A preliminary study of periodontitis and vascularcalcification compound model

MENG Yun1-2 DENG JingPan Ke-qing

【Abstract】Objective This experiment is desired to establish a compound model of chronic periodontitis and vascular calcification， so as to study the relation of periodontal and vascular calcification. Methods Forty male Wistar rats were randomly divided into： control group（group C）， periodontitis group（group CP），vascular calcification group（group VDN），compound group（group CP+VDN）.Every groups accepted the corresponding manages to establish the animal model. Eight weeks later， all the rats were sacrificed and the following items were observed： inflammatory factor in serum were tested， Hematoxylin-eosin staining（HE）staining of vascular tissue were taken to test.Results Through detection of periodontal tissue， serum and vascular tissue， animal models were successful. Histopathologic observation revealed： obvious inflammation of periodontal tissue was obversed in group CP and CP+VDN. The red Mineralized nodules deposition in group VDN and CP+VDN were higher than in group C and CP（P<0.05） by HE staining， and that in group CP+VDN was significantly higher than in group VDN（P<0.05）； Animals in group CP+VDN showed higher level of IL-1 in serum than that in group CP，VDN and C. Conclusion This study has demonstrated that periodontitis have some promoting effect on vascular calcification.

【key words 】vascular calcification；periodontitis；inflammatory cytokines；animal model

【CLC】R722.12 【Document code】B【Article No.】1004-4949（2015）03-0535-01

Periodontitis is a chronic inflammatory disease of tooth-supporting tissues caused by periodontopathic bacteria[1]， which is characterized by gingival bleeding， periodontal pocket formation， and destruction of the connective tissue attachment and the absorption of alveolar bone. Coronary artery calcification is part of the development of atherosclerosis； it occurs exclusively in atherosclerotic arteries and is absent in the normal vessel wall[2]. With recent clinical and basic research， however， there is increasing recognition that coronary calcification is an active， regulated process[3] .But there is far less information about any possible association between periodontitis and vascular calcification.

In the present study， we investigated serum inflammatory cytokines including IL-1 and HE staining in vascular tissue were examined in these four groups.

1. Materials and Methods

1.1Materials

Male Wistar rats（200±20g）were obtained from the Animal Center， animal experimental center of Affiliated Hospital of Qingdao University（Qingdao），and were housed under standard conditions（room temperature 20±4℃，humidity 60±10 %，lights from 6：00 to 18：00） and given standard rodent chow and water freely. Rat IL-1 were from Nanjing JianCheng Bioengineering Institute（Nanjing）. Vitamin D3 was from Jiangsu Wuzhong industrial Limited by Share Ltd Suzhou sixth pharmaceutical factory. Nicotine was from Sigma （St. Louis， MO， USA）.

1.2Animal models

Forty male Wistar rats were randomly assigned to 4 groups（n =4 each）： control group（group C），periodontitis group（group CP），vascular calcification group（group VDN），compound group（group CP+VDN）. Group VDN was induced by administration of vitamin D3 plus nicotine as described. Briefly， male Wistar rats were given vitamin D3 （300，000 IU/kg in arachis oil， intramuscularly） simultaneously with nicotine （25mg/kg in 5ml peanut oil， intragastrically） at 9：00 on day 1. The nicotine administration was repeated at 19：00.Group CP was silk ligature and inoculated with oral mixed bacteria to form the periodontal model， A3/0 silk suture was placed in a subgingival position around the maxillary first molars. Group C， given amount of saline intramuscular injection， pure peanut oil to fill the stomach to replace vitamin D3 and nicotine treatment respectively， meanwhile， inoculating of saline in the mouth. The periodontal treatment of VDN+CP group was group VDN plus group C. Animals were sacrificed to taken vascular tissue and serum samples after eight weeks. According to standardized procedures， vascular tissues were washed with PBS and stored at -80℃ until analysis. All tissues were maintained at 4℃ throughout preparation.

1.3Methods

1.3.1 Determination of serum parameters

Tissue homogenates were centrifuged for 15 min at 15，000 g， and then the clear supernatants were removed for analyses. Sensitive Interleukin-1 （IL-1） were determined by IL-1 ELISA kit. According to the manufacturers instructions， as described by our laboratory.

1.3.2 HE staining

The sections are placed in distilled water after hematoxylin staining solution for several minutes ， acidic water and ammonia separation ， each for a few seconds ， after the water rinse 1 hour into distilled water for a moment ， the 70% and 90% dehydrated alcohol for each 10 minutes into the alcoholic eosin stain for 2-3 minutes ， Dehydration， transparent ， were mounted. microscope， with photographs taken to demonstrate calcific nodules distribution[4].

1.4 Statistical analysis

Statistical analyses were performed by using SPSS 17.0 software for Windows. Comparisons across group and group involved unpaired Students t test. Data were expressed as mean±SD. P<0.05 was considered statistically significant.

2. Results

2.1 Serum inflammatory cytokine levels

Table 1 shows that all the rats in the CP group， in the VDN group and in the CP+VDN group had elevated levels of IL-1， all the rats in the CP+VDN group had elevated levels than in the CP group and the VDN group.

Table 1 Serum inflammatory cytokine levels

C n=8 CPn=9 VDNn=9 CP+VDNn=6IL-1 （mmol/L） 62.31±2.57 105.95±3.79* 98.21±4.56* 137.64±3.32*☆△note： compared with group C，*P<0.05；compared with group VDN， △P<0.05； compared with group CP，☆P<0.05.

2.2 HE staining

Fig.1 shows that red Mineralized nodules deposition can be seen in group VDN and CP+VDN， group CP+VDN was significantly higher than in group VDN. There were no significant mineralization depositions in group C and in group CP.

Group C Group CPGroup VDN Group CP+VDNFig.1 the performance of HE staining in every group

3. Dicussion

Large doses of vitamin D3 because of its cytotoxicity can increase calcium assay in cardiovascular tissue and the level of calcium and phosphorus in plasma[5].Nicotine has injury to the artery vascular tissue and enhanced the response of vitamin D3[6]. The combination of vitamin and nicotine can cause thelarge amounts of calcium deposition in the aortic tissue and formation of mineralized nodules， eventually leading to vascular calcification. The study shows that the mineralized nodules deposition of group VDN+CP was higher than in group VDN.

The inflammatory cytokines Interleukin-6 （IL-6）， IL-1 and tumor necrosis factor-α（TNF-α） are elevated in both chronic kidney disease and coronary artery disease and associated with increased cardiovascular mortality[7]. Periodontitis patients are reported to have higher serum IL-1， CRP and neutrophils in several studies[8]. These results suggest that periodontitis， as a chronic inflammatory condition， may contribute to increased serum inflammatory cytokines. These released cytokines can trigger the inflammatory enzymes and mediators and result in the destruction of periodontal tissue[9]. The above results were aggrement with this study. The study shows that all the rats in group CP+VDN， group CP and group VDN had elevated levels of IL-1， all the rats in group CP+VDN group had elevated levels than in group CP and group VDN. We can draw the conclusion that periodontitis and vascular calcification may promote each other through inflammatory factor.

In summary， we can conclude that periodontitis may have some promoting effect on vascular calcification， but we still cant known which route periodontitis have influence on vascular calcification. Further studies are necessary to determine the specific mechanisms between periodontitis and vascular calcification， so that more aggressive clinical guidance are necessary.

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