The Effects Of Diabetes On The Periodontal Tissues, Interleukin-6 Expression And Serum Hs-CRP Level In Rats

Backgroud:Diabetes mellitus(DM) is a systemic disease characterized by abnormal metabolic regulation of both glucose and lipids,resulting in hyperglicemia and hyperlipidemia,is a distructive desease in periontal support tissue,including gingiva,periondontal ligment,alveolar bone,and can progress to tooth loss.In spite of some controversies,a positive correlation between diabetes and periodontal disease has been demonstrated,with periodontal disease considered the sixth most common complication of diabetes mellitus.The bidirection relationship between periodontal disease and diabetes mellitus is reported by many researchers.Compared with the normal people,periodontal disease in diabetic patients presents higher severity and prevalence,higher plaque index(PlI),gingival index(GI),periodontal pocket depth (PD),attachment loss(AL) and higher levels of resorption of alveolar bone,and hence diabetes mellitus can be considered a very important risk factor to the development of periondontal disease.Experimental models have demonstrated that the prevalence and severity of bone resorption in periodontal disease induced by silk ligatures or bacterial inoculation was higher in the presence of diabetes.However,in absence of aggressive stimuli such as silk ligatures,the influence of diabetes on rat periodontal tissues is incompletely explored.The aim of this study was to evaluate the influence of diabetes mellitus on the periodontal tissues and inflammation factors(IL-6,hs-CRP) with SD rats.Objective:(1) To set up the model of type 2 diabetes mellitus SD rats;(2) To compare the periodontal tissues of the model group and normal group;(3) To compare the radiographic structure of bone of the model group and the normal group;(4) To compare the bone structure in light mirror of the model group and the normal group;(5) To compare IL-6 expression immunohistochemistry result of the bone in both the model group and the normal group;(6) To compare the levels of serum high-sensitivity C reactive protein of the model group and the normal group.Methods:1.SD rats was divided into the normal group and the model group randomly.At the 4^th week,Diabetes was induced in SD rats(n=28) by intravenous administration of Streptozotocin(55mg/kg),the normal group was injected with the same dosage of citrate buffer solution.The urine glucose and blood glucose were monitored.At 6,10,12,16 weeks from the beginning of the experiment,3%continal to anesthetize,drew blood 5～7ml,centrifuged at 3000r/min for 15 minutes,left the serum alone,preserved in -70℃ultra cold freezer.Picked up the jaw bones and thigh bones of the two sides,one was fixed in 4%formalin,the other was preserved in -70℃ultra cold freezer.2.The periodontal attachment:After anesthecinesia,observed the colour,shape,texture and the recession of the gingiva,probed the periodontal tissues with periodontal probe.3.Radiographic analysis of the bone:Removed the nerves and muscle on the thigh bones and jaw bones,did radiograph with digital dental film radiographic machine.Working condition:Voltage 63Kv,electric current 8mA,exposure time 0.080s. 4.Histomorphology of the bone:Fixed for twenty-four hours,chose half of the jaw bones and one side thigh bone,made the bone block with special machine, paraffin imbedded the bone block,dyeing with HE.5.IL-6 immunohistochemistry of the bone:The bone paraffin sections of the rats were deparaffinaged as routine,deactivated the endogenous peroxydase with hydrogen peroxide,did febrile antigen recovery with citrate buffer solution,added 5%bovine serum albumin on the slice.IL-6 were diluted according to 1:20,1:40, 1:80,1:100,incubated in the attemperator at 37℃for one hour;biotinylated goat anti-rat IgG was added on the slice and incubated at 37℃for twenty minutes;SABC was added and incubated at 37℃for twenty minutes;did coloration with DAB for 5 to 30 minutes,then observed in the light mirror.6.Measuring the serum high-sensitivity c-reactive protein of the rats: Immuno-scattered radiation turbidimetric method was used to measure the high-sensitivity c-reactive protein in serum of the SD rats.The paramete was set according to the instruction.The normal value of the high-sensitivity c-reactive protein is 0～1mg/L。Results:1.After intravenous administration of Streptozotocin(55mg/kg),the urine glucose and blood glucose were obviously changed.The urine glucose was increased to +++,the blood glucose was higher than 11.2 mmol/L.The type 2 diabetes mellitus rat models can be successfully set up by intravenous administration of Streptozotocin.2.The results of periodontal probing:During the experiment,the shape and texture in both the model group and the normal group weren't abnormal.The gingiva recession wasn't found.The depth of periodontal pocket remained the same level as the beginning of the experiment in the two groups.Compared with the normal group, the gingiva colour of the models became redder,the gingiva bleeding index was a little higher in model group at the later period of the experiment.3.Radiographic results of the bone:The height of the alveolar crest nearly remained the same during the whole period of the experiment in the normal group.At the 10^th week from the beginning,the alveolar bone didn't change obviously,the crest of ridge could be seen clearly in both of the two groups,but the substantia corticalis ossium of the thigh bone appeared thicker in the model group.At the end of the 12^th week,degradation of the bone density could be found both in the alveolar bone and the thigh bone,with the resorption of the bone trabecula.The transmission imagination presented in the radiographic result.4.The observation of the bone morphology:The structures of the cortical substance and the trabecula of the jaw bones and thigh bond were clear in normal group during the whole period of the experiment.The trabecula of bone extended to different aspects,different forms appeared to be interlaced.The height of the alveolar crest remained no change in both of the two groups.At the 12^th week of the experiment,the resorption of the thigh bone and jaw bones were obvious,the thickness of the cortical substance and the trabecula of bone became thinner.HE dyeing presented the osteoclast cells and absorption lacuna of the bone.The thickness of the cortical substance of bone and the number of the rabeculae of bone in both of the two groups were measured.The results of the normal group was considered to be the standard.In model group,the thickness of the cortical substance of bone became thinner at the 6^th week,with statistically significant(P=0.007);at the 10^th week,the thickness of the cortical substance of bone changed to be thicker,the result wasn't statistically significant(P=0.062),maybe result from the reactive hyperplasia of the periosteum.The thickness of the cortical substance of bone became thinner obviously at the 12^th week and 16^th week,the results were statistically significant(P=0.000,0.000).The number of the trabeculae of bone remained nearly the same at the 6^th week compared with the normal group,was not statistically significant(P=0.135).At the 10^th week,the number beginned to be reduced,the result was statistically significant(P=0.041).At the 12^th week and 16^th week,the number declined notably,was statistically significant(P=0.001,0.000).5.IL-6 immunohistochemistry of the bone:The result of IL-6 immunohistochemistry of the bone with normal rats exhibited brown-yellow all over the cavitas medullaris and trabeculae of bone.The pallide-flavens granulation presented in the cavitas medullaris and trabeculae of bone of the model group.There was no significance of difference between the normal group and model group,that meant the protein reaction of IL-6 of the bones with the two groups presented to be negative.6.Results of the serum high-sensitivity c-reactive protein of the rats:The average value of high-sensitivity C-reactive protein of the model group was 5.9479,notably higher than the normal group(the average value was 0.8580),was statistically significant(P=0.000).Compared with the normal group,the average value of the blood glucose and triglyceride were higher(the average value were 12.41796, 2.8032),was statistically significant(P=0.001,0.003).While the average values of the total cholesterol did not change so obviously between the two groups,was not statistically significant(P=0.401).Conclusion:1..The type 2 diabetes mellitus rat models can be successfully set up by intravenous administration of Streptozotocin.The urine glucose and blood glucose were obviously changed.The urine glucose was increased to +++,the blood glucose was higher than 11.2 mmol/L.2.During the whole period of the experiment,the periodontal tissues remained nearly the same as the beginning both in the two groups.The conclusion could be drew out that:in absence of aggressive stimuli such as silk ligatures and bacterial inoculation,diabetes,as a solitary etiological factor,could not be the cause of the periodontal disease.3.At the later period of the experiment,the resorption of the bone in model group was more than the normal group.While the height of the alveolar crest remained unchangable.4.From the intermediate stage of the experiment,the cortical substance of bone and the trabeculae of bone became thinner,and the number of the trabeculae of bone was less。HE dyeing presented the osteoclast cells and absorption lacuna of the bone,osteoporosis beginned to appear.5.The IL-6 immunohistochemistry results of the bone in both of the model group and the normal group were not different widely.It may hint the expression of IL-6 in bone was very little.6.The level of the serum high-sensitivity c-reactive protein notably higher in the model group.The appearance of the acute phase inflammation factor demonstrated the organism was in the inflammation state,and also was a dangerous signal of the occurrence of the periodontal disease.