Full Upregulation of α 1 and β 1 adrenergic receptors as an effective factor on hyposalivation of patients with OLP

Objectives: Oral lichen planus (OLP) is one of the common chronic, autoimmune disorders in oral cavity. Regarding the role of adrenergic receptors in mediating stress and that’s effect on salivary composition, the purpose of this study was to investigate salivary levels of α1- and β1-adrenergic receptors in OLP patients to response to this research question: Do agonist or antagonist of adrenergic receptors affect hyposalivation of OLP patients? Method: In this case-control study, stimulated and unstimulated saliva samples were collected from 33 patients and 33 healthy individuals. The salivary ow rate and levels of α1- and β1-adrenergic receptors were measured by ELISA assay. Data were analyzed using SPSS 25 and T-test. Results: The stimulated and unstimulated salivary ow rate was signicantly lower in OLP patients than healthy subjects. The α1-adrenergic receptors in the unstimulated saliva of patients was signicantly higher than in the healthy subjects (p<0.001). α1-receptor in unstimulated saliva in both groups of patients (p<0.001) and healthy subjects (p=0.006) was signicantly higher than stimulated saliva in the same groups. The level of β1-adrenergic receptors in the patients was signicantly higher in the unstimulated saliva (p=0.001) and lower in the stimulated saliva than in the healthy subjects (p=0.003). β1-receptor was signicantly higher in the unstimulated saliva than stimulated saliva in the patients (p<0.001). Conclusion: high levels of α1- and β1-adrenergic receptors in saliva of OLP patients reduce salivary ow rate by increasing the salivary proteins, mucin and saliva viscosity. Selective antagonist of α1- and β1-adrenergic receptors Can improve hyposalivation of OLP patients.


Introduction
Oral lichen planus (OLP) is one of the commonly diagnosed chronic autoimmune oral mucosal disorders, which occurs through attacking autoreactive cytotoxic T lymphocytes to basal layer of oral epithelium [1].
It is commonly seen in middle-aged women, which affects not only the oral mucosa but also the skin and other mucosa such as vagina, esophagus and larynx [2]. In addition to its common symptoms like pain and irritation, OLP is associated with a number of other symptoms including xerostomia [3]. Various studies have documented the association of lichen planus with hyposalivation and xerostomia [4][5][6][7]. The prevalence of xerostomia in OLP patients is higher than that of healthy people. According to research, hyposalivation is the most common cause of xerostomia [8,9]. In addition, changes in salivary composition, especially salivary proteins, are very important in the sense of xerostomia [10]. The etiology of OLP has not been fully understood so far. However, stress, anxiety and depression are considered as effective factors in the incidence and exacerbation of OLP courses [11]. Following stress, sympathetic nervous system and hypothalamic-pituitary-adrenal (HPA) axis are activated and then apply their effects to the body through their own speci c receptors. Adrenergic receptors (ARs) are subtypes of G proteincoupled receptors (GPCRs) that react to releasing catecholamines in response to stress. These receptors are divided into two groups of α and β. Changes in the level or type of adrenergic receptors on the surface of immune and non-immune cells have been reported in some autoimmune diseases, although no reports were found on OLP patients [12]. The sympathetic nervous system (SNS) in the salivary glands exerts its functions through the α1-and β1-adrenergic receptors, and the parasympathetic nervous system (PNS) operates through vasoactive intestinal peptide (VIP) and acetylcholine (ACh) [13]. The β-receptor has the central role in secretion of salivary proteins, and the α-receptor has less active in the secretion of salivary uid, electrolytes and proteins [13]. Regarding the role of stress, SNS and adrenergic receptors in the etiopathogenesis of autoimmune diseases, including OLP [11], , as well as considering the role of adrenergic receptors on the quality and quantity of saliva [14], the current study aimed to evaluate the salivary level of α1-and β1-adrenergic receptors in OLP patients. According to best our knowledge so far this topic has not evaluated.

Materials And Methods
The The unstimulated and stimulated saliva samples were collected from 9 am to 12 noon, from all healthy people and patients. They were asked to avoid eating 2 hours before sampling. When unstimulated saliva sampling, the participants were asked to sit on a chair and then spit into a plastic vial as often as possible within ve minutes. To get the stimulated saliva sample, the participants chewed the same size and shape of chewing gum for a minute. After throwing the chewing gum and swallowing the saliva in their mouths, they spit their saliva in the vial within ve minutes. The saliva ow rate was calculated by dividing the saliva content (in milliliters) by time (in minutes). Hyposalivation and xerostomia were detected when the stimulated saliva ow rate was less than 0.5ml/min, and when the unstimulated saliva ow rate was less than 0.1ml/min [15].

Laboratory methods:
The samples were centrifuged at 2000 rpm for 10 minutes to remove sputum and other additional salivary substances such as plaque and debris. The puri ed saliva (supernatant) was poured into 1-ml microtubes, kept at -20°C (as frozen), and sent to the laboratory to measure α1-and β1-adrenergic receptors. Measurements were performed using ELISA kit (ELISA kit for adrenergic beta1, ELISA kit for adrenergic alpha1, manufacturer: USCN, the USA) to determine the exact level of receptors in each person.
Data were analyzed by SPSS version 25 software using t-test at the signi cance level of p-value<0.05.

Results
In this case-control study, were enrolled 33 OLP patients with mean age of 36.2 years (12 males and 21 females) and 33 healthy subjects with mean age of 35.6 years (12 males and 21 females) as case and control groups respectively. The α1-adrenergic receptor level was signi cantly higher in unstimulated saliva of OLP patients to control group (p-value<0.001) although this result was not found in stimulated saliva (p-value=0.545). In Both case and control groups, levels of α 1 -receptor in unstimulated saliva were signi cantly higher than stimulated saliva in same group (p-value<0.001) (p-value=0.001) ( Table 1).
The β1-adrenergic receptor levels were signi cantly higher in unstimulated saliva (p-value=0.001) and signi cantly lower in stimulated saliva (p-value=0.003) of OLP patients.
In case group, β 1 -receptor was signi cantly higher in the unstimulated saliva than stimulated saliva (p-value<0.001), although this result was not found in the control group (p-value=0.918) ( Table 2).

Discussion
Autoimmune diseases, including OLP, have multifactorial pathogenesis. Genetic, hormonal and environmental factors seem to play a role in the development of diseases. Physical and emotional stresses are the main environmental factors in etiology of autoimmune diseases [16]. The two main hormonal pathways that are activated in response to stress alone or together are the Hypothalamicpituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). The SNS activation directly and indirectly releases epinephrine and norepinephrine through adrenal medulla [17]. Several studies have shown a positive and signi cant relationship between psychogenic stress and OLP [18][19][20][21], while others did not nd a meaningful relationship [22,23]. The adrenergic receptors bind to the catecholamines secreted in response to stress, and lead to their effects in various cells of the body. Change in the level and/or the type of expressed receptor on the cell surface plays an important role in the etiopathogenesis of the disease [17]. Based on the results of this study, the levels of α1-and β1-adrenergic receptors were signi cantly higher in unstimulated saliva of OLP patients compared to control group. In addition, the stimulated and unstimulated salivary ow rates were signi cantly lower in OLP patients to controls.
Various types of autoimmune diseases such as rheumatoid arteritis, systemic lupus erythematous can be associated with dysfunction of salivary glands (regardless of the presence or absence of Sjogren's syndrome) [24]. The occurrence of hyposalivation as a common nding in OLP patients have been con rmed in various studies [4-7, 24, 25]. The autonomic nervous system has a critical role in the secretion of saliva. The β-receptor, mediated by cAMP, has high and very high performance in protein and mucin concentrations of saliva respectively. The stimulation of these receptors leads to low, foamy and viscous saliva. Stimulation of α-receptors leads to increased protein concentrations due to the induction of intracellular Ca 2+ . The α-receptors have low effect on volume, mucin and viscosity of the saliva [14]. In pathogenesis of autoimmune diseases, including OLP, chronic stress and increased SNS tone seems to be involved in reducing the salivary ow rate by increasing salivary mucin production (β1-adrenergic receptor), increasing protein secretion (α1-and β1-adrenergic receptors), vasoconstriction and decreasing blood supply of salivary gland parenchyma. Although the increase in salivary protein components can increase partially the salivary volume, but elevated salivary concentrations and viscosity highly decline the salivary ow rate (volume/min). As we know, the salivary ow rate is the main criterion to evaluation of hyposalivation, not the volume of saliva. The changes in the quality of saliva (increased mucin and protein) are one of the most effective factors in the development of xerostomia even in cases where the patient has no hyposalivation [13]. The high production of protein in the secretory cell requires a lot of energy and leads to stress in the endoplasmic reticulum (ER). The secretory cell response to ER stress by activating hypoxic signals, producing reactive oxygen species (ROS), autophagy, and cell death through internal apoptotic processes. This phenomenon, if chronic, leads to less secretion units in the salivary glands, resulting in reduced saliva production. During apoptosis, the production of in ammatory cytokines, such as IL-6 and type-1 IFNs, and the release and secretion of endogenous antigens and chaperon proteins via apoptotic buds of salivary cells can even be involved in the pathogenesis of Despite the lack of previous studies on the correlation between adrenergic receptors and OLP pathogenesis, down regulation of muscarinic receptors in OLP patients have been con rmed [25]. It seems that the main role in reducing the salivary ow rate in the OLP patients is due to upregulation of SNS and downregulation of PNS. These two systems are not antagonists to each other in salivary secretion, but function together. The SNS has synergistic effects on secretory cells receiving parasympathetic bers [9,14]. It should be noted that the variable concentration of norepinephrine during SNS stimulation can differently stimulate the α1-and β1-adrenergic receptors [26]. In the present study β1adrenergic receptor in the unstimulated saliva signi cantly increased and decreased in the stimulated saliva among the OLP patients. Several studies have also shown the contradictory effects of adrenergic agonist and antagonist drugs on the salivary ow rate [27][28][29]. Also lowering entrance of sympathetic nerve bers to the parotid gland in comparison with other major salivary glands that cause to less expression of adrenergic receptors on the surface of the secretory cells of parotid gland [13], stimulation of saliva secretion through chewing instead of taste triggers that cause to preferably stimulation of PNS to SNS and more time opportunity for the exchange of acinar cells and ducts in unstimulated saliva to stimulated saliva [10,16] justify lower levels of adrenergic receptors in the stimulated saliva compared to the unstimulated saliva in both control and OLP groups. Upregulation of salivary adrenergic receptor in OLP patients, probably source both receptors in the secretory cells of salivary gland and elevated level of these receptors in blood cells. However, this phenomenon has not yet been investigated in the OLP patients, such Studies have shown increased expression of the α1-adrenergic receptor in peripheral blood mononuclear cells (PBMCs) during chronic in ammation in patients with juvenile RA, which led to an increase in IL-6 levels in these patients. These receptors are not expressed normally on the surface of the PBMCs [29][30][31]. Another study revealed that the adrenergic receptors on the surface of immune cells shifted from β2 to α1 in autoimmune diseases [29]. In healthy condition SNS, balance the prein ammatory and anti-in ammatory responses by stimulating different receptors. Chronic stress in genetically susceptible individuals led to an imbalance in the levels of adrenergic receptors on the surface of the immune and salivary cells that can be effective in reduced salivary ow rate and pathogenesis of autoimmune diseases such as OLP.

Declarations
Ethics approval and consent to participate: "Not applicable" Consent to publish: In this study there is no details, images, or videos relating to an individual person that which needs to be agreed to publish.
Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.