Cytolysin Of Enterococcus Faecalis In Promoting Periapical Periodontitis With Secondary Root Canal Infection In Rat Model

periapical periodontitis is one of common diseases in oral cavity, Root canaltherapy is the preferred effective treatment for periapical periodontitis. But, even aftercomplete root canal treatment, there are still cases of failure, which often present asprogressive periapical bone destruction and recurrent periapical abscess, eventuallyforming intractable periapical periarthritis. It has been found that the main cause of itis the persistent infection of bacteria in root canals. During recent years, a highdetection rate of Enterococcus faecalis in root canals has been observed in root canaltreatment failure cases.Enterococcus faecalis has a variety of virulence factors, whose expression can beadjusted according to the change of environmental factors. This character is the key ofEnterococcus faecalis to survive and cause infection after root canals treatment. It hasbeen demonstrated that many virulence factors of Enterococcus faecalis are relatedwith the development of periapical periodontitis. Cytolysin (Cyl) is one of the mainvirulence factors of Enterococcus faecalis. Although it has been confirmed that Cylhas strong pro-inflammatory effect in other infectious disease such as endocarditis,endophthalmitis, there are few reports about its role in pathogenesis of intractableperiapical periodontitis.Expression of cyl is affected by surrounding environment. There are manyfactors that can influence its expression. It is reported that Cyl expression level is upregulated by decreasing surrounding oxygen concentration, blood cells andincreasing surrounding cell density. Afeter root canal treatment, bacterial and celldensity decrease dramatically in root canals. There cells survive on nutrient fromserum of periapical tissue. And, oxygen concentration in root canals decreases sharply.Thus it is speculated that when Enterococcus faecalis enters the root canal, cylexpression may be regulated by these factors, promoting the bacteria survival andpathogenesis in treated root canal.Firstly, this study established animal model of rats with chronic periapicalperiodontitis infected by mixed bacteria was established by purposely exposure ofpulp cavity. Then, secondary root canal infections was induced by introduction ofhemolytic (contain cyl gene) or nonhemolytic (not contain cyl gene) Enterococcusfaecalis in root canals of rats accordingly. Subsequently, histopathological analysisand inflammatory factor expression measurement were conducted. The results werecompared between the hemolytic and nonhemolytic groups. Changes of cylexpression in each stage were also observed under in vitro and in vivo condition. Therole of Cyl was evaluated in periapical periodontitis for secondary root canalinfections by Enterococcus faecalis.Part Ⅰ: Histopathologic analysis of the effect of cytolysin in the inflammatoryprogress of rat periapical periodontitis models with secondary root canalinfections by Enterococcus faecalisMethods: Eighty four Sprague dawley rats were used in this study. Pulp cavities ofbilateral first maxillary molars of each rat were exposed to establish chronic periapicalperiodontitis with primary infection by mixed bacteria in oral cavity (E.faecalis notinvolved). Then the rats were divided into four groups randomly：Six rats wereallocated into disinfection group (Rats were fed for2weeks after root canalpreparation and subsequent filling in pulp cavity with FC sterilized cotton pellet）. Sixrats were allocated into chronic apical inflammation group (Rats were fed for8weeksbefore sacrifice). Thirty six rats were allocated into group of periapical periodontitiswith secondary root canal infections by hemolytic Enterococcus faecalis, and anotherthirty six rats were allocated into group of periapical periodontitis with secondary root canal infections by nonhemolytic Enterococcus faecalis (Two weeks after root canalpreparation and disinfection, root canals were infected by nonhemolytic Enterococcusfaecalis via respectively injection of ATCC29212and ATCC700802bacteriasuspension and then sealed off for1week,2weeks,3weeks,4weeks,5weeks and6weeks accordingly). For later two groups, six rats were sacrificed radamly at eachtime point. Histology biopsies were made. Histopathological analysis was conductedwith hematoxylin-eosin staining.Results: Normal healing process was observed in peri-apical lesion of disinfectiongroup. Mild inflammation and fibrocytes proliferation were presented in chronicapical inflammation group. Acute severe inflammation state with bleeding and boneresorption was observed in both hemolytic Enterococcus faecalis and nonhemolyticEnterococcus faecalis groups from the end of1st week to the end of3rd week,especially at the end of2nd week. Chronic inflammation state was observed from theend of the4th week. Periapical inflammation gradually subsided from the end of the4th to the end of the6th week. But there was still inflammatory cell infiltration. At theend of the6th week, the inflammation in nonhemolytic Enterococcus faecalis groupturned to be mild. However, the inflammation in hemolytic Enterococcus faecalisgroup was more serious.Conclusions: Although Cyl is not the main determinant to influence the progress ofinflammation, it may inhibit the healing process of periapical lesions infected withEnterococci faecalis and prolong the inflammation process.Part Ⅱ: Influence of Cytolysin on inflammatory cytokines expression in ratperiapical periodontitis models with secondary root canal infection byEnterococcus faecalisMethods: Sampling and slice method was as the same as that in part Ⅰ. At each timepoint, TNF-α, IL-1β and MMP-8expression was detected by immunohistochemicalstaining technique. The expression difference was compared between hemolyticEnterococcus faecalis and nonhemolytic Enterococcus faecalis groups.Results: The change of inflammatory cytokine expression in both hemolyticEnterococcus faecalis and nonhemolytic Enterococcus faecalis groups were similar as described below: There was a sudden jump in TNF-α expression from the end of the1st week. It reached a peak at the end of the2nd week. After that, TNF-α expressiondecreased gradually until the end of the6th week. Expression of IL-1β rose sharplyfrom the end of the1st week to the end of the3rd week and reached a high poiont,and then it fell till the end of the6th week. Expression of MMP-8experienced asteadily increase from the end of the1st week to the6th week. Disinfection groupwitnessed the lowest expression level of all three cytokines above. The inflammatorycytokine expression level was higher in hemolytic Enterococcus faecalis group thanthat in nonhemolytic Enterococcus faecalis group at all time points observed. Theinflammatory cytokine expression level in chronic apical inflammation group waslower than that in both hemolytic Enterococcus faecalis group and nonhemolyticEnterococcus faecalis group.Conclusions: Cyl may promote the expression of TNF-α, IL-1β and MMP-8so as tofacilitate destroy and the inflammation expansion of periapical tissue, although it isnot the only regulatory factor of these three cytokines.Part Ⅲ: Real-time PCR detection of cytolysin gene expression of Enterococcusfaecalis under different conditionMethods:(1) Real-time fluorescent quantitative PCR detection of cyl gene wasperformed on hemolytic Enterococcus faecalis (ATCC29212) suspension preparedunder aerobic and anaerobic condition respectively.(2) Sixty six Sprague dawley ratswere used in this study. Pulp cavities of bilateral first maxillary molars of each ratwere exposed to establish chronic periapical periodontitis with primary infection bymixed bacteria in oral cavity (E.faecalis not involved). Three weeks after, root canalpreparation and subsequent filling in pulp cavity with FC sterilized cotton pellet wereconducted on those teeth. Two weeks later, cotten pellet was removed and root canalswere re-infected by hemolytic Enterococcus faecalis via injection of ATCC29212bacteria suspension and then sealed. Six rats were randomly sacrificed after6hours,12hours,24hours,48hours,96hours,1week,2weeks,3weeks,4weeks,5weeksand6weeks respectively. Maxilla was resected and the teeth were removed. Then the extracted teeth were immersed in1ml TRNZOL for1hour then stored at－80℃.Eventually, Real-time fluorescent quantitative PCR detection of cyl gene wasperformed.Results: Expression of cyl gene in anaerobic environment was higher than that inaerobic environment at the most of the time. Six hours after root canals infected byhemolytic E.faecalis, cyl gene expression increased dramatically until it peaked at24th hour. Then, there was a rapid decrease in cyl gene expression from the48th hourto the1st week after infection. Afterwards, it rose steadily until the6th week. It isnoted that from the96th hour to the4th week, the gene expression was even lowerthan that under aerobic condition. In the5th and6th week, the expression wasincreased rapidly, and appeared highly expressed again in the6th week.Conclusions: Pure reduction of oxygen concentration in the surrounding environmentmay promote the expression of cyl. In the short term, sudden reduction ofenvironmental nutrition around has a strong role in promoting the expression level ofcyl. Being influenced by multiple factors, such as complex root canal environmentand nutritional deficiency, the gene expression of cyl is complicated.