periodontitis , a worldwide complication of diabetes ( known as diabetic periodontitis ) , is diagnosed by destruction of periodontal cementum , ligament , and alveolar bone . both types of diabetes in clinic , type 1 diabetes ( t1d ) and type 2 diabetes ( t2d ) , exhibit dramatically higher risk and severity of periodontitis [ 24 ] .
in recent years , researches are focused on the bidirectional communication between diabetes and periodontitis .
because of the complexity of clinical trials , establishing reliable animal models is particularly necessary . in diabetic periodontitis , significantly increased inflammatory cytokines could be found in serum and gingival cervical fluid .
previous studies have confirmed immune and inflammatory responses in the gingival epithelium play an initial role on the onset and development of diabetic periodontitis [ 7 , 8 ] .
protein tyrosine phosphatase nonreceptor type 2 ( ptpn2 ) is an intracellular tyrosine - specific phosphatase , which is expressed in epithelial cells , fibroblasts , or endothelial cells .
the biological function of ptpn2 is believed to vary in response to proinflammatory stimuli such as interferon - gamma ( ifn- ) , tumor necrosis factor ( tnf- ) , hyperosmotic stress , or hyperglycemia [ 10 , 11 ] .
moreover , proteins on janus family kinase ( jak)/signal transducer and activator of transcription ( stat ) pathway , an important signaling in inflammation , have recently been found to be targets of dephosphorylation by ptpn2 .
inactivation of those substrates by dephosphorylation lead to the negative regulation of signaling pathways involved in inflammatory responses induced by the proinflammatory cytokines like tnf- , which is also increased in serum and gingival cervical fluid of patients with periodontitis .
mice are usually used as the experimental animal models for the research of human disease , because of its unique host response .
a large number of researchers have studied the experimental mice models of chronic periodontitis and diabetes disease , respectively [ 15 , 16 ] .
but the research about the establishment and characteristics of the experimental diabetic periodontitis mice models is yet insufficient . in this study
, we established three mice models of experimental diabetic periodontitis and compared characteristics in metabolism and periodontal inflammation .
the levels of ptpn2 expression and key factors on jak / stat pathway were analyzed to reveal the potential biological reasons behind the differences .
twenty 4-week male db / db mice , 20 male tallyho / jngj ( th ) mice , and 40 male c57bl/6j ( c57 ) mice were fed commercial mouse food ( finely ground autoclaved low - fat diet ) and housed under a controlled environment ( temperature around 22c and relative humidity 4555% with a 12 - 12-hour light - dark cycle ) for the entire research process . at 6 week , 20 c57 mice were received streptozotocin ( stz , 55  mg / kg body weight ; sigma - aldrich , st .
louis , mo , usa ) for 5 days successively by intraperitoneal injection to establish diabetic mice ( stz - c57 ) .
animal treatment was approved by the ethics committee of animal welfare ( wccsirb-2015 - 133 ) .
porphyromonas gingivalis ( p. gingivalis ) w50 was acquired from the state key laboratory of oral diseases , sichuan university and cultured anaerobically in blood - agar ( oxoid , oxoid ltd .
, hampshire , england ) with hemin / menadione ( sigma - aldrich ) . at 6 weeks
, the mice in the infection groups were inoculated with p. gingivalis as the following methods : 10 colony - forming units of p. gingivalis were diffused in 100  l phosphate buffered saline ( pbs ) with 2% carboxymethylcellulose and then orally inoculated three times every second day .
the mice in shame - infection groups were infected with the equal volume of pbs ( 100  l ) .
fasting blood glucose was determined every 2 weeks in tails blood collected from tail veins of the mice after an eight - hour fast using the glucose meter ( onetouch ; lifescan , milpitas , ca , usa ) .
, the serum tumor necrosis factor - alpha ( tnf- ) levels were determined in tails blood collected as above in triplicate by elisa kit ( cusabio ; sino - american biotechnology , wuhan , china ) according to manufacturer 's instructions .
level of alveolar bone loss was determined using sem ( scanning electronic microscope ; zeiss evo , craic technologies inc . ,
the alveolar bone loss level was measured by the average area ( mm ) bordered as the cementoenamel junction , the mesial and distal line angles , and the alveolar bone crest on the lingual sides of the mandibular second molars .
maxillas of db / db and c57 mice were dissected after sacrifice and were made into section slides and stained with immunohistochemical ( ihc ) as presented previously . in short ,
both sides of maxillas were decalcified in 10% edta solution ( biorad , biorad laboratories , hercules , ca ) for 14 days and embedded in paraffin .
the paraffin - embedded tissues were cut into thin sections ( 4  m ) and stained with immunohistochemical .
the primary antibodies , anti - ptpn2 ( 1  :  50 ) , anti - jak1 ( 1  :  100 ) , anti - stat1 ( 1  :  100 ) , and anti - stat3 ( 1  :  100 ) , and the secondary antibodies ( 1  :  1000 ) were all from santa cruz biotechnology ( santa cruz , santa cruz biotechnology , inc . , santa cruz , ca ) .
the staining pictures were captured by an optical microscope ( nikon 80i , nikon ltd . ,
pancreas tissues of four types mice were dissected , rinsed in pbs , and fixed in 4% paraformaldehyde ( sigma - aldrich ) for 24 hours .
the paraffin - embedded tissue samples were cut into a series of 4  m section slides and stained with hematoxylin and eosin ( sigma - aldrich ) .
the morphological characteristics of pancreas tissues were observed in the images captured by an optical microscope ( nikon 80i ) .
data were shown as mean  standard error of the mean ( sem ) and analyzed with student t - test when comparing two groups or one - way analysis of variance ( anova ) test followed by snk - q multiple comparisons when comparing three or more groups using spss software ( spss 17.0 , chicago , il , usa ) .
the fasting blood glucose and body weight levels of all mice were determined every 2 weeks and changes over time are presented ( figure 1 ) . within the mice with p. gingivalis infection , db /
db mice exhibited hyperglycemia from the beginning of research and the fasting blood glucose was maintained at a high value as averagely 26 
th mice increased spontaneously and exhibited high glucose at 8 weeks old and then elevated continuously to a final value as approximately 25 
stz - c57 mice exhibited hyperglycemia 2 weeks after stz injection at 8 weeks old and continued to increase from weeks 810 .
the level maintained steady as evenly 25  mmol / l from weeks 1018 , while the fasting blood glucose level of c57 mice maintained normal from 6  mmol / l to 7  mmol / l for the entire experimental period .
the body weight level of db / db mice increased spontaneously from weeks 610 but decreased gradually after 10 weeks .
the body weight level of c57 mice was steady , but the level of stz - c57 mice in the infection groups decreased .
as shown in figure 2 , comparing infection groups with shame - infection groups , no statistical difference was observed in the fasting glucose levels among the four types of mice ( p > 0.05 ) , which indicated that p. gingivalis infection did not affect the metabolic feature in the mice .
however , the body weight of the db / db infected mice exhibited a gradual decrease since weeks 10 , while the shame - infection group continued to increase . at sacrifice
the body weight of infection group was dramatically higher than that of shame - infection group in db / db mice ( p < 0.01 ) .
the level of alveolar bone loss was significantly higher in injection groups compared to shame - injection groups ( figure 3 ) . within the infection groups ,
db / db mice exhibited more alveolar bone loss than those of th mice , stz - c57 mice , and c57 mice ( db / db mice versus th mice , stz - c57 mice , and c57 mice : p < 0.01 ) .
similar results could be found in the shame - infection groups ( db / db mice versus th mice , stz - c57 mice , and c57 mice : p < 0.05 ) . as shown in figure 4 , the levels of serum tnf- after sacrifice were higher in the infection groups compared to the shame - infection groups ( c57 mice :
p < 0.01 ; stz - c57 mice : p < 0.05 ; th mice : p < 0.05 ; db / db mice : p < 0.05 ) . within the infection groups , c57 mice exhibited less serum tnf- levels than those of diabetic mice ( stz - c57 , th , db / db mice versus c57 mice : p < 0.01 ) .
similar results could be observed in the shame - infected groups ( stz - c57 , th , db / db mice versus c57 mice : p < 0.01 ) .
no statistical difference was found in the serum tnf- levels among diabetic mice in neither the infection nor the shame - infection groups ( p > 0.05 ) . to further illuminate the underlying mechanism of the alveolar bone loss in these mice models
, we evaluated the protein expression of ptpn2 , jak1 , stat1 , and stat3 in gingival epithelium at sacrifice ( figure 5 ) .
c57 mice share the same genetic background with db / db mice and are often used as controls to study the diseases mechanism .
the results demonstrated that high glucose seriously reduced ptpn2 expression in gingival epithelia of diabetic periodontitis mice .
jak1 , stat1 , and stat3 expression levels were dramatically elevated in infection groups of both db / db and c57 mice . moreover , infected db / db mice exhibited the lowest ptpn2 expression compared to their shame - infection controls and c57 mice .
as shown in figure 6 , in c57 mice , round or oval islets cells clusters with clear border were located in the central part of islets and contained a large number of islets  cells with round nuclei . in db
/ db mice and th mice , the volume of islets cells clusters shrinked slightly ; vessels enlarge mildly , which were the histological appearances of type 2 diabetes , while in stz - c57 mice , the volume of islets clusters decreased dramatically .
vacuolar degeneration , necrosis , or disappearance in islets  cells and proliferous fibrotic tissue between islets  cells could be observed ; thus the islets appeared empty .
in this study , three mice models of diabetes periodontitis were established by p. gingivalis oral inoculation and the metabolic and periodontal features were compared .
we found no contrary impact could be observed between this periodontal infection and onset and severity of diabetes in both types 1 and 2 diabetic mice .
variations of ptpn2 and jak / stat pathway in gingival epithelia and different amount of serum tnf- may lead to the different inflammation response to periodontal pathogen .
currently , chemical induced and transgenic mice are two main types of diabetic models . db / db mice had a long history of utilization as t2d spontaneous model .
they shared the same genetic background with c57 , which often used as the genetic controls to explore disease mechanisms .
th mice are relatively new model for t2d characterized by glucose intolerance and hyperglycemia and show metabolic abnormalities .
chemical induced diabetic mice were generally induced by streptozotocin ( stz ) , which destroy pancreatic  cells , resulting in irreversible insulin - dependent diabetes mellitus ( t1d ) .
oral infection of periodontal pathogens has been generally used to establish periodontitis in experimental mice because of limited oral space .
p. gingivalis is widely implicated as an crucial etiological agent in the pathogenesis of periodontitis .
the selection of bacteria with different virulence is important in making sure of the alveolar bone loss .
p. gingivalis w50 is more aggressive in experimental mice for severe periodontal tissue damage because of its high virulence , compared to other strains such as p. gingivalis 381 and w83 [ 27 , 28 ] .
this research implied that p. gingivalis w50 could cause significant periodontal tissue destruction and alveolar bone loss in all infected mice , indicating that oral infection of w50 is an available method to establish periodontitis mice models with both types 1 and 2 diabetes .
the alveolar bone loss levels positively correlated with the severity of periodontal inflammation . in our study , we could observe that all the diabetes mice exhibited alveolar bone loss and db / db mice exhibited more compared to other diabetes mice in both the infection and shame - infection groups .
it proved that three types of diabetes mice could all exhibited the characteristic of diabetes periodontitis and db / db mice showed more severe periodontal infection than th and stz - c57 mice . on the infection groups of all mice , no effect of periodontal infection on the fasting blood glucose was observed .
however , the reverse change was found in the body weight of db / db mice between the infection and shame - infection groups . as db / db mice exhibited more severe periodontal damage , leading to the more difficulty in feeding than other mice with periodontitis
, we supposed that the reverse change was elicited by the more periodontal damage , resulting in less food - intake . in all
, the results revealed that periodontal infection could not affect the risk and severity of diabetes in both type 1 diabetes and type 2 diabetes mice .
prior research has proved that the serum tnf- levels positively correlate with fasting blood glucose level , which can not only destroy pancreatic  cells and reduce the sensitivity of insulin but also active the signaling pathways like jak / stat [ 30 , 31 ] . in our study , we observed that the serum tnf- levels of diabetic mice were higher compared to their normal controls , and the diabetic periodontitis mice showed higher serum tnf- levels than those of c57 mice in the infection group .
the results indicated that diabetes mice models could all represent the degree of diabetes periodontitis inflammation .
its crosstalk with inflammatory pathways such as jak / stat has been found in immune cells like astrocytes and macrophages
. researches about human macrophages also demonstrated the communication between ptpn2 and jak / stat signaling .
a recent research implied that the expression of ptpn2 decreased in gingival epithelial under condition of periodontal disease .
based on this research , we found the ptpn2 expression in the gingival epithelium negatively correlated with the severity of periodontal destruction and hyperglycemia .
moreover , the expression of jak1 , stat1 , and stat3 increased in the gingival epithelium of diabetic periodontitis .
jak / stat pathway is important in the progress of chronic human inflammatory diseases including diabetes .
the main proteins in this pathway ( jak1 , stat1 , and stat3 ) are proven to be enhanced in periodontal inflammation .
our result demonstrated that the interaction between jak / stat pathway and ptpn2 may contribute to the development of diabetic periodontitis .
however , further comprehensive studies are needed to clarify the accurate mechanisms . taken together , severe periodontitis with t2d could be observed in db / db , while mild t2d periodontitis was found in th mice .
the changes of inflammation - related protein expressions in gingival epithelia might lead to the differences in the mouse models .
 periodontitis is one of the severe complications in diabetic patients and gingival epithelium plays an initial role on the onset and progression of this disease . 
 however the potential mechanism is yet sufficiently understood . 
 meanwhile , the research on the correlational experimental animal models was also insufficient . here , we established periodontitis with type 2 diabetes in db / db and tallyho / jngj ( th ) mice and periodontitis with type 1 diabetes in streptozotocin induced diabetes c57bl/6j ( stz - c57 ) mice by oral infection of periodontal pathogen porphyromonas gingivalis w50 . 
 we demonstrated that periodontal infected mice with high blood glucose levels showed dramatically more alveolar bone loss than their counterparts , in which infected db / db mice exhibited the most bone defects . 
 no contrary impact could be observed between this periodontal infection and onset and severity of diabetes . 
 the expressions of ptpn2 were inhibited whereas the expression of jak1 , stat1 , and stat3 increased dramatically in gingival epithelia and the serum tnf- also significantly increased in the mice with diabetic periodontitis . 
 our results indicated that the variations of inflammation - related protein expressions in gingival epithelia might lead to the phenotype differences in the mice with diabetic periodontitis . 