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Review Article | DOI: https://doi.org/10.31579/2690-8794/165
1 Departement of Clinical Pathology, Faculty of Medicine, Brawijaya University, Kota Malang, Jawa Timur 65145, Indonesia.
2 Department of Dermatology and Venereology, Faculty of Medicine, Brawijaya University, Kota Malang, Jawa Timur 65145, Indonesia.
3 Doctoral Program of Medical Science, Brawijaya University, Kota Malang, Jawa Timur 65145, Indonesia.
*Corresponding Author: Kusworini Handono, Dhelya Widasmara, Hani Susianti, Thigita A Pandaleke (2023), Orthosiphon Aristatus Alleviates Skin Barrier Through Cytokines Regulation in DNCB- Induced Atopic Dermatitis Balb/C Mice Model, Clinical Medical Reviews and Reports, 5(3); DO
Citation: Kusworini Handono, Dhelya Widasmara, Hani Susianti, Thigita A Pandaleke (2023), Orthosiphon Aristatus Alleviates Skin Barrier Through Cytokines Regulation in DNCB- Induced Atopic Dermatitis Balb/C Mice Model, Clinical Medical Reviews and Reports, 5(4); DOI:10.31579/2690-8794/165
Copyright: Kusworini Handono, Dhelya Widasmara, Hani Susianti, Thigita A Pandaleke (2023), Orthosiphon Aristatus Alleviates Skin Barrier Through Cytokines Regulation in DNCB- Induced Atopic Dermatitis Balb/C Mice Model, Clinical Medical Reviews and Reports, 5(3); DOI:10.31579/2690-8794/165
Received: 05 June 2023 | Accepted: 22 June 2023 | Published: 30 June 2023
Keywords: orthosiphon aristatus; atopic dermatitis; 2,4-dinitrochlorobenzene; IgE; IL4; IL22; PGE2; NO
Background: to analysed the effect of Orthosiphon aristatus on repairing skin lesions by regulating cytokines such as IgE, IL4, IL22, PGE2, NO in DNCB-induced Atopic Dermatitis BALB/C Mice Model
Methods: used BALB/C Mice which were sensitized by DNCB for 21 days to developed atopic dermatitis model. Mice were administered oral Orthosiphon aristatus extract once daily (on last 14 days after 7 days of sensitization). The doses given were divided into 6 groups: 17.5 mg/kgbw, 35 mg/kgbw, 70 mg/kgbw, and 140 mg/kgbw. We analysed the levels of cytokines such as IgE, IL4, IL22, PGE2 obtained from the blood. Additionally, we also measured morphological skin lesion severity to confirmed the amelioration effect clinically.
Results: Administration of Orthosiphon aristatus extract reduced the skin lesion severity in all intervention groups. The clinical improvement was supported by decrease of IgE, IL4, IL22, and PGE2 in dose dependent manner.
Conclusion: Orthosiphon aristatus alleviates DNCB-induced Atopic Dermatitis BALB/C Mice Model clinically through downregulating of IgE, IL4, IL22, PGE2, NO levels.
Atopic Dermatitis (AD) is a chronic skin inflammation, recurrent, characterized by itching, arising in certain predilection sites, and associated with other atopic diseases such as allergic rhinitis and asthma. The clinical manifestations of AD are primarily characterized by pruritus, erythematous lesions in acute and lichenification in chronic state, and dry skin. Itch-scratch cycle in AD also triggers secondary infection [1]. The etiology of atopic dermatitis is not fully understood. It might be multifactorial, such as genetic, destruction of skin barrier, immunological, environment like food and aeroallergen. All of those induce a pathological pathway including inflammation and oxidative stress [2].
Atopic dermatitis begins with the destruction of the skin barrier. Destruction to the skin barrier causes increased production of keratinocyte cytokines such as Thymic Stromal Lymphoprotein (TLSP), IL33, IL1, IL6, IL8, and TNFα. These cytokines are able to stimulate formation of Th2, secrete IL4, IL13, and IL5, and produce IgE by B cells, increasing the expression of endothelial cell adhesion molecules, and eosinophil cell proliferation [3, 4]. Increase of IgE serum levels were found in the majority of AD cases and has a significant relationship with severity of AD [5]. Therefore, IgE is one of the criteria for diagnosing AD [3, 4]. In addition, Th2 and IL22 cells were reported to be involved in the inflammatory mechanism in AD. The cytokine IL22 is reported to cause epidermal hyperplasia and inhibit keratinocyte differentiation and filaggrin formation. Fillaggrin plays a role in the formation of Natural Moisturizing Factor (NMF) which keeps the skin moist. Interestingly, IL22 production is influenced by prostaglandin secretion. Previous studies reported an increase in Prostaglandin (PG) D2 and PGE2 in AD lesions followed by an increase in IL22 production [6, 7]. Robb et al (2018) showed that the DNCB-sensitized DA rats’ model had EP4 deficiency and increased IL22 production by T cells in lymph nodes [8]. PGD2 is major prostanoid produced by activated mast cells. The binding of PGD2 to the CTH2 receptor induces chemotaxis of Th2 cells, eosinophils, and basophils which exacerbates the inflammatory process in AD [6]. In addition to the inflammatory process, oxidative stress also plays a role in the mechanism of AD. ROS and NOS which increase either acutely or chronically cause damage to keratinocytes through the destruction of DNA, enzymes and cell membrane structures, thereby reducing the integrity of the skin barrier. Apart from originating from environmental free radicals, oxidative stress is also generated through inflammatory processes. During the inflammatory process, inflammatory cells such as macrophages secrete pro-inflammatory cytokines and nitric oxide (NO) [9, 10]. Therefore, nitric oxide (NO) is an important parameter of skin damage due to oxidative stress and inflammation in AD patients. Based on these mechanisms it can be concluded that effective therapy in AD is by repairing the skin barrier, inhibiting the inflammatory process, and reducing oxidative stress.
Therapeutic modalities currently available are systemic, topical, or phototherapy. The main purposes of the therapy are reduced the severity, prevent infection, and control in long term. Pharmacological therapy includes corticosteroids to treat inflammation, antibiotics for bacterial infections, antihistamines for pruritic symptoms, and calcineurin inhibitors to prevent the spread of eczema and reduce inflammation. Additionally, immunomodulation and phototherapy also been reported to have significant therapeutic effect [11, 12]. Long-term use of systemic corticosteroids in AD patients can cause side effects such as metabolic (hyperglycemia, hypertension, hyperlipidemia), gastrointestinal (gastric ulcers, gastritis, pancreatitis, ulcerative colitis), and Cushing's syndrome. Meanwhile, topical corticosteroids can have side effects on the skin such as skin atrophy, striae, purpura, telangiectasia, hypertrichosis, hypopigmentation, acneiform eruptions, and perioral dermatitis [1]. Antimetabolite groups such as mycophenolate mofetil, azathioprine can cause myelosuppression, especially in elderly users and those with kidney disorders, besides that, antimetabolite groups are also not recommended for use in pregnant women (Category D) [1]. Therapies that are currently being developed such as biologic agents have good effects but are expensive and require multiple injections, making them difficult to give to pediatric patients, and are less curative [13]. Therefore, there is demand of therapeutic modalities with minimal side effects and cost effectiveness, such as the use of bioactive compound from nature like Orthosiphon aristatus. Orthosiphon aristatus has antioxidant and anti-inflammatory activity which is played by phenolics, flavonoids, diterpenes, triterpenes [14, 15]. An in vitro study on murine macrophages conducted by Laavaola et al reported that the chloroform extract from Orthosiphon aristatus leaves, especially the eupatorine and sinestein contents, inhibited iNOS expression and reduced NO and PGE2 production, thereby reducing IL22 levels produced by Th cells [16]. Orthosiphon aristatus ethanol extract and its bioactive compounds (ursolic acid) has the most prominent inhibitory ability by suppressing LPS-induced production of NO and prostaglandin E2 (PGE2) by inhibiting ROS formation while reducing iNOS and COX2 expression in RAW 264.7 cells [17]. Therefore, Orthosiphon aristatus is a potential candidate for effective atopic dermatitis therapy with minimal side effects and cost effectiveness. This study uses Orthosiphon aristatus as an intervention for therapy of DNCB-induced atopic dermatitis BALB/c mice model. This study showed the clinical symptoms of AD pre and post intervention, and analszed serum IgE, NO, IL4, IL22, PGE2, and NO levels as parameters of atopic dermatitis. Additionally, this is the first study that observed the effect of Orthosiphon aristatus in atopic dermatitis.
Methods
Animals and experimental design
This research was conducted at the research and animal handling laboratory, Brawijaya University, Malang. This study used 36 BALB/C mice (aged 6 weeks) with 15–20 grams in weight. This model adopted a previous study (Son et al., 2019) which used 2,4 dinitrochlorobenzene (DNCB) to sensitize and made an atopic dermatitis model. Mice was maintained in a cage with room temperature (22 ± 20C), 40–60% of humidity for acclimatization in a week. They were maintained with food and drink ad libitum with a 12:12 hour dark-light cycle. After a week of acclimatization, their back hair was shaved and started to sensitize the skin with 200 µL of DNCB 1
Atopic disease like atopic dermatitis is a chronic inflammatory skin disease associated with other atopic diseases such as allergic rhinitis and asthma [19–22]. It is a genetic predisposition disease and common in pediatric patients. was frequently associated with food allergy at least in 40% of children [23]. The clinical manifestations of AD are primarily characterized by pruritus, erythematous lesions in acute and lichenification in chronic state, also dry skin [1]. Previous study showed that AD patient had sleep disturbance and depression, and also affect the quality of life of patients [18]. This study confirmed that skin lesion severity in positive control was worse than negative control significantly. These symptoms include erythema, hemorrhage, edema, excoriation/erosion, and scaling/dryness. Interestingly, these symptoms were alleviated with administration of Orthosiphon aristatus leaves extract. This study showed that skin lesion was healed after extract administration in dose dependent manner (Fig. 1). Additionally, this study analyzed serum markers to confirmed that beneficial effect of Orthosiphon aristate give impact in molecular level. Therefore, we measured serum levels of IgE, IL22, IL4, PGE2, and NO. These molecules had known play roles in atopic dermatitis pathophysiology.
The etiology of atopic dermatitis is not fully understood. It might be affected by multifactorial, such as genetic, destruction of skin barrier, immunological, environment like food and aeroallergen [2]. All of these risks cause skin barrier destruction through inflammation reaction and oxidative stress. These reactions increase production of keratinocyte cytokines such as TLSP, IL33, IL1, IL6, IL8, and TNFα, which are able to stimulate the formation of Th2, secretes IL4, IL13, and IL5. Additionally, Th2 stimulates B cells to secrete IgE antibodies, which increase expression of endothelial cell adhesion molecules, and eosinophil cell proliferation [3, 4]. On the other hand, inflammation involves hypersensitivity reactions associated with allergens. Furthermore, production of IgE and histamine are closely associated with this mechanism and become one of the diagnostic criteria of AD [3, 4, 21, 22]. In addition, IL22 plays a role in the inflammation reaction of AD. This cytokine causes epidermal hyperplasia and inhibits keratinocyte differentiation and filaggrin formation which disturbs skin moisturizing [6]. Interestingly, IL22 production is influenced by prostaglandin secretion. Previous studies reported an increase in Prostaglandin (PG) D2 and PGE2 in AD lesions followed by an increase in IL22 production [6, 7]. A study showed that the DNCB-sensitized AD rats’ model had EP4 deficiency and increased IL22 production by T cells in lymph nodes. PGD2 is the main proteinoid produced by activated mast cells. Its binding to the CTH2 receptor also induces chemotaxis of several immune cells which exacerbates AD inflammation process [6]. This study showed increase of IgE, IL4, IL22, PGE2, and NO levels in BALB/C mice after DNCB sensitizing-induced atopic dermatitis.
Currently, steroids, antihistamines, and immunosuppressives are commonly used to treat AD, but side effects often occur [24]. Recently, complementary and supportive therapy like herbal medicine, have been reported more frequently and globally. Although evidences are limited, some herbal medicines administered topically and orally effective for treating AD [25]. Orthosiphon aristatus leaves extract is one of important herbal medicines. Previous studies reported that more than 20 phenolic compounds were detected from Orthosiphon aristatus leaves, and the compounds that have pharmacological properties include caffeic acid, rosmarinic acid, sinensetin, eupatorium, and polymethoxylated flavones [14, 26]. Beneficial effects of phenolic compounds in Orthosiphon aristatus includes antioxidant [27], antibacterial [28], antifungal [29], antidiabetic [30], antiinflammatory [17], antimutagenic [31], and antiarthritic [32].
This study is the first study that confirmed beneficial effects of administration Orthosiphon aristatus in atopic dermatitis. This study showed that administration of the Orthosiphon aristatus leaves extract reduced IgE, IL4, IL22, PGE2, NO serum levels. It was parallel with improvement of the skin lesions. It reflected that this extract improved the inflammation and oxidative stress that induced AD. Anti-inflammatory effects of Orthisiphon aristatus extract have been found in previous studies. A study used a 200µg extract and showed the inhibitory effect of TPA (tetradecanoylphorbol)-induced inflammation in mice [33]. A series of experiments showed that Orthisiphon aristatus extract reduced NO production in LPS activated macrophage-like J774.1 cells. Lyckander and Malterud (1992) showed the effect of ethyl acetate from extract and 8 lipophilic flavonoids isolated from Orthosiphon aristatus (leaves) on the arachidonic acid oxidation catalyzed by 15-lipoxygenase [34]. The results showed inhibition effect on 15-lipoxygenase [IC50 value amounting to 0.018% (w/v)] in dose-dependent inhibition manner compared to quercetin (positive control). Additionally, Orthosiphon aristatus showed antioxidant activity based on β-carotene coupled and autooxidised linoleic acid mechanism and were comparable to quercetin and butylated hydroxyanisole [35]. Another study also marked inhibition mechanism of NO, PGE2, and ROS production, including iNOS and COX-2 gene expression in LPS-stimulated RAW 264.7 cells [17]. Even though, further research is still needed to assess the beneficial activity of this extract to control atopic dermatitis. Histological studies may be proposed to assess the degree of improvement at the microscopic structural level. Related to the role of Orthosiphon aristatus extract as atopic dermatitis therapy, both as supporting/adjuvant therapy, still needs further discussion.
Conclusion
We firstly reported the therapeutic effects of Orthosiphon aristatus leaf effect on atopic dermatitis by DNCB-induced AD-like lesion mouse models. Administration of Orthosiphon aristatus extract improved the progression of AD-like lesions severity. Additionally, it was demonstrated that the anti-atopic effects were exerted through down-regulating the serum level of cytokines such as IL4, IL22, IgE, PGE2, and NO. These effects suggested that Orthosiphon aristatus could be a valuable herbal therapy for atopic dermatitis.
Abbreviations
AD- atopic dermatitis
DNCB- 2,4 Dinitrochlorobenzene
IgE- immunoglobulin E
IL- Interleuikin
Inos- Inducible nitric oxide synthase
LPS- Lipopolysaccharides
NO- nitric oxide
NOS- nitrogen oxygen species
PGE- prostaglandin E
ROS- reactive oxygen species
Th- T-helper
TNF- tumor necrosis factor
TPA- tetradecanoylphorbol
TSLP- Thymic Stromal Lymphoprotein
Declarations
Availability of data and materials
The datasets generated during and/or analysed during the current study are available in Figshare: Dataset of Orthosiphon aristatus effect on DNCB-induced Atopic Dermatitis Mice Model. https://doi.org/10.6084/m9.figshare.22776173.
Acknowledgements
Not applicable
Ethics approval and consent to participate
All procedures performed in this experimental study involving animals were approved by the ethical committee of Faculty of Medicine, Brawijaya University with registration number 102-KEP-UB-2021
Consent for publication
Not applicable.
Competing interests
The authors have no confict of interest to declare.
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