Astragaloside IV prophylactic administration relieves recurrent allergic atopic dermatitis

Atopic dermatitis (AD) is a common allergic inammatory skin disease with high relapse rates and recurring severity. However, the underlying pathogenesis of AD recurrence is still unclear. Our previous study reported Astragaloside IV (AS-IV) administration in sensitization stage ameliorated the allergic inammation of AD. In this study, we aimed to evaluate the ecacy of AS-IV which prophylactic applied in remission phase of AD and to investigate the underlying mechanisms.


Abstract Background
Atopic dermatitis (AD) is a common allergic in ammatory skin disease with high relapse rates and recurring severity. However, the underlying pathogenesis of AD recurrence is still unclear. Our previous study reported Astragaloside IV (AS-IV) administration in sensitization stage ameliorated the allergic in ammation of AD. In this study, we aimed to evaluate the e cacy of AS-IV which prophylactic applied in remission phase of AD and to investigate the underlying mechanisms.

Methods
The e cacy of AS-IV with prophylactic administration in remission phase of AD was evaluated in a uorescein isothiocyanate (FITC)-induced AD relapse (AD-Re) model. Production of IL-4, IL-5 IL-13, IFN-γin mice ear tissues, IgE in serum were measured by ELISAs. Pharmacokinetic pro le of AS-IV was assessed by HPLC-MS. Protein and gene expression levels of TLRs and NF-κB were detected by WB and qRT-PCR, respectively.
Results: Prophylactic administration in remission phase of AD, AS-IV attenuated ear swelling, in ammatory cell in ltration, IgE production of ear homogenates in AD-Re mice. Levels of T helper (Th)2 cytokines including IL-4, IL-5, IL-13 but not Th1 cytokine IFN-γin AD-Re mice were inhibited remarkably with AS-IV preventively treatment. In addition, a different pharmacokinetic pro le of AS-IV was observed as applied in remission phase. Moreover, AS-IV prophylactic administration decreased the gene expression of NF-κB and TLR8 in AD-Re mice. Consistently, the proteins expression of TLR8, TIRAP and MyD88 in ear homogenates also reduced obviously in AS-IV treated mice.
Conclusions: Our nding indicated the disordered TLR8-mediated NF-κB pathway may play an important role in the pathogenesis of AD recurrence. AS-IV prophylactic administration in remission phase could regulate TLR8-mediated NF-κB pathway to against AD recurrence. These ndings further improved our understanding of the pathogenesis of AD recurrence and the pharmacological effects of AS-IV.

Background
Atopic dermatitis (AD) is a common allergic in ammatory skin disease, with a lifetime prevalence of up to 20% and substantial effects on quality of life [1]. Characteristic features of AD include intense pruritus and a chronic or chronically relapsing course [2]. It has been recognized the innate and adaptive immune systems play dynamic interrelated roles in the pathogenesis of AD, which can in turn favour epidermal barrier disruption in AD. Current treatments for AD include topical moisturizers and anti-in ammatory agents (such as corticosteroids, anti-histamines, leukotriene regulators, as well as novel immunomodulators). These approaches successfully control the symptoms and in ammation of AD, but barely reduce the recurrence rate of AD along with the inevitable side-effects. Therefore, we need to improve understanding the mechanisms of AD recurrence and develop effective prophylactic drugs to against AD relapse.
Our previous study elucidated that YPFS attenuated recurrent allergic in ammation of AD by repairing epithelial barrier defects in remission phase [13]. Furthermore, by means of bioactive components screening, we found AS-IV as a potent bioactive component of YPFS, could ameliorate the allergic in ammation by inhibiting alarmin cytokines such as thymic stromal lymphopoietin (TSLP) and IL-33 in sensitization stage of AD [14]. However, whether AS-IV could also effectively alleviate the recurrent allergic in ammation of AD was still unknown.
In this study, we compared the e cacy of AS-IV with three commonly applied medicines including Dexamethasone (Dex), Montelukast (Mon), and Loratadine (Lor) in a uorescein isothiocyanate (FITC)induced AD relapse (AD-Re) model. By investigating the pharmacokinetics pro les of AS-IV, and its effect on TLRs-mediated NF-κB signalling pathway, we attempted to unravel the underlying mechanism of AS-IV with prophylactic administration against AD recurrence.

Experimental AD relapsing model and medication
Male BALB/c mice of 6-8 weeks old were purchased from Shanghai SLAC Laboratory Animal Co. Ltd (SCXK-2012-0002, Shanghai, China). Mice were maintained at Nanjing University of Chinese Medicine under speci c pathogen-free conditions. All procedures and animals were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine and strictly performed according to the Guide for the Care and Use of Laboratory Animals. The FITC-induced AD-Re mouse model was established as reported previously [13]. Brie y, mice abdomens were shaved with an area of approximately 3×3cm 2 on day 0, topically sensitized with 80 μl of 1.5% FITC (Sigma-Aldrich, St. Louis, MO, USA) solution on days 1 and 2, and the right ear was treated with 20 μl of 0.6% FITC solution on day 6 (elicitation). The initial allergic in ammation was established on day 7 (24 h after elicitation). From day 7 to day 14, mice were given no treatment, allowing in ammation to subsided naturally, which con rmed by the ear swelling measurement with a thickness gauge (7301; Mitutoyo, Kawasaki, Japan). Mice were then preventively treated with AS-IV (12.5, 25, 50 mg/kg/day, i.g.), Lor (1.3 mg/kg/day, i.g.), Mon (1.3 mg/kg/day, i.g.), or Dex (0.67 mg/kg/day, i.p.) for 10 days in remission phase of AD-Re. Medications were terminated at indicated time points before re-challenge with 0.6% FITC on the right ear (to induce AD relapse). Ear swelling was calculated as the thickness difference between the left and right ears. Mice were then sacri ced and samples taken. Thymus index was calculated as the ratio of the weight of thymus glands to the body weight of mice.

Determination of cytokines and IgE production
Production of IL-4, IL-5, IL-13, and IFN-γ in mice ear tissue homogenates and IgE in serum were measured by ELISAs (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions.

Histology
Ear tissue were xed in 10% formalin immediately after mice were euthanized. Para n-embedded sections (4 μm) of the ear tissue were stained with hematoxylin and eosin (H&E) for histology analysis.

Pharmacokinetic study of AS-IV in vivo
To evaluate the pharmacokinetics of AS-IV in remission of AD-Re model, male BALB/c mice of 6-8 weeks old were divided into two groups (Control and AD-Re). Control: mice were treated with AS-IV (25mg/kg/day, i.g.) for 10 days; AD-Re: mice were treated with AS-IV (25mg/kg/day, i.
Quantitative real-time PCR Gene expression of TLR2, TLR3, TLR4, TLR8, TLR9 and NF-κB in ear tissue were detected by quantitative real-time PCR as described previously [15].

Statistical analysis
Data are expressed as means ± standard deviations (SD). One-way ANOVA analysis was used for multiple groups comparisons and the unpaired two-tailed Student's t-test was used for comparison between two groups, using GraphPad Prism 7 (GraphPad Software, CA, USA). A statistical value of p<0.05 was considered signi cant.

AS-IV attenuated recurrent allergic in ammation in AD relapse model
Based on the well-established FITC-induced AD-Re model [13], we rstly evaluate the effects of AS-IV on allergic in ammation relapsing as outlined (Fig 1a). Brie y, the initial AD allergic in ammation was established on day 7 (Fig 1b). From day 7 to day 14, mice were given no treatment, allowing the allergic in ammation to subsided naturally, and AD mice were then divided into 7 groups for further indicated medication treatments on day 14 (Fig.1C). AS-IV (12.5, 25, 50 mg/kg), Lor (1.3 mg/kg), Mon (1.3 mg/kg), or Dex (0.67 mg/kg) were administrated from day 14 in remission phase and terminated at 1 h before rechallenged on day 23 (Fig 1a). Ear swelling in AD-Re group increased remarkably at 6 h after re-challenge on day 23 (Fig 1d). Three commonly applied drugs Dex, Mon, and Lor, as well as three doses of AS-IV (12.5, 25, 50 mg/kg) signi cantly suppressed ear swelling and in ammatory cell in ltration in recurrent phase of AD compared to the untreated AD-Re group (Fig 1d and 1e). These results suggested that preventive administration of AS-IV and conventional medicines, which terminated with a short time prior to allergen re-exposure, effectively attenuated recurrent AD allergic in ammation.

AS-IV exhibited advantages in anti-recurrent allergic in ammation
Clinically, allergic in ammation relapsing occurs unpredictably, we then identi ed whether AS-IV administrated in remission phase of AD-Re could maintain a long-term anti-recurrent allergic in ammation e cacy. Medications were administrated as described above and terminated at 24 h before nal re-challenge on day 24 (Fig 2a). Ear swelling of AD-Re mice was measured at 4, 8 and 12 h after nal re-challenge. As shown in Fig 2b, ear swelling of AD-Re mice increased dramatically at 12 h after nal challenge. Three conventional drugs (Dex, Mon, and Lor) showed barely effect in AD-Re mice (Fig 2b). On the contrary, AS-IV (12.5, 25, 50 mg/kg) alleviated ear swelling and in ammatory cell in ltration notably compared to AD-Re mice (Fig 2b and 2c). In addition, serum levels of IgE in recurrent phase of AD also decreased signi cantly in AS-IV (25, 50 mg/kg) treated mice (Fig 2d). However, in contrast to Dex, AS-IV with three doses (12.5, 25, 50 mg/kg) showed no obvious effect on thymus index (Fig 2e). These results indicated AS-IV administration in remission phase of AD exhibited a superior effect in maintaining remission compared with conventional drugs.

AS-IV inhibited Th2 cytokines production in AD relapse model
Since AD is mainly mediated by type 2 immunity, the effects of AS-IV on T helper 2 (Th2) cytokines (including IL-4, IL-5, and IL-13) production from AD-Re mice were then evaluated. As shown in Fig 2f, IL-4 levels of ear homogenates reduced signi cantly with Dex or Lor treatment compared with AD-Re mice, but not as potent as AS-IV (25, 50 mg/kg) being capable to decrease to a similar level with control.
Furthermore, both IL-5 and IL-13 levels of ear homogenates attenuated remarkably with AS-IV (25, 50 mg/kg) administration compared to AD-Re mice (Fig 2g and 2h). In contrast with AS-IV, three conventional medicines showed no evident e cacy (Fig 2g and 2h). In addition, none of these medications showed notable effect on IFN-γ production (Fig 2i), which is a typical Th1 cytokine. These results indicated AS-IV preventive administration inhibited Th2 cytokines production in AD-Re model.

Pharmacokinetics of AS-IV in remission phase of AD relapse model
Previously, we have found that in remission phase of AD, although the allergic in ammation subsided, the underlying pathological changes were still existed [13]. We next evaluated the pharmacokinetic characters of AS-IV (25 mg/kg) with prophylactic administration in remission phase from AD-Re mice compared with control mice. As shown in Tab 1, the T max of AS-IV in control group and in remission phase of AD-Re group were both reached at 2 h. However, the AUC (0-t) and AUC (0-∞) of AS-IV in remission phase of AD-Re group increased signi cantly compared with control group (p<0.05). Consistently, the C max of AS-IV was raised in AD-Re mice with a statistical difference compared with control (Fig 3 and Tab   1). In addition, the CLz/F of AS-IV in remission phase of AD-Re mice decreased notably as opposed to control mice (p<0.05). The different pharmacokinetic pro les of AS-IV between AD-Re and control indicated that the pathological changes still existed in remission phase of AD, which con rmed with our previous ndings. It also suggested that AS-IV effectively attenuated the recurrent allergic in ammation might due to its regulation of certain underlying pathological processes of AD rather than drug retention, since the blood concentration of AS-IV had dropped to 1.661±1.83 ng/ml after 12 h administration.

AS-IV regulated TLR8-mediated NF-κB pathway in AD relapse model
In view of the pharmacokinetic pro les of AS-IV in remission phase of AD-Re, we then speculated that AS-IV administration in remission phase might regulate certain potential pathological process of AD. It has been widely recognized that alterations of TLRs play a potent role in susceptibility to AD. Therefore, we assessed the gene expression of TLRs in ear tissue with AS-IV (25 mg/kg) treatment in remission phase of AD-Re before nal re-challenge (Fig 4a). The gene expression of TLR2 in ear tissue was downregulated obviously with AS-IV treatment compared with AD-Re group, however no signi cant changes between AD-Re and control mice (Fig 4b). In addition, gene expression of TLR3, TLR4 and TLR9 in ear homogenates showed no detectable changes (Fig 4c, 4d, and 4f). Notably, gene expression of NF-κB in ear homogenates of AD-Re group increased signi cantly compared with control group, which decreased dramatically with AS-IV treatment (Fig 4g). Consistently, the gene expression and protein level of TLR8 increased in AD-Re group, but reduced obviously with AS-IV treatment (Fig 4e and 5h). Moreover, protein levels of TIRAP and MyD88 in ear tissue also reduced obviously in AS-IV treated mice (Fig 4h). These results suggested that AS-IV administration in remission phase of AD could regulate TLR8-mediated NF-κB pathway to against recurrence.

Discussion
Although topical corticosteroids as well as non-speci c immunosuppressive drugs control the symptoms and in ammation successfully, the recurrence of AD is still a clinical challenge. One of the reasons may due to the underlying pathogenesis of AD recurrence is still unclear. YPFS as a potent TCM against allergic disorder, applied in remission phase has been proven e cient through thousands of years of Chinese history [11,12]. Revealing the pharmacological mechanism of YPFS against AD recurrence, may help us to improve understanding the mechanisms of AD recurrence. We previously found that in remission phase of AD, although the major symptoms and in ammation subsided, the pathological changes still existed, especially the de ciency in tight junction protein (occludin and CLDN1) [13].
AS-IV has been shown to be effective in relieving allergic asthma [16,17]. As a representative bioactive compound of YPFS, we reported AS-IV ameliorated allergic in ammation in sensitization stage of AD by inhibiting TSLP and IL-33 production [14]. In this study, we rstly investigated the effect of AS-IV on anti-AD recurrence. Medications in remission phase of AD and re-exposure to allergens in a short time, AS-IV and conventional clinical drugs (Dex, Mon and Lor) alleviated the recurrent in ammation of AD signi cantly. However, with prolonged drug withdrawal, only AS-IV attenuated ear swelling and in ammatory cell in ltration as well as IgE production in recurrent phase of AD. AS-IV has been reported to down-regulate in ammatory cytokines such as TNF-α, IL-1β, and TGF-β [18]. However, the immune response in AD is skewed towards Th2-mediated pathway and can in turn favour epidermal barrier disruption [1]. Therefore, we then investigated the effects of AS-IV on Th2 cytokines production in AD-Re.
Unlike Dex, Mon, and Lor, AS-IV could remarkably damp the IL-4, IL-5, and IL-13 levels of ear homogenates in AD-Re mice, but showed feeble effect on IFN-γ production. These indicated AS-IV could speci cally decrease Th2 cytokines production in AD-Re model.
Recently, HPLC-MS becomes a powerful tool for qualitative and quantitative analysis of pharmaceutical ingredients. As a special saponin, AS-IV has low gastrointestinal tract absorption and bioavailability in rat and dog [19,20]. Interestingly, we found a different pharmacokinetic pro le of AS-IV (administration in remission phase) between AD-Re and control mice. It has been pointed out that the disease status can modify the pharmacokinetic characters of drugs [21]. Therefore, our results further con rmed the pathological changes still existed in remission phase of AD. It also suggested that AS-IV attenuated the recurrent allergic in ammation might due to its regulation of certain underlying pathological processes in remission phase of AD rather than drug retention.
Impairment of epidermal barrier function, for instance, due to de ciency in the structural proteins, have been recognized contributing to AD aetiology and clinical manifestation. We previously found the defects of tight junction protein including occludin and CLDN1 were observed in remission phase of AD. However, AS-IV appeared had barely effects on CLDN1 and occludin (data not shown) expression in AD-Re. In addition, TLRs play a fundamental role in detecting invading pathogens or damage and initiating the innate immune system of mammalian cells. Alterations of TLRs play a potent role in susceptibility to AD [22]. AS-IV presented a wide range of pharmacological effects through multiple pathways, however, most of them are related to TLR4-mediated NF-κB signalling pathways [8][9][10]. Therefore, we assessed several TLRs gene expression of epithelium in remission phase of AD-Re, and evaluated the effects of AS-IV on TLRs expression. As shown in Fig. 4, no obvious changes were observed in terms of TLR2, TLR3, TLR4 and TLR9 gene expression in remission phase of AD. In contrast, both gene expression and protein level of TLR8 increased in AD-Re group, but down-regulated with AS-IV treatment. Consistently, protein levels of TIRAP and MyD88, the adapter proteins of TLR8 in ear tissue also reduced obviously with AS-IV treatment. Moreover, the downstream transcription factor NF-κB showed a similar trend as TLR8. TLR8 was initially considered to be inactive in mice [23]. Until recently, the potential role of TLR8 in the generation of a critical immune response against bacterial infection and cancer has just begun to be uncovered [24]. Our nding indicated TLR8-mediated NF-κB pathway may also play an important role in AD recurrence pathogenesis. AS-IV administration in remission phase of AD could regulate TLR8mediated NF-κB pathway to against recurrence.

Conclusions
AS-IV administration in remission phase of AD attenuated the recurrent allergic in ammation of AD, along with inhibited Th2 cytokines production and presented a different pharmacokinetic pro le. Through regulating TLR8-mediated NF-κB pathway might be the potential mechanism of AS-IV against AD recurrence. These results further improved our understanding of the pathogenesis of AD recurrence and the pharmacological effects of AS-IV.
JZ and MH designed the experiments. XY and XW performed the experiments and collected data; JZ, XY, and XW prepared the gures and analyzed the data. HF assisted with data analysis. The manuscript was written, revised, and edited by JZ and MH.