key: cord-0014940-3tgbeyim authors: Zhang, Huiyuan; Zhang, Yameng; Chen, Xiaochun; Li, Juhong; Zhang, Ziyang; Yu, Haiyang title: Effects of statins on cytokines levels in gingival crevicular fluid and saliva and on clinical periodontal parameters of middle-aged and elderly patients with type 2 diabetes mellitus date: 2021-01-08 journal: PLoS One DOI: 10.1371/journal.pone.0244806 sha: ce819149da29481f9632a383fda4a789802d9d33 doc_id: 14940 cord_uid: 3tgbeyim OBJECTIVE: To analyze the effect of statins on cytokines levels in gingival crevicular fluid (GCF) and saliva and on clinical periodontal parameters of middle-aged and elderly patients with type 2 diabetes mellitus (T2DM). METHODS: Systemically healthy controls (C group, n = 62), T2DM patients not taking statins (D group, n = 57) and T2DM patients taking statins (S group, n = 24) were recruited. In each group, subjects (40–85 years) were subclassified into the h (periodontal health)group, the g (gingivitis)group or the p (periodontitis) group according to different periodontal conditions. 17 cytokines in gingival crevicular fluid (GCF) and saliva samples of each subject were measured utilizing the Luminex technology kit. Further, HbA1c (glycated hemoglobin), FPG (fasting plasma glucose), PD (probing depth), CAL (clinical attachment level), BOP (bleeding on probing), GI (gingival index) and PI (periodontal index) were recorded. Data distribution was tested through the Shapiro-Wilk test, upon which the Kruskal-Wallis test was applied followed by Mann-Whitney U test and Bonferroni’s correction. RESULTS: Levels of IFN-γ, IL-5, IL-10 and IL-13 in the saliva of the Dh group were significantly lower than those in the Ch group, while factor IL-4 was higher (p<0.05). Levels of MIP-3α, IL-7 and IL-2 in GCF of the Dh group were considerably higher than those in the Ch group (p<0.05), while that of IL-23 was considerably lower. Compared with the Cg group, levels of IFN-γ, IL-4, IL-5, IL-6, IL-10 and IL-13 were significantly lower in the saliva of the Dg group (p<0.05). Lower levels of IFN-γ, IL-5 and IL-10 were detected in the Sg group than those in the Cg group (p<0.05). At the same time, levels of IL-1β, IL-6, IL-7, IL-13, IL-17, IL-21 and MIP-3α in the gingival crevicular fluid of the Sg group were lower in comparison with the Dg group. In addition, lower levels of IL-4 and higher levels of IL-7 in GCF were identified in the Dg group than those in the Cg group, while in the Sg group, lower levels of IL-4, MIP-1αand MIP-3αwere observed than those in the Cg group (p<0.05). Lower levels of IFN-γ, IL-6, IL-10, IL-13 and I-TAC were found in the Sp group compared with those in the Cp group. The IFN-γ, IL-6 and IL-10 levels were lower in the Dp group than those in the Cp group (p<0.05). Meanwhile, in the Sp group, lower levels of pro-inflammatory factors IFN-γ, IL-1β, IL-2, IL-6, IL-7, IL-21 and TNF-α, in addition to higher levels of anti-inflammatory factors IL-4 and IL-5 in gingival crevicular fluid, were identified than those in the Dp group. Higher levels of IFN-γ,IL-1β,IL-2,IL-7,IL-21 and TNF-α and a lower level of IL-5 in the Dp group were identified than those in the Cp group (p<0.05). Moreover, statins were able to substantially reduce PD in T2DM patients with periodontitis, indicating an obvious influence on the levels of cytokines secreted by Th1 cells, Th2 cells and Th17 cells, as revealed by PCA (principal component analysis). CONCLUSION: Statins are associated with reduced PD and cytokines levels in the GCF and saliva of T2DM patients with periodontitis. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Periodontitis is a type of chronic, progressive, and infectious disease of periodontal supporting tissues predominantly caused by local irritating factors, such as plaque biofilms. Periodontitis has become one of the most common causes of tooth loss [1] , being related to systemic diseases, including diabetes [2] [3] [4] , cardiovascular diseases [5] , hypertension [6] , chronic kidney disease [7] , chronic liver disease [8] , peptic ulcers [9] and breast cancer [10] . Changes of periodontal local inflammatory factors are related to the progression of periodontitis [11] , which is a result of plaque microbial infection and immune responses triggered by plaque biofilms. As the cytoderm component of Gram-negative bacteria in the plaque biofilm, Lipopolysaccharide (LPS) is able to activate mononuclear macrophages, thereby upregulating the secretion of interleukins (IL), macrophage inflammatory protein (MIP), colony stimulating factor (CSF), interferon (IFN), transforming growth factor (TGF) and tumor necrosis factor (TNF) [12] . Increased levels of pro-inflammatory factors, for instance, interleukins-1β (IL-1β), interleukins-6 (IL-6), tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein (MIP-1α), as well as decreased levels of anti-inflammatory factors, namely interleukins-4 (IL-4), interleukins-5 (IL-5), and interleukins-10 (IL-10) are detectable in the local periodontitis lesions. These inflammatory factors synergistically interact with each other, thereby forming a complicated immune response that is critical in initiating and regulating host regulations of periodontal disease [13, 14] . Inflammatory factors render changes in periodontal tissues through different pathways. By illustration, pro-inflammatory factors can regulate the receptor activator of nuclear factor κB ligand (RANKL), which elevates the activity and number of osteoclasts and thus causes bone resorption [15, 16] . Fibroblast apoptosis is also induced by upregulating collagenolytic enzymes, prostaglandin E2 (PGE2) and chemokines [17] . Meanwhile, anti-inflammatory factors can downregulate pro-inflammatory factors and RANKL expression in activated T cells, thereby inhibiting osteoclastogenesis [18] . Type II diabetes mellitus (T2DM) is a type of metabolic disease caused by deficiency of insulin, where the pathogenesis thereof principally includes insufficient insulin production, insulin malfunction, and lack of insulin receptors on the cell surface, resulting in decreased glucose tolerance and increased blood sugar. A myriad of clinical and epidemiological studies have exhibited a two-way relationship between T2DM and periodontitis [4, 19] . The degree of periodontitis in T2DM patients is more serious than that in non-diabetic patients, particularly for those with poor blood sugar control and glycosylated hemoglobin. In parallel, increased levels of systemic pro-inflammatory mediators caused by periodontitis are not conducive to the blood sugar control of T2DM patients. Here, insulin resistance is aggravated, along with an increase in the risk of diabetes complications [2] . After periodontal treatment, FPG and HbA1c are majorly reduced in T2DM patients with periodontitis [20] [21] [22] . Dyslipidemia is one of the risk factors for the occurrence and progression of atherosclerosis in T2DM patients. The American College of Cardiology/American Heart Association cholesterol Guidelines (ACC/AHA) in 2013 recommended that T2DM patients must receive at least moderate statin treatment, this being primarily attributed to the fact that statins can lower the serum level of low-density lipoprotein (LDL). Moreover, statin treatment should continue even if the serum level of LDL is lower than 70 mg/dl, and the dosage of other statins should not be reduced. Said moderate statin treatment contributes to prevention against cardiovascular complications caused by diabetes, such as coronary heart disease and stroke [23] . Reports have articulated that, by stimulating bone regeneration and inhibiting osteoclast differentiation, non-surgical treatment in combination with topical application of statins can relieve periodontitis [24] . The reason for this is that statins can improve periodontal conditions by promoting bone regeneration and inhibiting osteoclast differentiation. Further, compared with only filling hydroxyapatite, a combination of simvastatin can substantially improve the bone regeneration of key mandibular defects in T2DM and osteoporosis rats by upregulating the vascular endothelial growth factor (VEGF) and bone morphogenic protein 2 (BMP-2) [25] . Simvastatin is able to abolish the upregulated MMP-1, which is involved in the destruction of periodontal tissues, in LPS and also the high glucose-induced gingival fibroblasts in vitro [26] . Principal Component Analysis (PCA) is a statistical method that applies dimensionality reduction in order to visualize data. Here, a group of potentially correlated variables can be transformed into a group of linearly uncorrelated variables through orthogonal transformation. Said group of variables after conversion is referred to as the principal component. In multiple studies, many factors related to a problem are often proposed for the purpose of analyzing the problem comprehensively. In this case, each variable reflects certain information related to the research in different levels. Feres et al. [27] adopted 40 kinds of oral microorganisms as the main component to distinguish aggressive periodontitis from periodontitis. Yet, no studies presently exist that have employed PCA to analyze the effects of statins on oral cytokines, especially with regard to the levels of cytokines secreted by different types of Th cells in patients with T2DM. Prior research has demonstrated the role of statins in improving local periodontal inflammation. Despite this, the role of statins in the treatment of periodontitis and gingivitis of T2DM patients remains unclear. The aim of the present study was to assess the effect of statins on changing inflammatory factors in gingival crevicular fluid (GCF) and saliva and on clinical periodontal parameters of middle-aged and elderly patients with T2DM. Eligible subjects (n = 143) treated in the Department of Stomatology and Endocrinology, Shenzhen Center for Chronic Disease Control from December 2018 to May 2019 were recruited. Informed written consent to voluntarily participate was received from every participant and a clear explanation of the research was provided. Furthermore, the stipulations outlined by the World Medical Association's Declaration of Helsinki and the CONSORT group were stringently followed throughout the course of conducting and reporting the present study. The present study was approved by the Ethics Committee of Shenzhen Center for Chronic Disease Control. Subjects who were within the age range of 40-85 years and had at least 20 teeth were eligible, and were classified into one of three groups: systemically healthy controls (C group, n = 62), T2DM patients not taking statins (D group, n = 57) or T2DM patients taking statins(S group, n = 24). In each group, patients were subclassified into the h group (periodontal health), the g group (gingivitis) or the p group (periodontitis) group according to the condition of periodontal diseases. The diagnostic criteria for periodontitis are premised on the 2017 WORLD WORKSHOP [28] . A patient is a periodontitis case in the context of clinical care if: 1) Interdental CAL is detectable at �2 non-adjacent teeth, or 2) Buccal or oral CAL �3 mm with pocketing >3 mm is detectable at �2 teeth and the observed CAL cannot be ascribed to non-periodontal causes, such as: 1) gingival recession of traumatic origin; 2) dental caries extending in the cervical area of the tooth; 3) the presence of CAL on the distal aspect of a second molar and associated with malposition or extraction of a third molar, 4) an endodontic lesion draining through the marginal periodontium; and 5) the occurrence of a vertical root fracture. The diagnostic criteria for gingivitis consist of: 1) Free gums and gingival papillae have become bright red or dark red; 2) Edema of gingival tissue, thickening of gingival margin and hypertrophy of the gingival papilla; 3) Gingival tissue has become fragile with a lack of elasticity; and 4) Dental plaque or dental calculus on the tooth surface. Patients without the above symptoms or clinical signs were included in the periodontal health group, with the grouping and baseline characteristics being listed in Table 1 . Exclusion criteria: (i) Subjects had other systematic diseases except for T2DM in the D and S groups (e.g. hypertension, hyperlipidemia, heart disease, rheumatoid arthritis, and tumor); (ii) Administration of antibiotics or periodontal scaling within 6 months; (iii) Taking medication long-term aside from statins and hypoglycemic drugs; (iv) Patients with aggressive periodontitis; (v) Pregnant and lactating women;(vi) smokers. Six sites of each tooth, except the third molar and the residual root covered by gingiva, were examined utilizing the Florida probe (USA), through which PD, CAL, BOP, GI and PI were recorded. Subjects were subclassified into the h group (periodontal health), the g group (gingivitis) or the p group (periodontitis) predicated on the probing results. In addition, BMI, FPG and HbA1c were recorded. Smoking and alcohol consumption were forbidden one hour prior to saliva collection, and any lipstick was removed. At 9:00 in the morning, each subject was requested to rinse the mouth thereof. Thirty minutes later, saliva collection was performed under a non-stimulation condition in an effort to ensure repeatability. Each subject was asked to open the eyes thereof, sit quietly and slightly tilt the head thereof forward, while oral movement was required to be as minimal as possible. Saliva was collected using a saliva collection tube (SARSTEDT, Genman) in accordance with the instructions. Upon centrifugation for 2 minutes and 1,000 � g, a clear saliva sample was yielded in the tube, which was preserved with the other samples at -80˚C for use. The examined area was isolated by cotton rolls after cleaning any debris. Three Whatman paper filters (Whatman, England) (2×10 mm) were inserted into the gingival sulcus or periodontal pocket of four first molars, respectively and held for 30 seconds. If the first molar was missing, the second molar in the area was detected. Filters were then placed in a 1.5 mL tube containing 500 μL of PBS, and stored at -80˚C for use. Statistical analysis SPSS 26 .0 was employed for statistical analysis, while data distribution was assessed by applying the Shapiro-Wilk test. The data did not follow a normal distribution, thus, non-parametric tests were applied. Differences observed in cytokines levels among the different groups were analyzed by either the non-parametric Mann-Whitney U test for binary variables(h group) or the respective Kruskal-Wallis test for those consisted of several independent groups(g group and p group). In the latter case, variations of statistical significance were further subjected to post hoc pairwise analysis by applying the Mann-Whitney U test and Bonferroni's correction. PCA was conducted to evaluate the differences among the subgroups. Here, PCA was first performed on the levels of the above cytokines in saliva and gingival crevicular fluid. Subsequently, the cytokines were grouped pursuant to the correlation thereof with Th1, Th2 and Th17 premised on prior research. Further, the PCA was conducted with Th1-related, Th2-related and Th17-related inflammatory factors in saliva and GCF as factors. BMI, FPG and HbA1c were compared in subgroups of periodontal health, gingivitis and periodontitis. As can be observed in Table 2 , no significant difference in BMI was detected between the Dh and the Ch group (p>0.05). BMI in the Sg group was similar to that in the Cg and Dg groups (p>0.05), but BMI in the Dg group was substantially higher than that in the Cg group (p<0.05). Significant differences in BMI were not observed among the Cp, Dp and Sp groups (p>0.05). FPG was significantly higher in the Dh group than that in the Ch group (p>0.05). FPG in the Dg group was similar to that in the Sg group, with FPG of both groups being higher than that of the Cg group (p<0.05). In addition, FPG in the Dp group was similar to that in the Sp group, with FPG of both groups being higher than that in the Cp group (p<0.05). In the periodontal health subgroups, the HbA1c level in the Dh group was substantially higher than that of the Ch group (p<0.05). Similar levels of HbA1c were detected in the Dg and Sg groups, which were higher than that in the Cg group (p<0.05). HbA1c was lower in the Cp group compared with that in the Dp and Sp groups (p<0.05), while the levels of the latter two groups were similar. No major differences in BMI, FPG and HbA1c were observed among the periodontal health subgroups (Ch, Cg and Cp groups) (p>0.05). In T2DM patients not taking statins, major differences in BMI, FPG and HbA1c were not found among the Dh, Dg and Dp groups (p>0.05), which was also the case for T2DM patients taking statins (Sp and Sg groups) as well (p>0.05). PD and CAL were compared in subgroups of periodontal health, gingivitis and periodontitis. The PD, BOP, GI and PI values are the average of each value in the above sites in each patient's mouth, while the CAL values are the maximum of the CAL values in the above sites in each patient's mouth. As exhibited in Table 3 , in subjects with healthy dental periphery and gingivitis, no significant differences in clinical parameters were identified (p>0.05). In the periodontitis group, PD in the Dp group was substantially higher than that in the Cp and Sp groups (p<0.05). No significant difference was found in the other clinical parameters among the Cp, Dp and Sp groups (p>0.05). p>0.05 indicated no significant difference between two groups; � p<0.05 indicated a significant difference and A