Angelica Yinzi Alleviates Pruritus-Related Atopic Dermatitis through Skin Repair, Antioxidation, and Balancing Peripheral μ- and κ-opioid Receptors

Background Angelica Yinzi (AYZ) is a Chinese traditional herbal formula reported to attenuate itches and inflammation caused by atopic dermatitis (AD). However, the underlying mechanism of AYZ in the attenuation of itchiness and inflammation remains unknown. Objective This study investigated the mechanism of AYZ in reducing itchiness in mice with 1-chloro-2,4-dinitrobenzene- (DNCB-)-induced atopic dermatitis. Methods Hematoxylin and eosin (H&E) and toluidine blue staining were used to evaluate pathological changes in skin tissue, while an enzyme‐linked immunosorbent assay (ELISA) was used to assess the cytokine levels in the skin. After that, qRT-PCR was performed to determine the mRNA levels of cytokines in the skin. Immunofluorescence and western blotting analysis were further used to assess µ-opioid receptor (MOR) expression and immunohistochemistry to assess the p-ERK, p-AKT, and κ-opioid receptor (KOR). Results The AYZ treatment alleviated the AD clinical symptoms, including decreasing the scratching frequency, the ear thickness, and the infiltration of mast cells, lymphocytes, inflammatory cells, and mononuclear cells. In addition, AYZ inhibited the expression of interleukin (IL)-13, thymic stromal lymphopoietin (TSLP), and reduced neuraminidase (NA), corticotropin-releasing factor (CRF), and reactive oxygen species (ROS) expression. Markers involved in itches, such as p-ERK and p-AKT, were significantly downregulated following AYZ treatment. Besides, AYZ significantly increased MOR expression and downregulated KOR in the epidermis and spinal cord. Conclusion Our findings imply that AYZ ameliorates pruritus-related AD through skin repair, antioxidation, and balancing peripheral MOR and KOR. The findings in this study lay a theoretical foundation for the control mechanism of peripheral itch.


Introduction
Atopic dermatitis (AD) is the most common chronic infammatory skin disorder characterized by scaly red rashes, pimples, desquamation, and severe pruritus [1].Pruritus is the most burdensome AD symptom, which, together with scratching, increases skin damage, leading to sleeplessness, fatigue, and poor life quality [2,3].Despite this, topical AD therapy does not control all AD symptoms, particularly pruritus, while the current systemic treatment raises a series of safety concerns.
Itch is considered a protective sensation and response to the environment.Scratching damages the epidermal barrier and facilitates an appropriate physiologic cascade associated with sensing the insult.Tis cascade includes multidirectional communication between the nervous and immune systems within the skin.Depending upon the insult, such as a contact allergen, or in association with a disease, for example, atopic dermatitis, a pathophysiologic itch may develop [15].Pruritus is mediated in the nonmyelinated Cfbers whose peripheral terminals are in the skin epidermis and dermo-epidermal junction.Specifcally, the aferent nerve fbers send signals to the second-order spinal neurons in the dorsal horn of the spinal cord, which project them to the contralateral spinothalamic tract and subsequently to the brain [16,17].Tese pruritus signals are passed on to the peripheral, spinal cord, and cortical by increasing the signals from the periphery while decreasing the neural circuits [18,19].Recently, the endogenous opioid system (EOS) and peripheral opioid system have been established to contribute in pruritus transmission.Besides, central nervous system neurobiological studies revealed that EOS is involved in central itch regulation [20][21][22][23].However, it is elusive how the peripheral opioid system transmits the pruritus signals between the skin and brain.

Methods
For the qualitative analysis, the Q-TOF-MS mass spectrometer (G6530) was coupled with electrospray ionization (ESI) and a diode array detector (DAD).Te samples were determined in the positive and negative modes using the following ESI parameters: dry air temperature of 350 °C; dry gas fow rate of 10 L/min; atomizing gas pressure of 35 psi; sheath temperature of 350 °C; sheath gas fow rate of 12 L/min; capillary voltage of 4000 V (positive mode) and 3500 V (negative mode).Te Agilent MassHunter (B.08.00) was used to collect the data.Subsequently, Qualitative Navigator (B.08.00) and Qualitative Workfows (B.08.00) were used for data analysis.

Animals and Treatments.
All animal procedures were reviewed and approved by the Animal Care and Use Committee of the Materia Medica Institute, China.Seventyfve adult male C57BL/6 mice (6 weeks old) were provided by the Hubei Provincial Center for Disease Control and Prevention (China).Tey were randomly assigned into fve groups (n � 15), which were the control, model, AYZ (6.24 g/ Kg), SDQP (0.96 g/Kg), and CHT groups (1.3 mg/Kg).Te AD-like immunological and skin lesions were induced by treating the mice with DNCB.Briefy, 1% DNCB dissolved in acetone-olive oil (AO, 3 : 1) was applied on approximately 8 cm 2 of dorsal skin after completely removing the hair, while 1% DNCB was applied on the face and the back of both ears after four days.All the mice except the control group were treated with 0.5% DNCB thrice weekly for three weeks (day 1-20) on the same areas on the dorsal skin.In addition, AYZ, SDQP, and CHT were dissolved in pure water and orally administered once daily for three weeks.Te SDQP and CHT were used as the positive controls and are traditional Chinese medicine and western medicine, respectively, which have been accepted for the clinical treatment of AD.
On day 21, all the mice were sacrifced.Te serum, lumbar (L2-L3) spinal cord contents, and lesions on the dorsal skin were collected for further analysis.Evidence-Based Complementary and Alternative Medicine

Evaluation of Ear Tickness and Scratching Behavior.
Te thicknesses of the right and left auricles were measured weekly using an electronic digital caliper during the experimental period.For elucidation, the duration of the scratching behavior was defned as the time spent rubbing the head, scratching the dorsal skin, nose, or face with the hind limbs over a 20-minute period captured using a digital camera facing the test box on the penultimate day of the experiment.

Cytokine Measurement.
Te NA, CRF, TSLP, and ROS were measured using standard ELISA kits from the serum samples according to the manufacturer's instructions.Te A values were determined at 450 nm.

Histological Analysis.
To investigate the efects of AYZ on DNCB-induced AD, we determined the skin thickness and mast cell infltration.Te dorsal skin samples were fxed in 4% formalin solution for 24 h, repeatedly rinsed, dehydrated, and embedded in parafn solution.Deparafnized skin sections were stained with H&E to determine the skin thickness and toluidine blue for mast cell infltration.

2.7.
Immunohistochemistry. Skin, lumbar, and spinal cord samples were deparafnized by consecutive washes with xylene and ethanol, and antigen was retrieved in citric acid bufer (pH 6.0) for 10 min.Te samples were then washed in PBS for 15 min and incubated with peroxidase blocking reagent for 35 min.Next, the samples were incubated with 3% BSA at room temperature for 30 min.Subsequently, the samples were incubated with the primary antibodies, namely: p-ERK (1 : 200 dilution, Immunoway, YP1197), p-AKT (1 : 100 dilution, Abclonal, AP0140), and KOR (1 : 100 dilution, Abcam, ab183825) overnight at 4 °C.Tereafter, they were incubated with the secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (DAKO, K5007) for 50 min.Te samples were then stained and visualized, and images were captured under a light microscope (Olympus, Japan).
2.9.Western Blotting.Te skin samples were lysed in RIPA bufer and then centrifuged at 12,000 rpm for 10 min at 4 °C.Te proteins in the lysates were detected using a bicinchoninic acid (BCA) protein assay kit following the manufacturer's guidelines.For each sample, 100 μg of protein was separated on an SDS-PAGE gel and then transferred onto nitrocellulose (NC) membranes (Millipore, Burlington, MA, USA).Te membranes were blocked with 5% BSA for 2 h at room temperature, then incubated with the primary antibodies, MOR, after which they were washed in three changes of PBS.Te membranes were then incubated with secondary HRP-conjugated anti-rabbit IgG antibody (1 : 200 dilution, Cell Signaling Technology, #4412) for 1 h at room temperature.Proteins were visualized by adding ECL (Termo), then scanning and imaging the membranes using the FluorChem FC3 system (ProteinSimple, USA).

Statistical Analysis.
All the treatments in all the assays were replicated thrice.Statistical analyses were performed using the SPSS version 22.0 statistical software.Te diferences among and between groups were performed by one-way analysis of variance (ANOVA) and Student's t-test, respectively.All data were presented as mean ± standard deviations (SD).A p value <0.05 was considered statistically signifcant.

Quality Control Analysis of AYZ.
Te chemical ingredients of AYZ were identifed by UHPLC/Q-TOF-MS in both positive and negative-ion modes.A base peak chromatogram of the PTQX granule extract was acquired for structural confrmation (Figure 1).Te authentic compounds were elucidated by their MS/MS fragmentation.Te quasi-molecular ions and fragment ions of compounds are listed in Table 1.

AYZ Attenuated DNCB-Induced AD-Like Symptoms.
Histologically, AYZ treatment signifcantly reduced the scratching frequency and the ear thickness (p < 0.05 and Evidence-Based Complementary and Alternative Medicine   2(a) and 2(b)).To further examine whether AYZ inhibits DNCB-induced AD-like skin infammation in mice, the infltrations of infammatory corpuscles were observed following the H&E and toluidine blue staining.Tere was a signifcant reduction in the infltration of the mast cells, which are the major efector cells in the pathogenesis of AD, with the treatment of AYZ.Similarly, the AYZ-treated group recorded a signifcant reduction in lymphocytes, infammatory cells, and mononuclear cell infltration into dorsal skin lesions compared to the model group (shown in Figure 2(c)).

Efects of AYZ on DNCB-Induced Cytokine Production in
Serum and ROS Level in Cutaneous Tissue.Te CRF expression levels in the model group were signifcantly increased compared to the control group (p < 0.01).In addition, the Hypothalamic-Pituitary-Adrenal (HPA) axis was activated.Moreover, cytokines were signifcantly downregulated following SDQP and CHT treatment.In addition, AYZ treatment signifcantly downregulated CRF and NA levels compared to the model group (p < 0.5; shown in Figure 3(a)).Te AYZ treatment also decreased the expression of ceramide, a lipid degraded by NA, which maintains moisture and the skin barrier function (p < 0.5) (shown in Figure 3(b)).Besides, the elevated cytokine levels produced by T2 cells in serum were also signifcantly decreased following AYZ treatment.However, NA was signifcantly increased in the DNCB-induced group compared to the control group (p < 0.01).Similarly, the TSLP expression levels in the DNCB group were signifcantly increased compared to the control group (p < 0.01).At the same time, the cytokines were signifcantly reduced following the AYZ treatment (p < 0.5) (shown in Figure 3(c)).Te ROS quantifcation in cutaneous tissue revealed that the ROS levels were signifcantly increased in mice treated with DNCB and in the model group compared to the control group (p < 0.01).Tis implies that oxidative stress was increased in DNCB-induced AD mice.Nonetheless, AYZ treatment signifcantly reduced the oxidative stress levels compared to the model group (p < 0.5; shown in Figure 3(d)).

Expression of MOR and KOR Proteins in the Epidermis and
Spinal Cord of Mice.Te MOR and KOR proteins were As: Angelica sinensis; Pra: Paeoniae radix alba; Cx: Chuanxiong rhizoma; Rg: Rehmannia glutinosa; Tt: Tribulus terrestris L; Sr: Saposhnikoviae radix; Ss: Schizonepetae spica; Pm: Polygonum multiforum thunb; Am: Astragalus membranaceus; Grr: Glycyrrhizae radix et rhizoma; and Gr: Ginger root.6 Evidence-Based Complementary and Alternative Medicine expressed in keratinocytes present in the epidermis, fbroblast-like cells, and nerve fber-like structures in the dermis.Tey were predominantly expressed in the upper epidermis.Te expression of MOR was signifcantly enhanced (p < 0.01) compared to the control group, and AYZ treatment reversed the increased MOR expression in the epidermis and spinal cord (p < 0.5) (shown in Figure 4(a), 4(b)).Te MOR expression in the skin was verifed by western blotting (shown in Figure 4(e)).In contrast, KOR expression in the model group was signifcantly decreased compared to the control group (p < 0.01).However, the KOR expression in the epidermis and spinal cord was reversed following AYZ treatment compared to the model group (p < 0.5) (shown in Figure 4(c), 4(d)).

AYZ Attenuated DNCB-Induced p-ERK and p-AKT Activation in the Spinal Cord.
To investigate the underlying mechanisms mediating the antiitch efects of AYZ, we assessed the p-ERK and p-AKT expressions in the spinal cord.Te DNCB treatment signifcantly activated the p-ERK and p-AKT in the spinal cord, while AYZ signifcantly inhibited their activation compared to the control, implying that AYZ attenuated DNCB-induced p-ERK and p-AKT activation in the spinal cord (p < 0.5; shown in Figure 5).

Efects of the AYZ on IL-13, MOR, and KOR mRNA
Expression in the Dorsal Skin Tissues.Following the DNCB treatment, IL-13 and MOR mRNA levels were signifcantly increased while KOR mRNA expression was signifcantly reduced (p < 0.01).However, AYZ treatment successfully reversed the mRNA levels altered by DNCB treatment (p < 0.5) (shown in Figure 6).

Discussion
Itch is a sensation of skin discomfort that usually leads to scratching behavior in terrestrial mammals [3,27], and this behavior can exacerbate skin damage.Itching and scratching induce sleep loss, severely afecting afected animals or individuals [26,28].Tis study revealed that AYZ alleviates pruritus-related AD by repairing the skin barrier, alleviating oxidative stress, and balancing peripheral MOR and KOR.
Te TSLP and IL-13, which are key factors in AD's pathogenesis, mediate pruritus, the main AD symptom.Itch disrupts the skin barrier due to the scratching, leading to exposure to pathogens and subsequently leading to infammatory fares [29].However, NA plays an important role in maintaining skin permeability through its cleavage enzyme ceramide [30].Ceramide is the main component    Evidence-Based Complementary and Alternative Medicine that forms the lipid membranes between keratinocytes with water-retaining potential that prevents water loss and pathogen infection [31].Te AYZ treatment reduced the ceramidase expression, leading to skin barrier repair function.
Oxidative stress is linked to the pathogenesis of skin, systemic, and metabolic diseases [32], whose one of the symptoms is itchiness [19,33].However, the spinal p-ERK activation is required for acute histaminergic itch and p-AKT activation for gastrin-releasing peptide (GRP)-induced itch [34].In the present study, AYZ reduced p-ERK and p-AKT activation in mice's spinal cord and dorsal horn, which implies that AYZ attenuated both histamine-dependent and -independent acute itch, possibly by decreasing the ROS accumulation in the skin.
Dry skin-induced pathological alterations during itchiness are processed in the peripheral nervous system and central nervous system, and subsequently manifest as exaggerated scratching behaviour, which is translated into surrogate markers of sustained stress due to the impairment of the HPA axis function [35].Subsequently, CRF signaling leads to an opioid receptor imbalance [36,37].In chronic pruritic diseases, such as atopic dermatitis, lichen simplex chronicons, and prurigo simplex, the epidermal MOR is signifcantly unregulated, which changes the epidermal nerve fber morphology [38].However, the MOR and KOR knockout mice scratch less [39].Te present study established that the MOR protein levels are higher in the epidermis and spinal cord in AYZ-treated mice, while the KOR protein levels are lower than in control.Tis is consistent with a previous study where a KOR agonist, nalfurafne hydrochloride, signifcantly suppressed the scratching behavior [40].Besides, Western blotting with an anti-MOR1 antibody recognizing the N-terminus yielded double bands, including MOR1D.Although the fndings in our study did not distinguish among MOR1 isoforms, they implied that the peripheral MOR, and specifcally MOR1D and KOR, were involved in itch-related behaviors in mice.
Itch is an active process initiated by MOR1D-mediated activation of GRPR [41].GRP is an itch-specifc peptide released from the primary aferents, activating the GRPR in the spinal cord in response to pruritic stimuli [42].Spinal opiates induce itch through MOR1D and GRPR heterodimerization [41].In addition, the efects of GRP are dependent on p-AKT activation.

Conclusion
In conclusion, AYZ inhibited histamine-dependent and -independent chronic itch in mice with DNCB-induced AD by skin repairs, antioxidation, and mediating the peripheral MOR and KOR balance.AYZ treatment repairs the skin barrier by reducing ceramidase expression and decreases ROS accumulation by reducing p-ERK and p-AKT activation.Te activation of the HPA axis function leads to the binding of MOR1D and GRPR heterodimerization in the spinal cord.AYZ inhibits the activation of the HPA, adjusting the balance of MOR and KOR.Tese fndings will contribute to the knowledge on efective treatment for pruritus-related AD.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Ethical Approval
Te experimental protocol for this study was approved by the Committee on the Ethics of Animal Experiments of the Hubei University of Chinese Medicine.All animals were maintained in accordance with the guidelines outlined by the Chinese legislation on the ethical use and care of laboratory animals.All eforts were made to minimize both animal sufering and the number of animals used to produce reliable data.Evidence-Based Complementary and Alternative Medicine

Figure 4 :Figure 5 :Figure 6 :
Figure 4: Immunofuorescence staining showing the MOR expression in the (a) epidermis and (b) spinal cord at ×200 magnifcation.Immunohistochemical staining showing the expression of KOR in the epidermis and spinal cord at ×200 magnifcation (c-d).(e) Te MOR proteins expression in the epidermis was determined using Western blot analysis.## p < 0.01, vs. the control group.* p < 0.05 vs. the model group.

10
Dong Rui Pharmaceutical Co., Ltd.(China).DNCB and olive oil were purchased from Shanghai McLean Biochemical Technology Co., Ltd.All enzyme-linked immunosorbent assay (ELISA) kits were obtained from Shanghai Fusheng Industrial Co., Ltd.

Table 1 :
Compounds detected and identifed in AYZ formula.