Comparative Assessment of Salivary Oxidative Stress, Antioxidant Activity and Psychological Stress Between Oral Lichen Planus Patients and Healthy Controls

Introduction

Lichen planus (LP) is a mucocutaneous lesion which includes a group of diseases and conditions that affect the skin as well as the mucous membrane.¹ Lichen planus (LP) is an autoimmune inflammatory disease of unknown etiology that affects mainly skin and oral mucosa, affecting approximately 1–2% of the general adult population, characterized clinically by polygonal, flat‑topped, skin colored or violaceous papular or plaque eruptions with overlying flat white scales (Whickmans striae).² It is derived from the Greek word “leichen” which means tree moss and Latin word “planus” meaning flat.³ Clinical subtypes of lichen planus are of two types: Cutaneous lichen planus (CLP) and Mucosal lichen Planus.²

The precise pathological mechanism of LP is not yet understood but studies have suggested that in chronic inflammatory diseases like lichen planus, increased production of reactive oxygen species (ROS) and lipid peroxides may play a role in the pathogenesis.⁴ Reactive oxygen metabolites lead to destruction and damage to cell membranes by lipid peroxidation. Malondialdehyde (MDA) is the principal and most studied product of polyunsaturated fatty acid peroxidation.⁵ MDA can thus be a suitable reflector of oxidative stress. Oxidative stress refers to the imbalance between oxidant and antioxidant levels. Thus, imbalance in this protective mechanism can lead to the damage of cellular molecules such as DNA, proteins, and lipids.² Antioxidants (both enzymatic and non-enzymatic) act as a physiological protective means against reactive oxygen species (ROS) and cytokine-associated toxicity in dermatological disorders.⁶ Total antioxidant capacity (TAC) shows a combination of all antioxidant activity than that obtained by measuring concentrations of individual antioxidants.

Lichen planus is a psychosomatic disease. Psychological stress plays a main etiological role in lichen planus. Patients with lichen planus often experience stressful events before the onset of the disease. Therefore, understanding the prevalence of psychiatric comorbidity helps in management and prognosis.⁷ Thus in this regard, the present study was aimed to compare and assess salivary oxidative stress and antioxidant activity with psychological stress in patients with oral lichen planus.

Materials and Methods

All the subjects were recruited from Department of Oral Medicine and Radiology, Dayananda Sagar College of Dental Sciences, Bangalore. Subjects were divided into two groups:

Group I - 26 subjects clinically and histopathologically diagnosed with oral lichen planus

Group II - 26 normal subjects

All the subjects were subjected to preformatted case record and psychological evaluation using Perceived Stress Scale. Only those subjects who volunteered for the study with the stress score ranging from 0-13 were considered for group II.

Inclusion Criteria

• For group I, subjects who were clinically and histopathologically diagnosed with OLP based on modified WHO criteria (2003) were considered.8

Clinical criteria

• Presence of bilateral, more or less symmetrical lesions.

• Presence of a lace like network of slightly raised gray-white lines (reticular patter n).

• Erosive, atrophic, bullous and plaque-type lesions were accepted only as a subtype in the presence of reticular lesions elsewhere in the oral mucosa.

• In all other lesions that resemble OLP but do not complete the above-mentioned criteria, the term “clinically compatible with” was used.

Histopathologic criteria

• Presence of a well-defined band like zone of cellular infiltration that is confined to the superficial part of connective tissue, consisting mainly of lymphocytes.

• Signs of liquefaction degeneration in the basal cell layer.

• Absence of epithelial dysplasia. For group II, only those subjects who volunteered with stress score ranging from 0-13 were considered.

Exclusion Criteria

• Patients undergoing treatment for oral lichen planus or those who had undergone treatment in the past three months.

• Patients suffering from systemic diseases like diabetes, hypertension, hypo or hyperthyroidism.

• Patients taking long term medications such as immunosuppressive agents, non-steroidal anti-inflammatory drugs or antipsychotic drugs over the past three months.

• Patients taking any supplementary vitamins over the last three months.

• Patients with history of any trauma or surgery over past one month.

Collection of Saliva

After completing the questionnaire, unstimulated whole saliva samples were collected from patients and controls by spit method. Subjects were asked to sit in an upright position and rinse their mouth with water for 30 seconds. Following this, they were asked to tilt the head down so as to allow the saliva to accumulate on the floor of the mouth. Then the patients were made to spit into a calibrated tube every 60 seconds for a period of five minutes. The salivary samples were stored at -70 degree in chemical engineering lab. On collection of ten samples, they were transported in a cold storage box to the lab for biochemical estimation. In the lab, the samples were centrifuged at 900 rpm for 10 min at +4⁰ C, supernatant was drawn and stored in small aliquots at -800 C until analysis. Competitive Enzyme Linked Immunosorbent Assay (ELISA) method was used to measure Salivary TAC and MDA levels.

The measurement of salivary MDA was carried out using lipid peroxidation assay kit, also called fluorometric/ calorimetric kit (Abcam, USA) and the measurement of TAC was done using TAC kit (Oxi Tec TM, Korea).

In the lipid peroxidation assay protocol, the MDA in the sample reacts with thiobarbituric acid (TBA) to generate a MDA-TBA adduct. The MDA assay is also referred to as a TBARS assay.

Lipid Peroxidation Assay Protocol Summary

- TBA solution was added to samples and standards, incubated at 95ºC for 60 min, cooled in an ice bath for 10 min

- Transferred to wells of microplate

- Analyzed with microplate reader for higher sensitivity. Precipitated with n-butanol, centrifuged, dried and resuspended the pellet before analysis.

TAC assay kit measures the total antioxidant capacity within a sample. Samples were compared to a known concentration of uric acid standard within a 96-well microtiter plate format.

TAC Assay Protocol

Each uric acid standard and sample was assayed in duplicates or triplicates. A freshly prepared standard curve was used each time the assay was performed.

1. Twenty microliters of diluted uric acid standards and saliva supernatant samples were added to a 96-well microtiter plate.

2. 180 µL of 1X Reaction Buffer was added to each well using either a multichannel pipette or a plate reader liquid handling system and were mixed thoroughly.

3. Initial absorbance was obtained by reading the plate at 490 nm.

4. To initiate the reaction, 50 µL of 1X Copper Ion Reagent was added to each well. It was then incubated for five minutes on an orbital shaker.

5. 50 µL of 1X Stop Solution was added to each well to terminate the reaction.

6. The plate was read again at 490 nm.

Results

Parametric tests (Unpaired t test, Pearson’s correlation test) were applied.

Salivary total antioxidant capacity levels in study and control groups

In the present study, the mean value of salivary TAC in 26 oral lichen planus subjects was 0.2299, with a standard deviation of 0.0649, whereas in 26 subjects of control group, the mean value of salivary TAC was 0.5042 with a standard deviation of 0.099 (Table 1, Graph 1).

Comparison of salivary TAC levels in study and control groups

In the present study, the mean value of salivary TAC was 0.2299 in lichen planus subjects, whereas in the control group it was 0.5042, with a mean difference of -0.274 which was statistically highly significant (p=0.00*) (Table 1, Graph 1).

Salivary MDA levels in study and control groups

In the present study, the mean value of salivary MDA in 26 oral lichen planus subjects was 60.43, with a standard deviation of 5.20, whereas in 26 subjects of control group, the mean value of salivary MDA was 26.52 with a standard deviation of 4.00 (Table 2, Graph 2).

Comparison of salivary MDA levels in study and control groups

In the present study, the mean value of salivary MDA in lichen planus subjects was 60.43, whereas in the control group it was 26.52, with a mean difference of 33.91 which was statistically highly significant (p=0.00*) (Table 2, Graph 2).

Psychological stress between study group and controls

Psychological stress assessment was done using perceived stress scale questionnaire. In the present study, the mean value for psychological stress in 26 oral lichen planus subjects was 28.69, with a standard deviation of 4.057, whereas in 26 subjects of control group, the mean value of psychological stress was 7.92, with a standard deviation of 1.831 (Table 3, Graph 3).

Comparison of psychological stress in study and control groups

In the present study, the mean value of psychological stress in oral lichen planus patients was 28.69, whereas in the control group it was 7.92, with a mean difference of 2.076 which was statistically highly significant (p=0.00*) (Table 3, Graph 3).

Correlation between TAC, MDA with psychological stress

In the present study, the p value for correlation of psychological stress with TAC in oral lichen planus patients was 0.88, whereas in control group it was 0.54 and for correlation of psychological stress with MDA in oral lichen planus patients, the p value was 0.13 and in controls the p value was 0.95 (Table 4).

Discussion

Lichen planus has multifactorial etiology which can affect the oxidant/antioxidant system. Apoptosis plays a hallmark criterion in lichen planus and indicates the reactive oxygen species (ROS) as essential mediators of apoptosis in the disease process.⁹ When ROS interacts with the polyunsaturated fatty acids in membranes or lipoproteins, the process of lipid peroxidation begins. Increased amount of lipid peroxidation provokes immune and inflammatory responses. The inflammatory cellular infiltrates in LP, which consists mainly of CD4+ lymphocytes, are a well-known source of ROS. Uncontrolled production of lipid peroxidation leads to oxidative stress, thereby damaging the cell integrity. Oxidative stress can also be evaluated by the measurement of antioxidant capacity in biological fluids.¹⁰ Antioxidant elements protect cell membranes against lipid peroxidation by reducing free radicals and their subsequent oxidative damages. Since antioxidants are highly variable, Total antioxidant activity (TAC) measures total capacity of antioxidants in biological fluids. Saliva is rich in several antioxidants and it has been shown that saliva includes many defensive mechanisms called the salivary antioxidant system.⁹ For these reasons, we used saliva as a diagnostic tool to measure TAC in our study group. There are several advantages for employing saliva as a diagnostic tool as its sampling is rapid, less expensive, non-invasive and it is a safe procedure for healthcare workers.¹²

Stress, as well as other psychological alterations, seems to modify and promote dysregulation of immune functions by altering cytokine balance and increasing Th2 response, which is associated with the development of autoimmune diseases.¹³ Studies have previously evaluated the relationship between psychological stress levels and onset of OLP. Thus, in our study we used perceived stress scale questionnaire to assess the stress levels. In chronic stress state, despite the hypersecretion of glucocorticoids, there is an increase in pro-inflammatory cytokine levels suggesting a hypofunctional state of glucocorticoid receptors on immune cells that fail to suppress key components of cellular immunity. This explains that psychobiological mechanisms and psychiatric comorbidity may enhance OLP pathogenesis and immunological reactivity.¹⁴ Thus TAC and MDA reflects stress. So, we correlated psychological stress with TAC and MDA in our study. We considered 26 oral lichen planus patients and 26 healthy controls in which 14 were females and 12 were males and 12 female and 14 male subjects were included in the control group. The age group of subjects ranged from 30-64 years in study group and 27-54 years in control group.

The present study revealed that salivary MDA levels were significantly higher in OLP than in controls (Table 2, Graph 2) which is consistent with previous studies conducted by Agha-Hosseini et al. and Ergun et al¹⁵. Salivary TAC was lower than the control group with a mean difference of -0.274 which was statistically highly significant (p=0.00*) [Table 2, Graph 1]. This is consistent with the study done by Celec et al., (2005), Canakci et al., (2009), Serafini et al., (2006).4 We also compared the salivary levels of TAC and MDA in OLP and in healthy controls. The mean value of salivary TAC in subjects was 0.2299, whereas in control group it was 0.5042 and the mean value of salivary MDA in subjects was 60.43 and in controls it was 26.52. In our study, on comparing salivary TAC and MDA values, TAC was found higher in controls compared to LP subjects and MDA was higher in LP subjects which is consistent with the study done by Abdolsamadi et al. ¹⁶ Thus, the synergistic association of oxidative stress and antioxidant activity may be involved in the redox balance which may prove to be useful in monitoring disease activity.

The present study also aimed to evaluate the extent of stress in patients with OLP by means of perceived stress scale questionnaire. The highest score of stress among the OLP patient group was 36 and the highest score in control group was 11, with a mean difference of 2.076 which is statistically highly significant (p=0.00*) (Table 3, Graph 3). This is consistent with the study done by Chaudhary et al., (2004), Pourshahidi et al., Ivanovski et al., Pokupec et al. ¹⁷

In our study, the correlation of psychological stress with TAC in oral lichen planus patients was 0.031, whereas in control it was 0.12 (Table 4). In controls, psychological stress to TAC value was more than the LP subjects. Correlation of psychological stress with MDA was also evaluated. We obtained a correlation of -0.3 in LP subjects and 0.012 in controls (Table 4). This indicates that salivary MDA may be only used as a marker of disease activity. There is no previous reported literature comparing salivary oxidative stress, total antioxidant activity and psychological stress in oral lichen planus patients. Our study aimed to compare these factors and, in our opinion, the results support that increased ROS and lipid peroxidation, and decrease of salivary and plasma antioxidant and increased psychological stress in LP may enhance the inflammatory response by immunological mechanisms to evaluate progress and monitoring process of disease.

Conclusion

By correlating psychological stress with TAC, we conclude that total antioxidant capacity of saliva can serve as an effective marker for stress but correlation of psychological stress with MDA was inconclusive. Thus, additional prospective studies with a larger sample size are necessary to further define the impact of psychological stress on TAC and MDA in OLP patients.

Conflict of Interest: Nil