Porphyromonas Gingivalis Enhances Blood-brain Barrier Permeability Through Mfsd2a/Caveolin-1 To Promote Alzheimer’s Disease-like Behavior And Pathological Changes

Objective:Numerous epidemiological studies have found that Periodontitis is one of the risk factors for Alzheimer’s disease（AD）.Porphyromonas gingivalis（P.gingivalis）is a key periodontal pathogen and its role and effect on AD have become a focus of research.Studies have confirmed that the main pathogenic mechanism of Periodontitis is the subgingival plaque biofilm bacteria directly entering the blood vessels,causing bacteremia.The blood-brain barrier（BBB）is a key structural and functional barrier with strong tight junction and low transcytosis,which can selectively regulate the entry and exit of macromolecules.BBB prevents pathogenic microorganisms and toxic substances from entering the central nervous system（CNS）effectively,simultancously,its dysfunction is one of the early markers of AD.Brain microvascular endothelial cells（BMECs）are important cells to study the mechanism of BBB injury.Studies have found that bacterial microorganisms can cause damage to the BBB function under specific circumstances,and can break through the BBB and enter the central nervous system,which is an important link and factor in triggering AD.BBB breakdown is mainly characterized by increased bulk flow transcytosis（transcellular pathway）and loss of tight junction（TJ）（extracellular pathway）in BMECs.Studies have found that the different mechanism of BBB breakdown may occur due to different types of bacteria.For example,Escherichia coli,Group B Streptococcus,Streptococcus pneumoniae,and Listeria enter the brain through a transcellular transport pathway,while Neisseria meningitidis can enter the brain by inducing specific cleavage of the TJ component.And a each bacteria has a different and multiple pathways for BBB disruption.Caveolaes are 50–100 nm invaginations in the plasma membrane.Caveolin-1（Cav-1）is obligatory for caveolae vesicle formation,which is highly expressed in BMECs.Major facilitator superfamily domain containing 2a（Mfsd2a）is an important regulatory molecule in MFS family.Mfsd2a establishes a unique lipid environment that inhibits caveolae vesicle formation in CNS endothelial cells to suppress transcytosis and ensures BBB integrity.At present,however,the specific effects of P.gingivalis on the permeability of BBB and its underlying mechanism remain unclear.Based on the above research background,the aim of this study was to verify the effect of P.gingivalis on the permeability of BBB and further to explore the underlying mechanism by P.gingivalis infection in SD rats and Transwell BBB model in vitro.This study is expected to reveal the effect of P.gingivalis on the biological function of BBB and its underlying mechanism,and further to analyze the transport pathway of P.gingivalis and its virulence factors into the brain.This study will provide a new strategy for the prevention and treatment of AD from the perspective of inhibiting P.gingivalis infection.Methods:1.The SD rats were injected intravenously with P.gingivalis through the tail for 8weeks.The low-dose group was injected with 200μL bacterial solution containing1.0×10³CFU P.gingivalis.The high-dose group was injected with 200μL bacterial solution containing 1.0×10⁸CFU P.gingivalis.The control group was injected with 200μL PBS.Morris water maze（MWM）and new object recognition（NOR）were conducted to detect the ability of learning and memory of AD in rats.Hematoxylin-eosin staining was used to observe histopathological changes of the hippocampus and cortex tissues in rats.The protein level of amyloid-βpeptide_1-42(Aβ_1-42)was detected by immunohistochemical staining（IHC）.2.Evans blue was used to detect BBB integrity after P.gingivalis treatment.Western blot was used to detect the Albumin and Fibrinogen protein levels in the hippocampus and cortex tissues.An in vitro Transwell BBB model was established,P.gingivalis（MOI10,100,or 500）was added into the upper chamber to treat BMECs for 24 h.Then P.gingivalis form the upper chamber to the lower chamber were detected by anaerobic culture method.The FITC positive BMECs after treatment with P.gingivalis was detected by flow cytometry.3.The differentially expressed m RNAs were screened by m RNA sequencing.GO and COG enrichment analysis of differentially expressed m RNAs were conducted.The ultrastructures of brain microvascular in the hippocampus and cortex and b End.3 were observed by using transmission electron microscopy（TEM）after P.gingivalis treatment.The protein level of Caveolin-1（Cav-1）in the hippocampus and cortex tissues was detected by immunohistochemical（IHC）and Occludin（OCLD）protein level in the hippocampus and cortex tissues were detected by western blot.Confocal laser scaning microscopy（CLSM）was used to observe the colocalization of Cav-1 and P.gingivalis.Si RNA interference technology was used to inhibit Cav-1（si Cav-1）.The quantity of P.gingivalis internalized into b End.3 after transfected with si RNA was evaluated by q RT-PCR.FITC positive cells were detected by flow cytometry after transfected with si RNA.Mass spectrometry was conducted to screening the virulence factors of P.gingivalis interacting with Cav-1.Protein-protein ducking was used to elucidate the 3D structure of Cav-1 and Rgp A.4.Cluster analysis was used to screening of key molecules regulating the permeability of BBB permeability.Western blot was used to detect major facilitator super family domain containing 2a（Mfsd2a）expression after P.gingivalis infection.The colocalization of Mfsd2a and CD31 was detected by CLSM in SD rats.Si RNA interference technology was used to inhibit Mfsd2a（si Mfsd2a）and plasmid was transfected into b End.3tooverexpress Mfsd2a（pc Mfsd2a）.After transfecting with si RNA and plasmic for 24h respectively,P.gingivalis was added.The quantity of P.gingivalis internalized in b End.3^{pc Mfsd2a}and b End.3^{si Mfsd2a}was detected by q RT-PCR.The positive cells of FITC after knockdown and overexpression of Mfsd2a were evaluated by flow cytometry.The protein expression of Cav-1 after transfected with Mfsd2a plasmid was detected by western blot.Results:1.P.gingivalis reduced the learning and memory ability of SD rats and promoted the pathological changes related to AD The escape latency time of MWM in the high group was significantly longer than that of sham group on the fifth day（P=0.017）,and there was no difference between the low group and sham group.The results of exploration test of MWM showed that the crossing times to the platform in the high group was significantly less than that in the high group（P=0.001）.A similar result was obtained in the NOR assay.The results of the NOR test showed that the preferential index for new objects in the high group was significantly lower than that in the sham group（P=0.001）,and there was no difference between the low group and sham group.The above results indicated that P.gingivalis is able to reduce the learning and memory abilities of rats.IHC results showed that P.gingivalis increased Aβ_1-42in the hippocampus tissues.2.P.gingivalis enhanced the permeability of BBB in rats and Transwell BBB model in vitro.Compared with the sham group,the deposition of Evans blue dye was higher,while did not have any effect in the low group.Western blot results showed that Albumin and Fibrinogen protein level in the hippocampus were significantly increased both in the high group and the low group compared with sham group（P=0.002,P=0.001,P=0.037,P<0.001）.P.gingivalis significantly increased Albumin and Fibrinogen protein level in the cortex in the high group（P=0.019,P=0.012）,while had no effect in the low group.TEM showed that P.gingivalis-like structure was able to be observed in the microvessels and parenchymal of hippocampus.The P.gingivalis colonies were able to be detected in the Transwell BBB model.These results indicated that P.gingivalis was able to enhance the permeability of BBB both in vivo and in vitro.3.P.gingivalis enhanced the permeability of BBB by evaluating transcytosis of BMECs（1）RNA sequencing suggested that the differentially expressed genes（DEGs）were enriched in membrane function.Cluster of orthologous group function annotation analysis showed that the DEGs were mainly enriched in intracellular transport,secretion,and vesicle transport pathways.（2）TEM was used to visualize transcytotic vesicles after P.gingivalis treatment.There were substantially increased numbers of transcytotic vesicles in both hippocampus and cortex in the SD rats after P.gingivalis treatment.The vesicles had a diameter range of50-100 nm in line with the diameter of normal caveolae structure.No obvious damage was observed in the microvascular structure in the hippocampus and cortex tissues.We further found P.gingivalis can be internalized into b End.3 and caveolae structure were able to be seen around P.gingivalis.IHC showed that P.gingivalis increased Cav-1expression in both hippocampus and cortex（P=0.039,P=0.021）.Western blot showed that P.gingivalis had no effect on OCLD protein expression in both hippocampus and cortex tissues（P=0.094,P=0.369,P=0.535,P=0.989）.After transfected with si RNA,the quantity of P.gingivalis internalized in b End.3^{si Cav-1}and FITC positive cells in b End.3^{si Cav-1}were significantly decreased compared to the si-control group（P=0.03,P=0.017）.（3）CLSM revealed that Cav-1 was colocalized with P.gingivalis in b End.3.Moreover,P.gingivalis promoted Cav-1 expression on cell membrane in a time dependent manner.Mass spectrometry showed that P.gingivalis arginine-gingipains（Rgp）was the highest score virulence factors of P.gingivalis interacting with Cav-1.Protein-protein ducking analysis showed that Cav-1 and Rgp A were relatively closely bound,and parts sites were strong hydrogen bonding.4.P.gingivalis enhanced the permeability of BBB by regulating Mfsd2a/Cav-1pathway.P.gingivalis inhibited the key regulator of transcytosis,Mfsd2a,protein expression（P=0.04）.CLSM results showed that P.gingivalis significantly inhibited Mfsd2a expression colocalized with CD31.After transfection with the si RNA,the percentage of FITC-positive cells was remarkably higher than that in the si-control group and the quantity of P.gingivalis internalized in b End.3^{si Mfsd2a}was higher than that in the si-control group（P=0.001,P=0.028）.After transfection with the Mfsd2a plasmid,the percentage of FITC-positive cells was remarkably lower than empty vector control group and the quantity of P.gingivalis internalized in b End.3^{pc Mfsd2a}was lower than that in the empty vector control（P=0.005,P=0.01）.Western blot showed that overexpression of Mfsd2a in b End.3 suppressed Cav-1 expression and reversed P.gingivalis-increased Cav-1 expression（P=0.004）.Conclusions:1.P.gingivalis was able to decreased the ability of learning and memory in SD rats and promoted the onset and pathological changes of AD.2.P.gingivalis significantly promoted transcytosis of BMECs and increased Cav-1protein expression and inhibited Mfsd2a protein expression.Mfsd2a/Cav-1 may be the key pathway governing BBB and BMECs permeability induced by P.gingivalis.3.Cav-1 may be able to binded with P.gingivalis-Rgp,thereby mediating P.gingivalis and Rgp transporting and promoting the onset of AD.