The Impact Of Beta-Amyloid On The Development Of Periodontitis Via Ism1 In Alzheimer’s Mice

Objective:Periodontitis is a common,chronic inflammatory disease that affects the periodontium（alveolar bone,cementum,periodontal ligament,gingiva）,and eventually leads to pathologic migration of teeth and tooth loss.As a pathogen of periodontitis,porphyromonas gingivalis cause changes in the intestinal flora,leading to increased permeability of intestinal epithelium,and cause systemic inflammation leading to endotoxemia,which increases the risk of atherosclerosis,adverse pregnancy outcomes,rheumatoid arthritis,aspiration pneumonia,and cancer.Therefore,periodontitis is closely related to human health and quality of life.Alzheimer’s disease is the most common neurodegenerative disease in the elderly,with cognitive dysfunction and memory impairment.The main pathological features are abnormal deposition of amyloid protein in the brain and the formation of neurofibrillary tangles.Researchers have proposed that chronic periodontitis may be one of the highest risk factors for Alzheimer’s disease,and some studies have shown that Alzheimer’s disease can further increase the susceptibility to periodontitis.A large number of studies have found that periodontitis promotes the progression of Alzheimer’s disease,but the effect of Alzheimer’s disease on periodontitis and the specific mechanism is still unclear.Therefore,exploring the mechanism affecting the occurrence and development of periodontitis with Alzheimer’s disease is of great guiding significance for developing new therapeutic drugs and improving the oral health of patients with Alzheimer’s disease.Lots of studies have confirmed that Amyloid beta is the core factor of AD formation and disease development.The increased production and deposition of Amyloid beta（Aβ）in the brains of AD patients triggered a pathological cascade that led to the formation of neurofibrillary tangles（NFTs）,gliosis,inflammatory changes,synaptic damage,and loss of neurotransmitters.The purpose of this study was to investigate the effects of Aβon periodontal tissue during the progression of periodontitis,to provide theoretical guidance and ideas for future treatment of Alzheimer’s disease patients.Methods:Part Ⅰ:Specific Pathogen Free mice aged 16 weeks randomly divided into 4groups（1）AD model mice control group,（2）AD model mice with periodontitis,（3）WT mice control group,（4）WT mice with periodontitis.The distance between enamel junction and alveolar bone crest was analyzed for the alveolar bone resorption by Micro-CT three-dimensional reconstruction.Hematoxylin and Eosin（H&E）staining detected the resorption of alveolar bone and the destruction of periodontium.The expression of Aβand COX-2 in periodontal tissue were detected by immunofluorescence.The expression of Aβ_1-40and Aβ_1-42in mouse brain and alveolar bone was detected by enzyme linked immunosorbent assay（ELISA）.Osteoclasts were detected and counted by Tartrate resistant acid phosphatase（TRAP）staining.RT-qPCR was used to detect the expression of inflammation cytokines in the gingival tissues around the second molar.Part Ⅱ:Isolation and culture of mouse bone marrow mesenchymal stem cells（mBMSCs）from mice aged 18 weeks by whole bone marrow adherent method.This part of the experiment was divided into two groups:（1）AD model mice mBMSCs;（2）WT mice mBMSCs.The expressions of bone formation-related genes（Col1a1,Runx2,Sp7,and Ocn）and bone formation-related proteins（COL1A1,RUNX2,and OCN）in the two groups were detected by RT-qPCR and Western blot respectively after 3-,7-and 14-days’osteogenic induction.Subsequently,to further verify the differences in osteogenic differentiation ability between the two groups,ALP staining was performed after 7 days of osteogenic induction,and alizarin red staining was performed after 21 days of osteogenic induction.Subsequently,we used CCK-8 assay to screen suitable Aβ_25-35concentration,and 20μM Aβ_25-35carried out follow-up in vitro experiments.This part of the experiment was divided into two groups:（1）WT mBMSCs,（2）WT mBMSCs+20μM Aβ_25-35.Then,after 3,5,and 7 days of osteogenic induction,the expression of bone formation-related genes（Col1a1,Runx2,Sp7,and Ocn）and bone formation-related proteins（COL1A1,RUNX2,and OCN）in the two groups were detected by RT-qPCR and Western blot respectively.Part Ⅲ:RNA was extracted for transcriptome sequencing to screen out the differential genes.The expression levels of Ism1 in AD and WT mBMSCs and control and Aβ_25-35groups were further verified by RT-qPCR.Subsequently,to further verify the effect of Ism1on the osteogenic differentiation ability and of mBMSCs stimulated by Aβ_25-35,siRNA transfection was used to knockdown the Ism1 in mBMSCs.RT-qPCR and Western blot were used to verify the knockdown efficiency.This part of the experiment was divided into4 groups:（1）NC osteogenic induction group;（2）Aβ+NC osteogenic induction group;（3）si-Ism1 osteogenic induction group;（4）Aβ+si-Ism1 osteogenic induction group.Alkaline phosphatase staining and alizarin red staining were used to detect the effect of Ism1knockdown on the osteogenic differentiation ability of mBMSCs with Aβstimulation.Subsequently,the expression of bone formation-related genes（Col1a1,Runx2,Sp7,and Ocn）was detected by RT-qPCR.Statistical analysis:All in vitro experiments were repeated at least 3 times,and in vivo animal experiments were repeated at least 3 times,and the experimental results were expressed according to the mean±standard deviation.LSD-t test was used for statistical analysis of the two groups of data conforming to normal distribution.Non-parametric Mann-Whitney U test was used to analyze the data that did not conform to the normal distribution.p<0.05 indicated a statistically significant difference.Results:Part Ⅰ:Micro-CT 3D reconstruction measurement results showed that alveolar bone resorption in the AD model mice periodontitis group was significantly increased compared with that in the control group,and was significantly higher than that in the wild-type mice.In the AD model mice periodontitis group,the distance between CEJ-ABC was significantly increased,BV/TV,Tb.N,Tb.The femoral cancellous bone was significantly decreased,and Tb.Sp was significantly increased.H&E staining showed that alveolar bone resorption increased significantly in the AD model mice periodontitis group,and periodontal tissue destruction was the most serious.Immunofluorescence results showed that the Aβfluorescence intensity was higher in the AD model mice periodontitis group than in the control group,followed by the AD model mice control group.Meanwhile,the fluorescence intensity of COX-2,an inflammation-related indicator,was also the highest in the AD model mice periodontitis group,followed by the wild-type mice periodontitis group.The RT-qPCR results showed that the gingival tissue of the AD model mice periodontitis group showed a higher expression of TNF-α,IL-6,and IL-1β.ELISA results showed that the expression of Aβ_1-40and Aβ_1-42in brain tissue and alveolar bone of the AD model mice periodontitis group was significantly higher than that of the wild-type mice group.Part Ⅱ:RT-qPCR and Western Blot results showed that the osteogenic differentiation ability of AD model mice-derived mBMSCs was significantly weaker than that of wild-type mice.The results of ALP and alizarin red staining further confirmed the above conclusions.After Aβtreatment,the expression levels of bone formation-related genes（Col1a1,Runx2,Sp7,and Ocn）and bone formation-related proteins（COL1A1,RUNX2,and OCN）of mBMSCs in WT mice were decreased.ALP and alizarin red staining and microscopy result also confirmed that Aβsignificantly inhibited the osteogenic differentiation ability of mBMSCs.Part III:Transcriptomic sequencing results showed that after Aβtreatment,Isthmin-1（Ism1）was highly expressed in mBMSCs.Meanwhile,RT-qPCR results showed that the expression level of Ism1 in mBMSCs derived from AD model mice was higher than that of mBMSCs in WT mice.The efficiency of Ism1knockdown was verified by RT-qPCR.RT-qPCR results confirmed that Ism1 knockdown alleviated the inhibitory effect of Aβtreatment on the expression of Col1a1,Runx2,Sp7,and Ocn bone formation-related genes.ALP staining showed that Aβtreatment significantly inhibited the osteogenic differentiation ability of mBMSCs,and Ism1knockdown mitigated this inhibitory effect.Alizarin red staining also showed that Aβtreatment inhibited the mineralization ability of mBMSCs,and the number and area of calcium nodules decreased significantly.Ism1 knockdown reduced the inhibition effect of Aβtreatment on the formation of calcium nodules.Conclusion:Compared with wild-type C57BL/6 mice,the periodontitis alveolar bone destruction was more severe in AD model mice.β-amyloid was deposited in alveolar bone in AD model mice.β-amyloid deposition inhibited the osteogenic ability of mesenchymal stem cells in mice.β-Amyloid peptide stimulating increase the expression of Ism1 in mesenchymal stem cells.Knocking down Ism1 may effectively rescue the osteogenic differentiation ability of mBMSCs.