Key words: Oral Lichen Planus, Lycopene, Anti- oxidant, Pain, Malondialdehyde

INTRODUCTION

Oral lichen planus (OLP) is a chronic inflammatory mucocutaneous disease of unknown etiology that affects 0.5 – 4% of the adult population; with a higher incidence in middle-aged females [1,2]. OLP may appear as reticular, papular, plaque like, atrophic, and bullous-erosive. The erosive and atrophic forms are often symptomatic and need effective therapeutic interference [3], because clinically these categories are related to oral cancer development [4].

However, the basic mechanisms directing OLP toward the development of oral cancer have not been clearly addressed yet. Reactive oxygen species (ROS) were reported as a corner stone in the inflammation-mediated carcinogenesis [5]; through their interaction with polyunsaturated fatty acids (PAFs) in membranes or lipoproteins, leading to lipid peroxidation.

The management of this disease is aiming at pain alleviation and remission of the symptoms. This could be achieved through the use of different corticosteroids. However, despite the efficacy of corticosteroids, many side effects are to be considered before using it [19]. Some cases are also resistant to corticosteroid therapy. Therefore, searching for new effective treatment modalities with fewer side effects is considered a real clinical need.

The uncontrolled production of lipid peroxides may lead to oxidative stress, with notable destruction to cell integrity. Numerous markers have been recommended to observe the lipid peroxidation process. Malondialdehyde (MDA) is a common product of PAFs peroxidation that is increased secondary to oxidative stress [6].

Currently, the ability of oxidative stress coupled with the diminished antioxidant enzyme expression has been suggested in the pathogenesis of OLP [7]. Moreover, decreased serum carotenoid's levels have been reported in patients with LP [8]. In 2011, a notable lower serum level of lycopene was monitored in symptomatic OLP cases [9].

Lycopene (LPN) is a red-colored carotenoid that has a variety of therapeutic properties like inhibition of cancer cell proliferation, antioxidant activity, inducing phase II, interference with growth factor stimulation, control of transcription and restoration of gap junctions. Lycopene has also a unique antioxidant activity via its physical and chemical quenching ability of singlet oxygen [10]. However; the direct evaluation of this anti-oxidant role in OLP wasn’t assessed. Hence, the present study evaluated the salivary malondialdehyde (MDA) as a quantitative indicator for the anti-oxidant capacity of the treatment as well as evaluation of the effectiveness of oral LPN compared with prednisolone (PSN) in the management of symptomatic OLP-patients.

SUBJECTS AND METHODS

Study design

A randomized double blinded clinical trial was applied on 40 patients suffering from symptomatic OLP. The design of this trial (50CCT21-021) was accepted by the Ethics Committee of Al-Azhar University (Girls Branch). This treatment protocol was applied in accord with the Declaration of Helsinki

Participants

Eligibility criteria

Inclusion criteria: Patients suffering from symptomatic OLP (Atrophic/Erosive). The included cases were having oral lesions of at least 10 mm in their widest dimension. Patients were free from any systemic disease except for OLP [11].

Exclusion criteria: Lichenoid reactions, malignant lesion or infective oral involvement. In addition, patients who received topical therapy for OLP in the last 2 weeks or systemic therapy in the last 4 weeks [12]; or any lichenoid inducing drugs. Pregnant or breast feeding women were also excluded.

Study setting

The participating patients were recruited in a consecutive order from the Oral Medicine clinic, Faculty of Oral and Dental Medicine, Al-Azhar University (Girls Branch), Cairo, Egypt. This study was performed between August 2017 and October 2018.

Allocation concealment

Randomisation

In this randomized and controlled intervention clinical attempt, all the participating patients were randomly assigned, using a computer-generated table to one of the following equal treatment groups (20 patients each) in a double-masked design. Group 1 was assigned for patients who received oral LPN. Group 2 included those patients who received a systemic PSN.

Blinding

• This is a double randomized trial where patients were blinded about the treatment groups. Each drug was provided as coated tablets in an opaque containers denoted as A and B treatment.
• The treatment was delivered by the dental practioner who had no access to the treatment codes denoted on the container.
• Assessor blinding: Post- operative assessment was carried out by blinded examiner.

Intervention

Pre-treatment measures: A detailed history was taken from each patient including: the systemic condition, duration as well as disease and drug history using diagnostic chart. Giving verbal and written oral hygiene instructions. The aim of the study, the steps, and the treatment plan were explained and clarified for each participant. Oral mucosal incisional biopsies measuring 5-7 mm; including a healthy and integrate tissue zone, were taken from the most representative lesion area. The definite diagnosis of OLP cases (Figure 1) was assessed in accordance with the modified criteria of OLP [13].

Application of intervention

• Patients in group [1] received systemic treatment with lycopene (LYCOPENE 10mg 60 Soft gels, Biovea, Egypt), twice daily for eight consecutive weeks.
• In group [2], patients received oral prednisolone capsules 20 mg (Wysolone, WyethIndia) two capsule per day (total dose was 40 mg) in morning for eight consecutive weeks of Then the prednisolone dose was tapered to 30 mg/day for two weeks, then to 20 mg/day for next two weeks and finally to 10 mg/day for the last two weeks. Both drugs were administered with unique drugcode to mask the identity of drugs.

Outcomes assessment

Each patient was evaluated immediately before active treatment (T0), then during the treatment course, after 2 weeks (T1), 4 weeks (T2), and 8 weeks (T3) [14] after completion of therapy. The following scores were used in this study:

Table1: Paired t-test values for Clinical score at different study times

Table2: Paired t-test results comparing the VAS at different study times

Table3: Mean and standard deviation for Clinical score values VAS at different study times

Table4: Paired t- test results comparing the MDA values at different study times

Table5: Means and standard deviation values for MDA at different study times

Primary outcome

Clinical scoring (CS): The representative lesion was characterized by being the most severe and clear lesion, and diagnosed by inspection using CS [15] as following:

• 0 means no lesion/normal mucosa.
• 1 means mild white striae⁄ no erythematous area.
• 2 means white striae with atrophic area <1 cm 2.
• 3 means white striae with atrophic area more than 1 cm 2.
• 4 means white striae with erosive area <1 cm 2.
• 5 white striae with erosive area more than 1 cm 2.

Secondary outcomes

Visual analogue scale (VAS): It consisted of a 10-cm horizontal line marked 0–10 (0 no pain; 10 most severe pain experienced). Each patient was asked to mark the scale at the follow-up time points [16].

Saliva collection for malondialdehyde (MDA) assessment: Five milliliter of unstimulated whole salivary samples was expectorated in dry plastic vials. Salivary samples underwent the following steps:

• Centrifuged at 2000 g for 10 min.
• The supernatants were stored at -70οC until further analysis.
• Salivary samples were taken at the same time of day (10–12 am) and at least 2 hrs after the last food or drink intake.

Measurement of saliva MDA

The salivary levels of MDA were determined by a method based on reaction with thiobarbituric acid (TBA) at 90–100οC [17] and expressed as nano gram/ ml.

In this clinical trial, both the patient and investigator were blind to the treatment intervention, one investigator make all the clinical evaluation steps. Another blind investigator makes the biochemical assessment. 

Sample size calculation

Our research design was based on similar studies [18]; the chosen primary outcome was CS. The secondary outcomes were: VAS and salivary MDA. A power analysis was designed to have adequate power to apply a 2-sided statistical test of the research hypothesis (Null hypothesis) that there was no difference between the two groups. Using alpha (α) level of 0.05 (5%) and Beta (β) level of 0.20 (20%), a post-study power indicated that this study had approximately 80% power and significance level = 5; with the predicted minimum sample size (n) was a total of 30 cases i.e. 15 cases in each group. 5 cases were extra-taken in each group to accommodate for any future case loss. Done by IBM™ SPSS™ Sample Power™ Version 3.0.1.

Statistical analysis

Data management and statistical analysis were done using Statistical Package for Social Sciences, Version 21.0 (SPSS, IBM) for Windows. Comparisons between the base line and after treatment at different study times interventions and over time were done by paired T- test. For pain score as well as clinical score, both were expressed as median and range and differences between the 2 groups was tested by the Student- T test was used for changes overtime. Adjustments of the p-value for multiple testing were performed using Bonferroni method.

P value < 0.05 will be considered significant.

RESULTS

Demographic data

The age ranged from 46 to 62 years with a mean of 52.1±4.2. Regarding the gender, 12 (60%) out of the 20 patients enrolled in the test group were females and 8 patients (40%) were males.

Clinical Score results

A statistically significant difference was noticed comparing the clinical score values before and 2 weeks after treatment and through all the follow up time 4 and 8 weeks after treatment as mentioned in (Table 1). The highest mean of clinical score was reported before treatment 3.6 ± 0.88 while the lowest mean of clinical score was noted in week 8 after treatment 1.15 ± 0.74 (Table 1) (figure 1-3).

Visual analogue score results

A statistically significant difference in VAS values was found comparing before and 2 weeks after treatment and through all the follow up time 4 and 8 (Table 2). The highest mean value for pain score was reported before treatment 7.65 ± 0.74. The least mean values of pain score were on week 8 after treatment 2.95 ± 0.82 (Table 2).

Salivary malondialdehyde (MDA)

There was a statistically significant difference in MDA values before and 2 weeks after treatment and through all the follow up time 4 and 8 weeks, 4 and 8 weeks after treatment. The least mean values were on week 8 after treatment 312.95 ± 51.4. The highest mean was reported before treatment 395.55 ± 7.8 (Table 3).

Significant at a p- value < 0.05

Table 1: Comparison between clinical score values between the two test groups at different study times.

Table 2: Comparison between visual analogue score of pain values between the two test groups at different study times.

Significant at a p- value < 0.05

Table 3: Comparison between Salivary malondialdehyde values (nmol/L) between the two test groups at different study times.

Significant at a p- value < 0.05

Figure 1: A Clinical photograph showing oral lesion of erosive lichen planus (Black arrows) in 47 years old female patient. A bilaterally distributed lesion was seen in her buccal mucosa.

Figure 2: A histologic view showing liquefaction of basal layers (White arrows) and inflammatory cellular infiltrates (Red arrows) in the papillary dermis (H&E X200).

Figure 3: A Clinical photograph showing oral lesion of erosive lichen planus (A) in 47 years old female patient. A marked improvement was noticed at the end of the follow up period (B), in the test group.

DISCUSSION

Among many alternative treatments used to spare corticosteroids in OLP, herbal medicine could be of value as palliative treatment to help decrease the symptoms of the disease and overcome the side effects of corticosteroids [20]. Given that the oxidative stress is believed to have a crucial effect in the pathogenesis of OLP, the use of anti-oxidants would be implied to induce remission in OLP patients [21].

Lycopene is a plant extract that has been used to treat many diseases with oxidative stresses playing a role in their pathogenesis [22]; including OLP [23]. Therefore, the current study evaluates the use of 10 mg of lycopene per day for 8 weeks in management of symptomatic OLP cases. Clinical score was measured before, 2, 4, and 8 weeks after treatment to evaluate the remission of oral signs. Visual analogue scale of pain was evaluated at the same time intervals; along with the salivary malondialdehyde (MDA) as a quantitative indicator for the anti-oxidant capacity of the treatment. The reaction of malondialdehyde with thiobarbituric acid has been widely employed in the spectrophotometric detection of malondialdehyde in several biological samples [24].

The present study reported reduction in clinical score of oral mucositis and pain in 2 weeks after the use of lycopene. These results are in accordance with the findings of Saawarn et al. in 2011 [25]. The authors compared lycopene 8 mg per day for 8 weeks to identical placebo on a sample of 30 symptomatic OLP patients and reported that lycopene has a valuable effect in treating OLP. They also added that oxidative stress may have a potential role in disease pathogenesis.

Moreover, the results of Shekhawat et al. in 2016 were in line with our findings. Their study included 50 symptomatic OLP patients that were randomly assigned into two groups; one received 8 mg lycopene per day while the other group had levamisole in a dose of 50mg 3 times/day for 3 successive days per week; for 8 weeks. Pain was assessed 2, 4 and 8 weeks after treatment and lycopene has shown a more potent and faster therapeutic effect.

Recently, Kushwaha et al. [26] used oral lycopene capsules (4 mg/day) for 8 successive weeks with symptomatic OLP patients. The assessment was done at 0, 2, 4, 6, and 8 weeks intervals to record the clinical and symptomatic improvement in OLP lesions. The authors reported as an effective therapy in relieving patients’ symptoms.

Moreover, the use of lycopene in our study revealed a notable decrease in the monitored salivary MDA levels along the duration of study. This result can be explained in the light of its ability to scavenge free radicals [27]. It is worth noting that this was the first clinical report to provide data about the effect of systemic lycopene administration and the oxidative stress biomarker in treating OLP patients. Given that lycopene deficiency has been reported in symptomatic OLP cases and lycopene supplementation significantly decreased the salivary MDA parallel with clinical improvement, a decreased lycopene levels are suggested to have a role in the etio-pathogenesis of OLP for future research. Additionally, lycopene can be successfully used as a sole treatment to manage OLP cases, and as a corticosteroid sparing as recommended by various researchers [19, 22].

Nevertheless, a special concern should be paid to the used dose of lycopene to be used as a corticosteroid sparing line. In this regard, Kushwaha et al. [28] have compared the use of lycopene (4 mg/day) with the systemic prednisolone (40 mg/day) in OLP patients for eight consecutive weeks. The authors reported a significant difference in pain reduction in the prednisolone group. This finding might be attributed to the used dose of lycopene (4 mg), in their model.

In accord, Devaraj et al. [29] examined the immune-modulatory effects of different doses (0, 6.5, 15, or 30 mg lycopene/day for 8 weeks) of purified lycopene supplementation on the oxidative stress biomarkers in healthy volunteers. The authors concluded that purified lycopene delivered a decrease in DNA oxidative damage at the high dose. The aforementioned speculations can explain the good clinical and biochemical results of our study; with further confirmation of the used regimen of lycopene (10 mg/day for 8 weeks); owing to its dose dependent effect.

A possible limitation can be addressed of our study; the relatively short evaluation period. However, this time frame was taken by various researchers [23, 25, & 26]. Indeed, longer follow up clinical trials are needed to further understand the effectiveness of various therapeutic protocols in treating OLP cases, as recommended by Gupta et al., 2017 [30].

CONCLUSION

According to our data; the oral use of lycopene has a promising clinical advantage in treating symptomatic OLP patients via its immune-modulatory effect on the expression levels of the salivary MDA. This finding indirectly substantiates the hypothesis of the potential role of oxidative stress in the pathogenesis of lichen planus. Further studies should be conducted to assess the maintenance effects of oral lycopene.

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