Chronic Apical Periodontitis Exacerbates The Formation Of Atherosclerosis In ApoE^-/- Mice By Affecting Gut Microbiota And Intestinal Barrier

Background:Chronic apical periodontitis（CAP）refers to a chronic inflammatory response of the periapical tissue caused by a long-standing infection in the root canal,manifested by formation of inflammatory granulation tissue and destruction of alveolar bone.CAP can affect distant organs and is considered a potential risk for systemic diseases,including cardiovascular disease（CVD）,which is pathologically based on atherosclerosis（AS）of medium and large vessels.Epidemiological evidence suggests a correlation between CAP and CVD,but the causal relationship between CAP and AS and the specific mechanisms have not been clarified.Objective:The effect of CAP on atherosclerosis（AS）and gut microbiota was studied by establishing a mouse model of CAP induced by Porphyromonas gingivalis（P.gingivalis）in apolipoprotein E knockout(apoE^-/-)mouse.And explore the effects of gut microbiota and their metabolites on the process of AS.Materials and methods:1.Forty 7-week-old male apoE^-/-mice were randomly divided into CAP and control（Con）groups.The mice in the CAP group were anesthetized and the pulp cavities of the bilateral maxillary first and second molars were exposed,sterile cotton balls containing 10⁸ colony forming units（CFU）/m L P.gingivalis were placed.Mice were euthanized 12 weeks after surgery,and periapical lesions were evaluated by micro-CT.Aortic arch and aortic root oil red O staining was used to count the percentage of plaque area and volume.Serum lipids,endotoxin（ET）and serum inflammatory factor levels were measured.The feces of mice were collected and 16s r RNA was used to detect gut microbiota,and liquid chromatograph-mass spectrometer（LC-MS）was used for untargeted metabolomics and bile acid metabolomics.The distal colon of mice was stained with hematoxylin-eosin staining（HE）and Alcian blue-Periodic acid Schiff stain（AB-PAS）,and the secreted proteins,paracellular proteins and extracellular regulatory proteins were detected by immunofluorescence and Western Blot.The expression of secreted proteins,paracellular proteins and Extracellular regulated protein kinases（ERK）signaling pathway proteins was examined by immunofluorescence and Western Blot.2.Twenty 7-week-old male apoE^-/-mice were randomly divided into the CAP and Con groups.Treatment according to the Experiment one method.At 9 weeks postoperatively,intestinal permeability was measured by using fluorescein isothiocyanate-dextran 4（FD-4）and the percentage of plaque area was counted in the aortic arch by Oil Red O staining.3.Fecal microbiota transplantation（FMT）was used to verify the causality.Thirty 7-week-old male apoE^-/-mice were randomly divided into FMT-CAP and FMT-Con groups.After antibiotic pretreatment,feces from the CAP group were transferred to the FMT-CAP group and feces from the Con group were transferred to the FMT-Con group.After12 weeks of treatment,mice were euthanized and the aortic arch was stained with oil red O to measure the percentage of AS lesions.Feces were collected for 16s r RNA sequencing.4.Twenty-eight 5-week-old male apoE^-/-mice were randomly divided into CD-CAP and CD-Con groups under Chow-diet（CD）.Treatment according toExperiment one.After 17weeks,the faeces were collected for 16s r RNA sequencing.Results:1.ApoE^-/-mice infected with P.gingivalis in the pulp cavity showed widening of the periodontal ligament,periapical lesions showed the transmissive images,and increased percentage of AS plaques at the aortic arch and aortic root in the CAP group（P<0.05）.Serum ET levels increased in the CAP group,while the levels of interleukin-10（IL-10）decreased（P<0.05）.Theαdiversity of the gut microbiota decreased and theβdiversity changed,the results of linear discriminant analysis showed an enrichment of Erysipelotrichaceae and Odoribacteraceae in the CAP group.The results of AB-PAS staining showed that the expression of neutral mucus was reduced in the CAP group.The expression of intestinal mucus protein MUC-2 was reduced,and the expression of paracellular junctions Zonula occludens-1（ZO-1）,Occludin,Claudin was reduced,and ERK signaling pathway was activated.The CAP group was clearly distinguished from the Con group in orthogonal partial least squares discriminant analysis（OPLS-DA）.Univariate analysis presented the reduced cortisone and increased phosphorylcholine,taurodeoxycholic acid and taurocholic acid in the CAP group.2.FD-4 levels in the serum of apoE^-/-mice increased after 9 weeks of pulp cavity infection with P.gingivalis,along with an increase in the plaque percentage of AS in the aortic arch.3.Theαdiversity of the gut microbiota of mice in the FMT-CAP group decreased,the Odoribacteraceae family was enriched in the FMT-CAP group,and the percentage of plaque area of AS in FMT-treated apoE^-/-mice increased.4.The CD-conditioned feeding of the CAP model also caused changes in diversity and composition of microbiota in apoE^-/-mice.Conclusions:1.CAP formed by molar pulp cavity infection with P.gingivalis in apoE^-/-mice could aggravate the extent of AS lesions by increase serum ET levels and decrease expression of anti-inflammatory factor IL-10,and affecting the abundance and composition of gut microbiota CAP may reduce the expression of paracellular junction protein as well as mucin in the colon through ERK signaling pathway and changes the composition of untargeted metabolism and bile acid metabolism in intestinal metabolites of mice involved in promoting AS.2.The increase in intestinal permeability was positively correlated with the extent of AS lesions.3.FMT experiments verified that altered gut microbiota in the CAP group could act as an independent factor to exacerbate the extent of AS lesions by affecting gut microbiota disorders in apoE^-/-recipient mice,which not modelled with CAP.4.Excluding the effects of a high-fat diet,CAP still induced alterations in gut microbiota.