Experimental Study On The Promotion Of Distant Metastasis Of Porphyromonas Gingivalis By Diabetes Mellitus

Background and PurposePeriodontitis is a chronic inflammatory disease in periodontal tissue caused by plaque biofilm.It is characterized by destruction of connective tissue and alveolar bone.In severe cases,it can cause tooth loosening,displacement and even loss,with a high global incidence rate.Porphyromonas gingivalis(P.gingivalis)is one of the main pathogenic bacteria of periodontitis.It secretes outer membrane protein,peptidyl-arginine deaminase,gingival protease,outer membrane vesicles and other virulent factors to participate in the occurrence and development of periodontitis.Patients with periodontitis can swallow 1 ×1012～1 × 1013 P.gingivalis,in addition,P.gingivalis itself can enter the blood circulation through the broken part of the inner wall of the periodontal pocket to reach the distant organs.The probability of bacteremia in patients with periodontitis after daily oral hygiene maintenance and periodontal treatment is as high as 17%～100%.Many studies have found that P.gingivalis can be detected in different tissues,such as brain,liver,placenta,blood vessels,heart,kidney,spleen,etc.,indicating that P.gingivalis has the ability of distant metastasis and can be implanted in and affect distant organs.Periodontitis is closely related to a variety of systemic diseases,of which the two-way relationship between diabetes and periodontitis is the most positive.Diabetes is a metabolic disease characterized by hyperglycemia,which is caused by insulin secretion defect or impaired biological function or both.Its long-term existence will lead to damage to various organs and tissues,especially chronic damage and dysfunction of kidney,heart,eyes and other tissues.However,whether diabetes affects the distant metastasis ability of P.gingivalis has not yet been studied.Therefore,this experiment simulates the way in which P.gingivalis enters the body through the oral gastrointestinal tract and periodontal pocket blood circulation pathways during daily life and periodontal system treatment in periodontitis patients by gavage and tail vein injection of P.gingivalis bacterial solution in mice aiming to study whether diabetes affects the distant metastasis ability of P.gingivalis.At the same time,look for the relatively susceptible organs of P.gingivalis.This study provides a new research idea for the possible interaction between periodontal disease,diabetes and other systemic diseases.Materials and Methods1.Culture and identification of P.gingivalis0.3～0.5 mL of bacterial liquid had been taken into 5～10 mL of BHI liquid culture medium,and the liquid culture medium was put into anaerobic bag and cultured in 37℃ anaerobic tank(N2:CO2:H2=80:10:10)for 2～3 days.After every 2～3 generations of bacteria,single colonies were selected for purification to ensure the purity of bacteria for subsequent experiments.2.Long-term infection of mice P.gingivalis32 male C57BL/6N 6-week-old mice were randomly divided into four groups:control group,diabetes group,P.gingivalis group and P.gingivalis+diabetes group.After one week of adaptive feeding,P.gingivalis group and P.gingivalis+diabetes group were given tail vein injection and intragastric administration of P.gingivalis bacterial liquid(1×109 CFU/mL per kg of mass)once every other day.The control group and diabetes group replaced P.gingivalis bacterial liquid with the same amount of sterile physiological saline for 8 months.3.Establishment of diabetes mouse modelThe mice in diabetes group and P.gingivalis+diabetes group were fed with high sugar and high fat diet(crude protein content≥15%,crude fat content≥12%)for one month after fed with ordinary diet for one week.After 1 month,1%streptozotocin was injected into the left lower abdominal cavity at a dose of 30 mg/kg body weight once a day for 4 days.4.The influence of diabetes on the ability of P.gingivalis distant metastasis and the analysis of P.gingivalis susceptible organsThe number of P.gingivalis in each organ of each group was detected by realtime fluorescent quantitative PCR to analyze whether diabetes affects the ability of P.gingivalis distant metastasis,and searched for organs relatively susceptible to P.gingivalis.Immunofluorescence method was used to detect the colonization of P.gingivalis in the tissue sections of various organs in each group.Results1.P.gingivalis was successfully cultured,which was spherical and rod-shaped under microscope,and showed smooth black colony in BHI solid medium.2.We had mastered the technology of tail vein injection and intragastric administration.During the experiment,we ensured that each mouse was injected with the same amount of P.ginivalis bacterial solution or sterile physiological saline,and the operation time of each mouse was controlled within 1 min,and successfully established the long-term load P.ginivalis model of mice.3.Blood was taken from the tail vein,and random blood glucose≥16.7 mmol/L was measured to successfully establish diabetes model mice.After that,blood glucose was monitored every 7 days.The blood glucose level of diabetes mice remained stable throughout the experiment,and was higher than or equal to 16.7 mmol/L.4.The number of P.gingivalis in each organ of each group was detected by realtime fluorescent quantitative PCR absolute quantitative method.There was no statistical difference in the number of P.gingivalis in each organ between the control group and the P.gingivalis group,and between the control group and the diabetes group.The number of P.gingivalis in liver,kidney,brain,lung,and colorectum:P.gingivalis+diabetes group was higher than diabetes group and P.gingivalis group,and there was statistical difference.The number of P.gingivalis in the heart,spleen,small intestine and stomach four organs:P.gingivalis+diabetes group was higher than diabetes group and P.gingivalis group,but there was no statistical difference.Compare the number of P.gingivalis in each organ horizontally,the number of P.gingivalis detected in the heart was the lowest and the number of P.gingivalis detected in the colorectum was the largest.ConclusionThe presence of diabetes promotes the distant metastasis of P.gingivalis to liver,lung,brain,kidney and colorectum.The colorectum is a relatively susceptible organ of P.gingivalis.This study provides a new research idea for the possible interaction between periodontal disease,diabetes and other systemic diseases.