone resorption by reducing osteoclast differentiation in classical osteoporosis model.Currently,the NF-κB signaling pathway is an indispensable part of the regulation of the inflammatory response and bone resorption.Targeting the inhibition of the NF-κB signaling pathway can alleviate various inflammatory bone diseases,including periodontitis.Therefore,we speculate that AP may participate in the regulation of periodontitis inflammation and bone remodeling through the NF-κB signaling pathway.However,no studies have yet been found on the effects of AP on periodontitis after reviewing the literature.Therefore,in this study,AP were extracted and subjected to amino acid composition analysis.The obtained AP were used in a mouse experimental periodontitis model.Techniques such as Micro-CT,H&E staining,TRAP staining,and real-time fluorescent quantitative PCR(qRT-PCR)were used to detect the effects of AP on osteoclast differentiation in mouse periodontitis at the tissue pathology and molecular biology levels.The study aimed to explore the effects and mechanisms of AP on alveolar bone resorption in mouse periodontitis.Additionally,an in vitro RANKL and Porphyromonas gingivalis lipopolysaccharide(Pg-LPS)induced RAW264.7osteoclast model was established,and experimental techniques such as histological staining,qRTPCR,and Western Blot were used to investigate the changes in the expression of related genes and proteins during RAW264.7 osteoclast differentiation induced by inflammation and the effects and mechanisms of AP on osteoclast differentiation in an inflammatory environment.Methods:In this study,AP were extracted using enzymatic hydrolysis and their amino acid composition was analyzed to obtain stable AP.48 C57BL/6 mice were induced with experimental periodontitis using the silk ligature method and randomly divided into four groups: a blank control group(Control),a periodontitis model control group(NC),a low-dose AP treatment group(250 mg/kg),and a high-dose AP treatment group(500 mg/kg),with administration via gavage.Samples were collected at 1 and 2 weeks after treatment for the following analyses: 1)vital organs were isolated from the mice and their paraffin sections were subjected to H&E staining to evaluate the safety of the AP;2)Micro-CT scanning,H&E staining,and TRAP staining were performed to detect the effect of AP on alveolar bone resorption under inflammatory conditions;3)qRT-PCR was used to detect the gene expression of inflammatory factors Tnf-α,Il-1β,Il-10 and osteoclast markers Trap,Nfatc1,Ctsk in periodontal tissues.The in vitro study used the CCK-8 method to detect the effect of different working concentrations of AP on the proliferation of RAW264.7 cells.RAW264.7 cells were stimulated with Pg-LPS,a dominant pathogenic bacterium in periodontitis,and induced with RANKL to simulate osteoclast differentiation in the periodontal inflammatory environment.TRAP staining,F-actin staining,qRT-PCR,Western Blot,and other experimental techniques were used to evaluate the effect of AP on Pg-LPS-stimulated RAW264.7 osteoclast differentiation.Subsequently,the mechanism by which AP affects RAW264.7 osteoclast differentiation under inflammatory conditions was explored using Western Blot.Results:1.Antler polypeptide was successfully extracted and appeared as a white powder.Amino acid composition analysis showed that its amino acid content met the requirements of pharmacopoeia.2.Antler polypeptide e reduced the mRNA expression of Tnf-α and Il-1β and promoted the mRNA expression of anti-inflammatory factor Il-10 in the periodontal tissue of experimental mice with periodontitis.3.Antler polypeptide reduced the formation of osteoclasts in the periodontal tissue of experimental mice with periodontitis and inhibited alveolar bone resorption.4.The working concentration of antler polypeptide with the most significant proliferative effect on RAW264.7 cells was 75 μg/m L.5.Antler polypeptide significantly reduced the number and area of TRAP-positive multinucleated cells in RAW264.7 cells and reduced the size of F-actin rings,resulting in the loosening of F-actin ring structure.At the same time,it inhibited the expression of osteoclast- related factors such as TRAP,NFATc1,CTSK,and MMP-9 genes and proteins in RAW264.7 cells.6.Antler polypeptide may act via the NF-κB pathway during Pg-LPS stimulated RAW264.7 osteoclast differentiation process.Conclusion:1.Antler polypeptide can effectively alleviate the inflammatory response of experimental mouse periodontitis and inhibit the alveolar bone absorption of experimental mouse periodontitis.2.Aantler polypeptide can inhibit the osteoclast differentiation of RAW264.7 cells under inflammatory conditions,and this inhibitory effect may be achieved by regulating the NF-κB signaling pathway.