| As a global health problem, obesity threat to human health seriously. According to World Health Organization(WHO), obesity indicators are usually quantified by the BMI (body mass index), BMI> 30 kg/m2 equivalent to obesity .The main characterization of obesity is the excessive accumulation of adipose tissue and its pathological expansion. In addition to storing energy, adipose tissue is also an active secretory organ,whose ability in secretion of inflammatory mediators and signaling factors play an important role in inflammation and immune regulation of the body.The possible link between periodontitis and obesity is supported by a series of Clinical Studies. Periodontits,as a new threat to global healthare, is supposed to be a risk factor to numbers of obesity related metabolic diseases such as diabetes and cardiovascular diseases. Although the biological mechanisms linking periodontitis and obesity are still clear.,emerging theory supposes that impaired immune response could one of that. A few animal studieshave found that periodontal infection has an effect on obesity related impaired immune response.As one of the important innate immune cells, macrophages have plasticity and versatility, which can be polarized to M1, M2 and other different phenotypes and show different functionsaccording to different microenvironment.Spleen is one of the main settlement sites of macrophages in the body, which contains a large number of lymphocytes and macrophages.Beside that,it is the center of cellular immunity and humoral immunity.At present,the specific association mechanisms between periodontitis and and the immune status of obese body is still uncertain,.But increasing data suppose that they have similar innate immune response and chronic low-grade inflammatory state.Based on the impaired immune response theory,we aimed to explore the host immune response, including systemic and splenic immune response,to periodontal infection and obesity in the our study,. The main contents of this study are structured into three sections as follows.Section 1. Establishment of diet induced obesity and experimental periodontitis mice model1. ObjectivesTo establish experimental periodontitis mice model and induced obesity on them by high-fat-diet; to evaluate the preservation status of obese and experimental periodontitis on mice;2. MethodsForty-four C57BL/6J mice (16 week) were randomly divided into low fat (LF,n=22) or high fat (HF, n=22) diet groups. To induce obesity,the mouse in HF group were fed by high fat diet.At the same time,as control, the mouse in LF group were fed by low fat diet. The diet last for 16 weeks (DIO16 w).At the end of 16 weeks, the mouse in each group was divided into sham-ligation (C, as control) groups or ligation (P, as periodontitis group). The duration of ligating was 10 days. Body weight was measured weekly as obesity related parameters. Weight and weight percentage of epididymal adipose tissue, perirenal adipose tissue and mesenteric adipose tissue were measured at 10 days’ ligation (n=27-36 for DIO16 w mice).HE staining (n=3-5) was used to count the Leukocytes infiltration amount in periodontal3. ResultsBy analyzing body weight in the two diet groups, it was found that HF(high fat diet)group had significantly increased body weight compared with LF(low-fat-diet) group at each week from week 1 to 16 (P<0.01).Compared to LF group HF group saw 33.5%(9.3 g) body weight increased.Apparently, obesity was induced in DIO16 w.Section 2. The impact of periodontal infection and obesity on host immune response in splenic Macrophage.1 Objective:To investigate the influence of pg infections on the Macrophage phenotye and mRNA expression for pro-and anti-inflammatory cytokines (interleukin-10, interleukin-lbeta,and tumor necrosis factor alpha) (Immune Functions) in splenic of obese mice.2. Methodsforty-four C57BL/6J mice (16 week) were randomly divided into low fat (LF,n=22) or high fat (HF, n=22) diet groups. To induce obesity,the mouse in HF group were fed by high fat diet.At the same time,as control, the mouse in LF group were fed by low fat diet. The diet last for 16 weeks (DIO16 w).At the end of 16 weeks, the mouse in each group was divided into sham-ligation (C, as control) groups or ligation (P, as periodontitis group). The duration of ligating was 10 days. Body weight was measured weekly as obesity related parameters. Weight and weight percentage of epididymal adipose tissue, perirenal adipose tissue and mesenteric adipose tissue were measured at 10 days’ligation (n=27-36 for DIO16 w mice).HE staining (n=3-5) was used to count the Leukocytes infiltration amount in periodontal.mRNA expression for pro-and anti-inflammatory cytokines (interleukin-10, interleukin-lbeta, and tumor necrosis factor alpha) in spleen were examined by real time PCR (n=4-5). The splenic mononuclear cells were isolated and cultured, and the percentage of type M1 and M2 marcophages were indentified using F480,CD11c,CD206 fluorescent antibody and measured by FACS(fluorescence-activated cell sorter).3 Result:Compared to that of the control group, the expression of IL-1βmRNA was significantly decreased in the HFC group(0.59±0.23) and HFP group (0.28±0.04) group (p<0.05) while the expression of IL-10 in the HFC group(4.73±0.29) and HFP group (4.03±0.29) is higher than that of control group.The expression of IL-1∞in HFP group is higher than that in LFP group(0.84±0.05).The percentages of M1 and M2 macrophages respectively. The percentage of Ml macrophages in the HFC [(4.04±1.01)%], HFP[(5.22±2.33)%] and LFP [(2.51+0.67)%] group significantly decreased compared with the control group (p<0.05) while the percentage of M2 macrophages in the HFC (44.81%±18.12%) and (33.38%±11.75%) increase significantly compared with the control group (p<0.05)。Compared to the control group, the ratio of M1 to M2 macrophages. Phenotype decreased In other three groups (P<0.05)4 Conclusion:These data indicate that, in Pg infection mice, high fat diet (HFD)-induced obesity reduce M1/M2 ratio of splenic Macrophages by increasing the percentage of M2 macrophages and up regulating the expression level of anti-inflammatory cytokines IL-10 which inhibit the macrophage M1 polarization.Section 3. The impact of periodontal infection on Serum cytokine in obese state1 ObjectivesTo investigate the changes of serum inflammatory cytokines in the spleen macrophages of mice, and to investigate the effects of obesity and periodontal pathogens on systemic immune function in the spleen and the whole body.2. MethodsBy using the mildly obese DI016w mice, this section aimed to explore the impact of periodontal infection on systemic immune response in Serum cytokine forty-four C57BL/6J mice (16 week) were randomly divided into low fat (LF,n=22) or high fat (HF, n=22) diet groups. To induce obesity,the mouse in HF group were fed by high fat diet.At the same time,as control, the mouse in LF group were fed by low fat diet. The diet last for 16 weeks (DIO16 w).At the end of 16 weeks, the mouse in each group was divided into sham-ligation (C, as control) groups or ligation (P, as periodontitis group). The duration of ligating was 10 days. Body weight was measured weekly as obesity related parameters. Weight and weight percentage of epididymal adipose tissue, perirenal adipose tissue and mesenteric adipose tissue were measured at 10 days’ligation (n=27-36 for DIO16 w mice).HE staining (n=3-5) was used to count the Leukocytes infiltration amount in periodontal.mRNA expression for pro-and anti-inflammatory cytokines (interleukin-10, interleukin-lbeta, and tumor necrosis factor alpha) in spleen were examined by real time PCR (n=4-5).Evaluation of systemic immune response:Serum cytokine (including TNF-α, IL-1β, IL-6, IL-10) response was measured by protein arrays (n=4-7).3 Result:Comparison of serum inflammatory factor (IL-1, TNF-a, IL-10) differences in the reaction of (n= 4～7), one-way ANOVA analysis showed that 16 weeks in obese mice IL-1 beta, IL10, TNFa concentration relative to weight group normal significantly up-regulated (IL-1 beta:F= 547.5, P< 0.05 IL-10:F= 11.557, P< 0.05; TNF-a:F=17.76, P< 0.01) and 16 weeks and in the obese state, mouse periodontitis of TNFa and IL-10 concentration compared with obese mice was significantly inhibited (TNF-a:F=6.60, P< 0.01; IL-10:F=17.88, P< 0.01)4 Conclusion:The obese mice spleen IL-1 beta, IL-10, serum level of TNFa increased. The obese state of periodontal pathogens infection of mice TNF-a, IL-10 concentration compared with obese mice was significantly inhibited, suggesting that obesity combined periodontitis status in mice there was a certain degree of tolerance.Section4. Detection of relative expression level of periodontitis macrophage inflammatory factor in spleen1 ObjectivesTo investigate the changes of serum inflammatory cytokines in the spleen macrophages of mice, and to investigate the effects of obesity and periodontal pathogens on systemic immune function in the spleen and the whole body.2. MethodsBy using the mildly obese DI016w mice, this section aimed to explore the impact of periodontal infection on systemic immune response in Serum cytokine forty-four C57BL/6J mice (16 week) were randomly divided into low fat (LF,n=22) or high fat (HF, n=22) diet groups. To induce obesity,the mouse in HF group were fed by high fat diet.At the same time,as control, the mouse in LF group were fed by low fat diet. The diet last for 16 weeks (DIO16 w).At the end of 16 weeks, the mouse in each group was divided into sham-ligation (C, as control) groups or ligation (P, as periodontitis group). The duration of ligating was 10 days. Body weight was measured weekly as obesity related parameters. Weight and weight percentage of epididymal adipose tissue, perirenal adipose tissue and mesenteric adipose tissue were measured at 10 days’ ligation (n=27-36 for DIO 16 w mice).HE staining (n=3-5) was used to count the Leukocytes infiltration amount in periodontal.mRNA expression for pro-and anti-inflammatory cytokines (interleukin-10, interleukin-1beta, and tumor necrosis factor alpha) in spleen were examined by real time PCR (n=4-5).Under sterile conditions, the mice were sacrificed and the spleen was separated and frozen in liquid nitrogen after grinding cracking spleen, spleen in real-time fluorescent quantitative polymerase chain reaction (PCR) to detect M1 macrophages anti-inflammatory type cytokines Tnfa, IL-1 beta, M2 macrophage related cytokines IL-10 mRNA relative expression level differences.3 Result:Real-time fluorescence quantitative PCR method for detection of splenic macrophage related cytokines mRNA:compared with the control group, the obese state M1 macrophage inflammatory cytokine IL-1 mRNA expression levels were decreased (HFC=0.59+0.23, P< 0.05) and il-10mrna (4.03+0.29, P< 0.05) expression level increased. MRNA (+,) expression levels of M1 (+0.29, p<0.05) were decreased at the same time (0.28+0.04, p<0.05), while the expression levels of IL-1 (+4.03, IL-10mRNA) were decreased in obesity complex periodontal pathogens. Compared with the simple periodontitis group (0.62+0.28, p<0.05), the expression of mRNA (IL-1) in the obese group was decreased (p<0.05), and the expression level of IL-10mRNA was increased.. |
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