Protective effects of an aqueous extract of Protaetia brevitarsis seulensis larvae in an ovalbumin-induced murine model of asthma

ABSTRACT Asthma is a disease-related allergic response characterized by the accumulation of inflammatory cells in airway tissue. Protaetia brevitarsis seulensis larvae are known for their nutritional value and have been used in food; however, it is unknown whether they exert protective effects in a murine ovalbumin (OVA)-induced asthmatic model. We investigated the protective effects of an aqueous extract of P. brevitarsis seulensis larvae (PBE) against OVA-induced asthma by performing histopathological analysis and measuring inflammatory mediators in bronchoalveolar lavage fluid using enzyme-linked immunosorbent assay and flow cytometry. Asthmatic symptoms were attenuated by PBE, as indicated by decreased inflammation in lung tissue and decreased levels of inflammatory cells, inflammatory cytokines, and Th2 cell activation. Additionally, four out of six major metabolites isolated from P. brevitarsis seulensis attenuated Th2 cell activation, indicating that PBE attenuates asthmatic inflammation by regulating Th2 cell activation. Therefore, our findings support PBE as a pharmacological candidate for asthma treatment.


Introduction
Asthma is characterized by airway obstruction, hyper-responsiveness, and inflammation, and almost 7.5% of the global population suffers from asthma (McCracken et al., 2017).As its incidence continues to increase in children and adults, an additional 100 million patients with asthma are estimated to be diagnosed by 2025 (Bahadori et al., 2009).Inflammation is a major hallmark of several diseases, and its manifestation in allergic asthma is characterized by airway eosinophil accumulation (Liu et al., 2013).Therefore, therapeutic approaches for inflammatory diseases, such as asthma, have been developed to eliminate or mitigate inflammatory cells (Murdoch & Lloyd, 2010).Th2 cytokines, such as interleukin (IL)-4, -5, -6, and -13, are related to allergic reactions and a heightened Th2 immune response is also connected to allergic diseases (Ngoc et al., 2005).Furthermore, in the case of asthma, activation of Th2 lymphocytes has been detected in the airway, and Th2-mediated cytokines are associated with eosinophilic inflammation (Brightling et al., 2002).
Historically, humans have used insect components, such as bodies, larvae, eggs, eggshells, and secretions as chemical materials, food, and medicines (Feng et al., 2009).The use of diverse insect species has been recorded for a long time, and various resources used for therapeutic purposes have been obtained from insects (Lokeshwari & Shantibala, 2010).Protaetia brevitarsis seulensis (Coleoptera: Scarabaeidae: Cetoniinae) is a whitespotted flower chafer found in Eastern Asia, and its larvae are used in functional food (Lee et al., 2017) and traditional medicine (Yeo et al., 2013).This insect is known to possess a high protein content (44-58%) (Baek et al., 2021), and its larvae have been registered as a food ingredient by the Ministry of Food and Drug Safety of Korea (Nam, Kang, et al., 2022).Various effects of P. brevitarsis seulensis larvae have recently been reported, including protection against hepatotoxicity (Lee & Bae, 2021), antioxidant activity (Choi et al., 2021), and anti-obesity effects (Ahn et al., 2019).
Furthermore, therapeutic effects of PBE were previously detected on benign prostatic hyperplasia model (Seo et al., 2021), osteoporosis (Choi et al., 2023) and protective effects on testis damage induced radiation therapy (Nam, Kang, et al., 2022) and neurodegenerative disease (Lee et al., 2021) were also demonstrated.
Despite studies on the protective effects of P. brevitarsis seulensis larvae against inflammation, the effects of the same sample on asthma have not yet been investigated.In this study, we aimed to demonstrate the anti-asthmatic effect of P. brevitarsis seulensis larvae in an ovalbumin (OVA)-induced asthmatic model.In addition, the anti-asthmatic effects of specific metabolites from P. brevitarsis seulensis were evaluated.

Preparation of the aqueous larval extract
Dried P. brevitarsis seulensis larvae were obtained from Kwang Myong Dang Co. (Ulsan, Republic of Korea) and morphologically and genetically identified as previously described (Lee et al., 2021).The samples (manufacturer's no.: K2281201707) were deposited in the Korean Herbarium of Standard Herbal Resources (index herbarium code: KIOM) at the Korea Institute of Oriental Medicine, Naju, Republic of Korea (medicinal ID:2-18-0111).Dried P. brevitarsis seulensis larvae (887.4 g) were ground, and an aqueous extract was obtained with 15 L distilled water by reflux extraction (100 ± 2°C) for 3 h.The extract was filtered, vacuum-evaporated, freeze-dried to yield 242.8 g of total extract (27.4%), and stored at −20°C for further analysis.

Animal experimental procedure and design
Seven-week-old female specific pathogen-free BALB/c mice were obtained from Dooyeol Biotech (Seoul, Republic of Korea) and maintained at 22 ± 2°C (humidity 55 ± 15%) with a 12 h light-dark cycle for 1 week.Food and water were provided ad libitum.After acclimatization and quarantine, mice were divided into five groups.The groups were as follows: normal control (sham), OVA-induced asthma group (OVA), OVA with 5 mg/kg dexamethasone-treated group (DEX), OVA with 200 mg/kg sample-treated group (PBE200), and OVA with 400 mg/kg sample-treated group (PBE400).All animal care and experimental procedures were approved by the Animal Care Committee of the Korean Institute of Oriental Medicine (20-002).Aluminum hydroxide (2 mg) and OVA in 50 µg phosphate-buffered saline were injected into BALB/c mice for 1 week to generate an animal model of asthma.After the first injection (i.p) of OVA solution, 25 µg OVA (50 µL) was administered intranasally on d 14, 15, 16, and 17.Mice were anesthetized 18 h after the last OVA treatment, and blood was collected from the abdominal aortic vein.The samples were orally administered at doses of 200 and 400 mg/kg for 7 d (d 11-17).DEX (5 mg/kg) was used as the control, using the same procedure.

Histology
Lung tissues were isolated from the animals and fixed in 10% formaldehyde for histological analyses.Fixed tissues were washed in water for 8 h, embedded in paraffin, and sectioned into 4 μm sections.Histological changes in the lung tissue were evaluated using hematoxylin & eosin staining to detect lung inflammation and periodic acid Schiff staining (IMEB Inc., San Marcos, CA, USA) to detect changes in mucus production.The sample slides were observed using a Pannoramic DESK slide scanner (3DHISTECH, Budapest, Hungary).

Bronchoalveolar lavage fluid collection and differential cell counting
Mice were anesthetized with alfaxalone (85 mg/kg; Jurox Pty Ltd., Rutherford, New South Wales, Australia) and 10 mg/kg xylazine (Rompun®; Bayer Korea, Seoul, Republic of Korea).Bronchoalveolar lavage fluid (BALF) was collected from each mouse by injecting cold phosphate-buffered saline (1 mL) into the lungs using a tracheal cannula.Isolated BALF samples were used for cell counting by transferring them onto slides using Cytospin (1,500 rpm, 10 min; Hanil, Deajeon, Republic of Korea).Glass slides were stained with Diff-Quik solution (Sysmex Suisse AG, Horgen, Switzerland) for cell counting.

In vitro Th2 polarization assay
Th2 differentiation was detected as previously described (Nam, Lee, et al., 2022).A naïve CD4+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to isolate splenic CD4+ T cells from splenocytes.Isolated cells were incubated with anti-CD3/CD28-coated particles with or without IL-2, IL-4, and anti-INFγ antibodies.During Th2 polarization, PBE samples and isolated metabolites were treated.After 5 d, Th2 differentiation and activation were analyzed by examining the proportion of GATA3+ CD25+ T cells.The isolated cells were cultured with PBE (200 and 400 µg/ mL) and the six isolated metabolites (50 µM) under Th2 polarization conditions.

Statistical analyses
All data are expressed as mean ± standard deviation.All statistical analyses were performed using GraphPadPrism 8.0 (GraphPad Software, San Diego, CA, USA).One-way analysis of variance followed by the Student-Newman-Keuls post-hoc test was performed to prove significant differences.In all cases, the differences were considered statistically significant at p < 0.05.

PBE attenuated histological changes in lung tissue
The anti-asthmatic effects of PBE were evaluated using an OVA-induced airway inflammation model.Hematoxylin & eosin staining confirmed a dose-dependent decrease in immune cell accumulation in the airways of the PBE-treated group compared with that in the OVA-induced inflammation group (Figure 1A).Furthermore, increased goblet cell hyperplasia and mucus overproduction were observed in the OVA-induced inflammation group as the inflammation progressed (Figure 1B).In contrast, PBE treatment inhibited inflammation in a dose-dependent manner.DEX, used as a positive control, attenuated inflammation compared to that in the OVA-induced inflammation group in histopathological analysis.

PBE reduced the number of inflammatory cells in BALF
BALF was isolated from an animal model to determine the composition of inflammatory cells, which significantly increased the number of cells in BALF in the OVA-induced inflammation group (Figure 2).The PBE-and DEX-treated groups showed significantly decreased total cell counts compared with the OVA-induced inflammatory group ( #### p < 0.001 compared to the sham group.*p < 0.05, ***p < 0.005 compared to the OVA group) (Figure 2A).Among the various inflammatory cells, the eosinophil count was confirmed to have the same tendency as the total number of cells (Figure 2B and 2C).PBE and DEX treatment significantly reduced the number of eosinophils induced by OVA in BALF, and PBE treatment had a dose-dependent tendency.

PBE reduced inflammatory cytokines in BALF
The levels of the major inflammatory cytokines, IL-4 and −5, were significantly elevated in the BALF of the OVA treatment group (Figure 3) compared with that in the sham group.Both cytokines were decreased in the PBE and DEX groups, and all values showed significant differences when compared with those in the OVA-induced inflammation group ( #### p < 0.001 compared to the sham group, ***p < 0.005 and ****p < 0.001 compared to the OVA group).

PBE attenuated CD4+ T cell activation in the lung tissue
In previous studies, we confirmed that PBE treatment regulates allergic inflammation by attenuating eosinophils and cytokines.Therefore, we evaluated Th2 cell activation to determine the anti-asthmatic effect of PBE.In the OVA-induced inflammatory group, a significantly increased percentage of activated CD4+ T cells were detected compared with that in the sham group (Figure 4) ( #### p < 0.001 compared to the sham group, ***p < 0.005 and ****p < 0.001 compared to the OVA group).PBE treatment resulted in a dose-dependent decrease in the CD4+ T cell population compared with that in the OVA-induced inflammation group, and DEX treatment also revealed the same tendency.2

PBE regulated Th2 cell activation in spleen tissue
As the inhibitory effect of PBE on CD4+ T cell activation was confirmed, we chose CD25 + and GATA3 + cell populations as subsequent targets.When we measured the CD25 + cell population after PBE treatment, the population decreased in a dose-dependent manner compared with that in the Th2 Group (Figure 5A).This tendency was also observed in GATA3 + cells (Figure 5B).For GATA3 + , all PBE-treated groups were significantly different from the Th2 group ( #### p < 0.001 compared to the sham group, *p < 0.05, ** p < 0.01 and ****p < 0.001 compared to the OVA group).

Discussion
Although asthma is caused by multiple biological mechanisms and many triggers can induce bronchoconstriction with allergic responses, allergic sensitization is considered a major cause of asthma progression (Diao et al., 2017;Ishmael, 2011).The Th2 response triggered by allergen inhalation leads to the expression of Th2-type cytokines.Released cytokines, including IL-4 and −5, induce bronchoconstriction, mucus overproduction, and recruitment of inflammatory cells, such as eosinophils, as well as their activation and survival.In this study, we investigated the efficacy of PBE against asthma and its underlying mechanisms using a well-established allergic airway inflammation model induced by OVA sensitization.First, we obtained histopathological evidence of the effect of PBE on asthmatic inflammation.Increased inflammatory cell accumulation and goblet cell hyperplasia were detected in the airways of an OVA-induced asthma model because of asthmatic inflammation.Thus, PBE treatment attenuated OVA-induced airway inflammation in a dosedependent manner.BALF is a sample obtained by flushing the lungs with physiological solutions used to study respiratory diseases (Ho et al., 2013).It was found that the accumulation of inflammatory cells, particularly eosinophils, was closely associated to asthmatic inflammatory response and its severity (Walford & Doherty, 2014;Xu et al., 2020).To confirm that PBE treatment inhibited the recruitment of inflammatory cells, especially eosinophils, differential counts were performed using BALF.In this study, significantly elevated eosinophil levels were observed in an OVA-induced asthmatic disease model, and a dosedependent attenuating effect of PBE was detected.These data confirmed the inflammatory response in the OVA-treated model and the effect of PBE on this disease.
To investigate if the effects of PBE on asthmatic inflammation were similar to what was previously reported, we determined the effect of PBE on inflammatory cytokines (IL-4 and -5) in BALF.Significantly increased levels of IL-4 and -5 were detected in the OVA-induced asthmatic inflammation model, whereas decreased levels were observed in the PBE-treated groups.IL-4 and −5 are Th2 cytokines necessary for IgE production and eosinophil development, respectively (Huang et al., 2023;O'Byrne et al., 2004).
We investigated the effect of PBE on T cell activation in mouse tissues to prove the hypothesis that the effect of PBE on asthmatic inflammation is related to Th2 cell activation.A dose-dependent decrease in activated CD4+ T cells was confirmed in the PBE-treated groups when compared to the OVA-induced asthmatic inflammation group, which showed significantly increased levels of activated CD4+ T cells.CD4+ T cells act as important regulators in inflammatory diseases and activate Th2 cells by encountering specific antigens.GATA3 is a specific transcription factor for Th2 cells (Lu et al., 2011) and is an important element that promotes Th2 cell differentiation by regulating Th2 cytokine genes (Yagi et al., 2011).In the present study, significantly lower CD25 + and GATA3 + population levels were detected in the PBE-treated group than in the Th2 group.Based on these results, we confirmed that PBE treatment attenuated asthmatic inflammation by regulating major Th2 cell activation targets.
To determine the effect of PBE on asthmatic inflammation, six major metabolites were isolated based on the UPLC fingerprint of P. brevitarsis seulensis (Lu et al., 2011).Flow cytometric analysis indicated that four PBE metabolites, namely adenine (1), hypoxanthine (2), and uridine (3), significantly suppressed Th2 cell activation.These three major-metabolites attenuated Th2 cell activation in the asthmatic inflammation model.
In conclusion, we confirmed that the effect of PBE on asthmatic inflammation occurs via the regulation of Th2 cell activation.The anti-asthmatic effects of PBE are considered to be derived from the complex metabolic action of the metabolites in PBE.The three major-metabolites, adenine, hypoxanthine, and uridine, are considered powerful effectors; however, their detailed metabolism in asthma requires further investigation, highlighting a limitation to our present study.In the case of uridine, it was demonstrated that it has therapeutic effects in asthma by suppressing the MAPK and NF-κB pathways (Luo et al., 2021); the contribution of adenosine was also studied in asthma, and it was related to its receptors (Brown et al., 2008).A further limitation to our study is that we only focused on inflammatory marker levels to evaluate the effect of PBE on asthma.Despite this, our study is valuable because it demonstrates the effect of PBE and its isolated metabolites on an asthmatic animal model, including inflammatory cell composition and Th2 cytokines.Therefore, our findings support the use of PBE as a

Figure 1 .
Figure 1.Histopathological changes in lung tissue due to the effects of Protaetia brevitarsis seulensis larvae extract (PBE).Paraffin sections of lung tissue were stained using hematoxylin & eosin (A) and Periodic acid-Schiff staining (B).Sham, normal control group; OVA, ovalbumin-induced airway inflammation group; PBE200, OVA group treated with 200 mg/kg of PBE; PBE400, OVA group treated with 400 mg/kg PBE; DEX, OVA group treated with 5 mg/kg dexamethasone.