Evaluation of anti-inflammatory effect of statins in chronic periodontitis.

OBJECTIVES: Statins are the group of lipid-lowering drugs commonly used to control cardiovascular and cerebrovascular diseases. Statins have potential anti-inflammatory effect by blocking the intermediate metabolites of the mevalonate pathway. The objective of this study was to evaluate the anti-inflammatory effect of statin medication in chronic periodontitis patients.
MATERIALS AND METHODS: Thirty patients of age group between 40 and 60 years were selected from the outpatient pool of Department of Periodontics, Thaimoogambigai Dental College and Hospital, Chennai. Thirty patients selected were grouped into two groups, Group-I consists of patients with generalized chronic periodontitis and on statin medication and Group-II consists of patients with generalized chronic periodontitis. Clinical parameters were recorded and gingival crevicular fluid (GCF) samples were analyzed for interleukin (IL)-1β using commercially available enzyme-linked immunosorbent assay.
RESULTS: The mean GCF IL-1β levels in generalized chronic periodontitis patients who are on statin medication (Group-I) were lower than the generalized chronic periodontitis patients without statin medication (Group-II).
CONCLUSION: Reduction of GCF IL-1β levels in statin users indicate that statins have anti-inflammatory effect on periodontal disease.

Introduction

Periodontitis is a multifactorial chronic inflammatory disease characterized by destruction of tooth-supporting tissues. The progression of periodontal destruction involves complex interaction between periodontal bacteria and cells of immune system.[1] The complex cytokine network that mediates the immune response includes proinflammatory cytokines, anti-inflammatory cytokines, and specific cytokine receptors.[2] Cytokines play an important role in the initiation, progression, and the host modulation of periodontal disease.[3]

Statins, 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, prescribed to prevent cardiovascular and cerebrovascular diseases. The effectiveness of statin medication is based on their capacity to reduce serum cholesterol levels, primarily low-density lipoprotein (LDL) cholesterol. Statins inhibit HMG-CoA reductase and decrease the production of mevalonate, geranyl pyrophosphate, and farnesyl pyrophosphate, and subsequent products on the way to construction of the cholesterol molecule. Thus, statins could inhibit inflammation, by inhibition of the cholesterol pathway and intracellularly interfering with Ras superfamily protein function.[4]

Interleukin (IL)-1 is found in two active forms, IL-1α and IL-1β encoded by separate genes. Both are potent proinflammatory molecules and are the main constituents of what was once called osteoclast-activating factor. The proinflammatory effects of IL-1 include stimulation of endothelial cells to express selectins that facilitate recruitment of leukocytes and induction of prostaglandin E2 by macrophages and gingival fibroblasts.[5]

Gingival crevicular fluid (GCF) provides a noninvasive means of studying the host response factor by change of constituents in the fluid. The inflammatory exudate from gingival microcirculation crosses inflamed periodontal tissue and en route collects molecules of potential interest from the local inflammatory reaction. Therefore, the fluid offers a great potential source of factors like inflammatory mediators, tissue break down products, and host derived enzymes that may be associated with tissue destruction.[6] Increase in the levels of inflammatory mediators in GCF may be of diagnostic value in evaluating periodontal disease status.[7] Although GCF IL-1β levels in periodontal disease have been studied extensively,[89] ours could be the first study carried out to know the influence of statin medication on the inflammatory mediator especially IL-1β. Statins also have shown to have anti-inflammatory effect.[10] The aim of our study was to know the effect of statin medication on the inflammatory mediator GCF IL-1β levels in chronic periodontitis subjects.

Materials and Methods

The present observational cross-sectional study was carried out after approval from Dr. MGR Educational and Research University's ethical committee. All the volunteers were informed about the aim and the methods of the present study and gave written consent to participate. Subjects were enrolled in the study from July 2012 to September 2012.

Thirty patients of age group between 40 and 60 years were selected from the outpatient pool of Department of Periodontics, Thaimoogambigai Dental College and Hospital, Chennai. The selected subjects were grouped into two; Group-I consists of subjects with generalized chronic periodontitis and on statin medication and Group-II consists of subjects with generalized chronic periodontitis. Generalized chronic periodontitis is defined by having the following criteria, subjects with clinical attachment loss of 3−5 mms in more than 30% of sites. Other inclusion criteria were subjects with minimum number of 15 teeth, plaque index and gingival index scores of 2−3 for both the groups. Group-I subjects were on atorvastatin medication with the dosage of 20 mg/day for a minimum period of 6 months. Exclusion criteria included diabetes mellitus, smokers, and subjects on long-term steroid medications and underwent periodontal treatment in the past 6 months. A full mouth periodontal examination was carried out which included plaque index, gingival index, and clinical attachment level (CAL).

GCF Collection

All GCF samples were collected from the site with maximum CAL in the forenoon at the same time of the day, to allow for circadian variation seen in GCF volume. A calibrated volumetric pipette of 5 μL capacity was placed extracellularly for collection of GCF [Figure 1]. The sample of 2 μL capacity was collected for 20 min. The collected sample was then transferred to a sterilized plastic vial with the help of air spray. The sample was transported to lab and the vial was stored at −71°C.

Figure 1

Micropipette for collecting GCF

Biochemical Analysis

GCF samples were analyzed for IL-1β using commercially available enzyme-linked immunosorbent assay (ELISA) (Avibion human IL-β ELISA Kit, Ani Biotech). Analyses were performed according to the manufacturer's protocol [Figure 2]. The GCF IL-1β values were obtained from ELISA reader [Figure 3]. Results were calculated using the standard curves created in each assay. The total amount of cytokines in GCF was expressed as pictogram (pg/ml).

Figure 2

Microtitre plates

Figure 3

ELISA reader

Statistical Analysis

Data were expressed as mean and standard deviations. Data analysis was carried out using SPSS as software for statistics. Independent sample t-test was done for intergroup comparison of clinical parameters and GCF IL-1β levels.

Results

A total of 30 patients were included in this study. Of these, 16 were males and 14 were females. Independent sample t-test was done for intergroup comparison of clinical parameters and GCF IL-1β levels.

The mean scores of GCF IL-1 β levels in Group-I and Group-II were 180.73 ± 32.15 and 308.20 ± 27.73 pg/ml, respectively. On comparison of mean GCF IL-1β levels of Groups I and II, GCF IL-1β levels in generalized chronic periodontitis subjects who were on statin medication (Group-I) were lower (180.73 ± 32.15) than the generalized chronic periodontitis subjects (308.20 ± 27.73) without statin medication (Group-II). The difference was statistically significant (P < 0.001) [Table 1 and Figure 4]. The mean values of plaque index and gingival index scores of Group-I and Group-II were 2.6 and 2.6 and 2.5 and 2.6, respectively. The mean values of CALs of Group-I and Group-II were 4.1 and 3.9 mms, respectively. On comparison of mean values of plaque index score, gingival index score and CALs of Group-I and Group-II, the difference was statistically non significant, [Tables 2 and 3]. Group-I and Group-II subjects were selected in such a way that both groups have similar amount of clinical parameters like gingival inflammatory status, plaque score, and clinical attachment loss.

Table 1

Mean GCF interleukin-1β levels in patients of chronic periodontitis

Figure 4

Mean GCF IL-1b levels in Groups-I and II

Table 2

Mean plaque index scores in patients of chronic periodontitis

Table 3

Mean gingival index scores of patients of chronic periodontitis

Discussion

Periodontal disease process is site-specific and has multifactorial origin where periodontal pathogens, host response, genetic, systemic, and behavioral risk factors interplay to develop the disease process. Hence, various measures have been taken to include microbial, immunologic, systemic, and genetic factors, in addition to traditional clinical and radiographic parameters, for assessing patient's periodontal status. Inflammatory mediator levels within the GCF have been considered as subclinical marker of progression and severity of periodontitis.[11]

In our study, we used micropipette of known internal diameter for collection of GCF. GCF in the crevice migrates into the tube by capillary action and because the internal diameter is known the volume of fluid collected can be accurately determined by measuring the distance the GCF has migrated. Advantage of this technique is that it provides undiluted sample of native GCF whose volume can be accurately assessed as given by Griffiths.[12]

In our study, Group-I and Group-II subjects were selected in such a way that both groups have similar amount of clinical parameters like gingival inflammatory status, plaque score, and clinical attachment loss, and their mean values between two groups were statistically nonsignificant.

In this study, the mean GCF IL-1β levels were lower in chronic periodontitis patients on statin medication than chronic periodontitis patients without statin medication which is similar to study reported by Sakoda et al.,[10] who found that simvastatin reduces IL-1-mediated production of inflammatory mediators IL-6 and IL-8 in cultured human epithelial cell lines. Our study could be the first study to assess the GCF IL-1β levels in chronic periodontitis patients who are on statin medication.

Anti-inflammatory effect of statins has been proved from our study. Numerous reasons have been quoted to this effect of statins. One reason could be that statins deplete the isoprenoids, which inhibit the signalling pathway for IL-1- and IL-6-mediated inflammation as stated by Omoigui et al.[13] Isoprenoid precursors are necessary for the posttranslational lipid modification (prenylation) and are needed for the function of Ras and guanosine triphosphatases (GTPases). These GTPase proteins such as Ras, Rho, Rac, and Rab (particularly Rho) are intracellular signalling proteins which, when activated, are involved in receptor coupled transduction of signals from extracellular stimuli to cytoplasm and the nucleus. Blockages of isoprenoids by statins affect the cell signalling pathway thereby reducing cytokine expression.[14]

Another reason for the anti-inflammatory effect of statins could be due to the beneficial effects of statins which is mediated through the lowered LDL cholesterol levels, which has been suggested to have anti-inflammatory properties as given by Matsuura et al.[15] Sangwan et al.,[16] in his study found that hyperlipidemic patients are more prone to periodontal disease and statins have positive impact on periodontal health.

Periodontitis is widely accepted as a risk factor for cardiovascular disease due to elevated inflammatory mediators in periodontal lesions and consequently increased serum levels.[17] The administration of statins to cardiovascular patients may have additional benefits through inhibition of inflammation in oral tissues. Meisel et al.,[18] described statins as effect modifiers in the relationship between periodontitis and its inflammatory counterparts in the systemic circulation. Statins could attenuate the inflammation-related systemic effects.

Numerous studies have showed the protective effect of statins on periodontal infections. Lindy et al.,[19] reported that patients on statin medication exhibited fewer signs of periodontal injury than subjects without statin regimen and Cunha-Cruz et al.,[20] showed the association of statin use with reduced tooth loss rate in chronic periodontitis patients. These finding indicate that statins might have beneficial effect not only in periodontal disease but also in cardiovascular disease.

Simvastatin and atorvastatin, as a local drug delivery, have shown to have several advantages in the treatment of periodontal diseases.[2122] The antioxidant and anti-inflammatory properties of locally delivered statins could further facilitate healing of periodontal intrabony defects. However, long-term clinical studies in human subjects are required to evaluate the potential benefits of statins in periodontal regenerative therapy.

Conclusion

Statins are now among the frequently prescribed medication and are currently used by about 25 million people worldwide. The safety and tolerability of statins support their use as a first-line treatment for hypercholesterolemia. Myopathy and its serious complication rhabdomyolysis are the potential effect of therapy with the available statins, but occur very rarely. The population treated with statins is likely to increase dramatically in near future, because they are now recommended also for people with low or normal cholesterol level, especially as a secondary prevention. The results of our study proved the anti-inflammatory effect of statins on chronic periodontitis. Patients of chronic periodontitis receiving statins would have additional benefits, if the anti-inflammatory effect of statins is confirmed through longitudinal further studies.