Porphyromonas Gingivalis Accelerates Atherosclerosis Through Regulating BMAL1 Expression

Background: Atherosclerotic cardiovascular diseases(ACVDs)are the leading cause of mortality worldwide.Atherosclerosis,which is now considered as a chronic inflammatory process,is the pathological basis of ACVDs.In addition to risk factors associated with the cardiovascular system itself,pathogenic bacteria such as the periodontitis-associated Porphyromonas gingivalis(P.gingivalis)are also closely correlated with the development of atherosclerosis,but the underlying mechanisms are still elusive.Circadian clock disturbance is also a risk factor for atherosclerosis,and there is an interaction between bacterial infection and circadian clock,but the correlation between periodontal bacteria and circadian clock has not been reported.Objectives: To elucidate the mechanisms of Porphyromonas gingivalis(P.gingivalis)-accelerated atherosclerosis and explore novel therapeutic strategies of ACVDs.Methods: Clinical atherosclerotic artery samples were collected and PCR was used to detect the presence of periodontal pathogens,especially P.gingivalis.Apo E-/-mice were used as atherosclerosis model.Bmal1-/-Apo E-/-mice and conditional endothelial cell Bmal1 knockout mice(Bmal1fl/fl;Tek-Cre mice)were constructed.Mice were treated with P.gingivalis by tail vein injection for 16 weeks.The areas of atherosclerotic plaque were measured by oil red O staining,Arterial oxidative stress levels were detected by DHE staining,immunohistochemical staining and ELISA were used to detect the expression of pro-inflammatory factors,q RT-PCR and Western blot were used to detect the expression of circadian clock genes.Then to evaluate the effect of P.gingivalis and circadian core gene BMAL1 on the formation of atherosclerotic plaque,as well as the relationship between them.Endothelial cells were cultured in vitro,and exposed to P.gingivalis.Transcriptome sequencing,lentivirus infection,Ch IP,Co-IP,Luciferase and other molecular biology experimental techniques were played to further study the role of BMAL1 in the process of P.gingivalis-promoted atherosclerosis,and explore the interaction between P.gingivalis and BMAL1,elucidate the underlying intrinsic molecular mechanism.Results: In the samples of clinical atherosclerosis,several periodontal pathogens DNA were detected,and the detection rate of P.gingivalis was the highest.P.gingivalis can accelerate atherosclerosis progression by triggering arterial oxidative stress and inflammatory responses in Apo E-/-mice,accompanied by the perturbed circadian clock,especially BMAL1 downregulation.Circadian clock disruption boosts P.gingivalis-induced atherosclerosis progression.The mechanistic dissection shows that P.gingivalis infection activates the TLRs-NF-κB signaling axis,which subsequently promote p65 phosphorylation and nuclear entry,followed by the recruiting of DNMT-1 to methylate the BMAL1 promoter and thus suppresses BMAL1 transcription.The downregulation of BMAL1 releases CLOCK,which bind with p65 and phosphorylates it to promote NFKB2 transcription,further enhances NF-κB signaling,elevating oxidative stress and inflammatory response in human aortic endothelial cells.Besides,the mouse model exhibits that joint administration of metronidazole and melatonin serves as an effective strategy for treating ACVDs.Conclusions: P.gingivalis accelerates atherosclerosis via the NF-κB-BMAL1-NF-κB signaling loop.Melatonin and metronidazole are promising auxiliary medications toward ACVDs.