The effective approach to discover innovative drugs will ask natural products for answers because of their complex and changeable structures and multiple biological activities. Inhibitory kappa B kinase beta (IKKβ), known as IKK2, is a key regulatory kinase responsible for the activation of NF-κB through its phosphorylation at Ser177 and Ser181 to promote the phosphorylation of inhibitors of kappa B (IκBs), triggering their ubiquitination and degradation to active the nuclear factor kappa-B (NF-κB) cascade. Chemical inhibition of IKKβ or its genetic knockout has become an effective method to block NF-κB-mediated proliferation and migration of tumor cells and inflammatory response. In this review, we summarized the structural feature and transduction mechanism of IKKβ and the discovery of inhibitors from natural resources  and chemical synthesis . In addition, the biosynthetic pathway of novel natural IKKβ inhibitors and their biological potentials were discussed. This review will provide inspiration for the structural modification of IKKβ inhibitors based on the skeleton of natural products or chemical synthesis and further phytochemistry investigations. In 1986, Sen and co-workers focused on the work to discover novel nuclear factors that regulated the expression of immunoglobulin G (IgG) in B cells NF-κB comprises five members of the family Rel, such as RelA p65), RelB, c-Rel, NF-κB1 (p50), and NF-κB2 (p52) , RelB, cBecause activated IKKβ can rapidly phosphorylate IκBs, chemical inhibition of IKKβ or its genetic knockout (KO) has become an experimental approach to block NF-κB mediated proliferation and migration of tumor cells and inflammatory response Figure  (PDB: 4KIK), IKKβ contains the N-terminal kinase domain , the C-terminal kinase domain , the ubiquitin-like domain , the scaffold dimerization domain , and the NEMO-binding domain . Compared with IKKα, IKKβ has a ULD following the KD C, which is necessary for IKKβ functional activity and is important for its substrate specificity Figure ) The IKK complex includes IKKα, IKKβ, and IKKγ (NEMO), of which IKKα and IKKβ are the catalytic subunits, and IKKγ is the regulatory subunit Single nucleotide polymorphisms (SNPs) are the most abundant form of deoxyribonucleic acid (DNA) variation in the human genome, which influences disease-related genes and non-coding RNAs via enhancing the promoter activity and specific nuclear protein-binding affinity Figure 2), including canonical pathway depended on IKKβ and non-canonical pathway depended on IKKα There are two principal pathways to activate NF-κB , diabetes, non-alcoholic fatty liver disease (NAFLD), are associated with IKKβ via blocking the NF-κB cascade.Cancer is a terrible disease that has existed for more than 200 million years and first appeared in humans more than one million years ago. Cancer, different from infectious, parasitic, and other diseases caused by many environmental factors, is mainly caused by abnormal changes of its own normal cells under the long-term action of internal and external factors Pancreatic cancer, first described in 1761, belongs to the member of malignant tumors with a high mortality 16), MLN120B, and shikonin, have been used in the treatment of other cancers as well in vitro and in vivo via preventing the IKKγ-binding domain (NBD) of IKKβ from entering the hydrophobic pocket of IKKγ , resulting the inhibition of proliferation, migration, and invasiveness In addition, IKKβ inhibitors, e.g. abietic acid, alantolactone , a natural product from Tripterygium wilfordii, inhibited the IKKβ activity to attenuate the Aβ cytotoxicity, triggering the neuroprotective effect in AD Alzheimer's disease (AD) is a neurodegenerative disease represented by impaired learning, memory, and cognitive abilities Spinal cord injury (SCI) is a permanent motor and sensory deficits characterized by the invasive degeneration of the spinal cord tissue caused by the mechanical trauma to the neurons and surrounding vasculature in vitro, but also attenuated insulin resistance and enhanced plasma levels of adiponectin in the high-fat diet (HFD)-induced animal model Metabolic diseases are becoming increasingly common in modern society, such as diabetes, NAFLD, and non-alcoholic steatohepatitis (NASH) due to people's lifestyles and irregular circadian rhythms ob/obLep obese mice 26) decreased insulin resistance and lipid abnormalities and increased the adiponectin level to attenuate the adiposity As a major public health issue, diabetes belongs to one of the family metabolic diseases characterized by hyperglycemia caused by insulin resistance S-glutathionylation was the potential mechanism of NAFLD Besides, emerging evidence has indicated the overexpression of IKKβ in liver diseases, including NAFLD, NASH, and liver fibrosis b]pyridazin-3-yl]-2-fluoro-N--4-fluoropyrrolidin-2-yl])methylbenzamide th day of gestation because of massive apoptosis of hepatocytes, therefore, these clinical trials have to be terminated Chemical inhibition of IKKβ is to the benefit of many other diseases, such as osteolysis, arthritis, premature birth, radiation injury, and allergy 57) in 2001, therefore, its development has become a hot topic. To date, IKKβ inhibitors have been found from various pathways, including natural products and chemical synthesis, herein, advances of IKKβ inhibitors are summarized.IKKβ potentials have been paid more and more attentions in the prevention and treatment of various diseases as the discovery of the first IKKβ inhibitor SPC839 , naturally occurred in Tanacetum parthenium, belongs to the family of 5,10-seco-eudesmane type sesquiterpenoids, and its inhibitory effect towards IKKβ was first reported by Benjamin in 2001 2), could covalently bind to Cys179 of IKKβ, and inhibit its activity, triggering its protective effect toward atherosclerosis in HFD-fed ApoE-/- mice Sigesbeckia glabrescens is a species of the genus Sigesbeckia first recorded in Xinxiu Herba written by Su Jing (Tang Dynasty) 3, Figure 4) significantly displayed an inhibitory effect against IKKβ via covalently binding to amino acid residue Cys46 in the KD N region of IKKβ with the olefinic bond of an α,β-unsaturated lactone 4), a natural sesquiterpenoid isolated from the genus Helianthus, displayed an anti-triple-negative breast cancer effect 4 , Wang et al. found that 4 could bind to IKKβ through the covalently binding of Cys179 in IKKβ 5, Figure 5), towards IKKβ, and optimized its structural to afford a library of its analogues, such as 6-106 and 7 displayed significantly inhibitory effects Parthenolide , isolated from Sphaeranthus indicus, is a sesquiterpene lactone with a 6/6/5 skeleton and possesses various bioactive activities 11) could suppress LPS-mediated NF-κB activation 12), a pseudoguaiane-type sesquiterpenoid from the genus Inula, could induce the IKKβ degradation to suppress the NF-κB pathway, thus causing apoptosis of colon cancer cell 13), zerumbone (14), and 1,6-O,O-diacetylbritannilactone (15) against IKKβ as well Inula helenium known as “Tu mu xiang” in Chinese, and found that alantolactone (16), isoalantolacton (17), and dehydrocostus lactone (18) exerted antitumor activities against glioblastoma through targeting IKKβ 7-Hydroxyfrullanolide , isolated from Ainsliaea macrocephala, displayed an anti-inflammatory potential in Raw264.7 cells after exposure to LPS 19) and linked a biotin to afford a probe Bio-19. Using this probe to fish the binding proteins, Lei et al. found that ainsliadimer A (19) could selectively suppress IKKα/β via covalently binding to Cys46 in the activation loop of IKKα/β, meanwhile ainsliadimer A (19) had a good affinity (Ki = 30.25 nM) and specific reactivity (Kinact = 7.74 min-1) with IKKβ For the sesquiterpenoid dimer, Lei's group found that ainsliadimer A  displayed a remarkable inhibitory activity against TNF-α-medicated NF-κB activation. The investigation on its action mechanism demonstrated that 17-acetoxyjolkinolide B (20) could suppress the phosphorylation of TNF-α-medicated IκB and the nuclear translocation of p65 via inhibiting IKKβ (IC50 = 300 nM) Alternaria brassicicola found alterbrassicene A (21) sharing a 5/9/4-fused carbocyclic skeleton with a rare fused 2-cyclobuten-1-one motif Figure 9. It is worth noting that alterbrassicene A (21) displayed an inhibitory effect against IKKβ (IC50 = 2.48 μM), and it could bind to KD N and C lobes of IKKβ through hydrogen bond interactions with Leu21 and Arg31 22, Figure 10) is a characteristic ent-labdane diterpenoid of Andrographis paniculata (Burm. f) Nees, a traditional medicinal plant in China, and possesses multiple bioactivities, such as antitumor and anti-inflammatory effects 22) inhibited the IKKα/β activity to reduce the increase of TNF-α mediated ICAM-1 expression and the activation of TNF-α mediated NF-κB 23), a diterpene quinone from traditional Chinese herb Salvia miltiorrhiza, has multiple potentials on various diseases Sagittaria trifolia resulted in 11 diterpenoids, and subsequent study on anti-proliferative effects demonstrated the inhibitory potential of sclareol (24) toward IKKβ 25) was reported as well, and the result of molecular docking demonstrated its mechanism of action towards IKKβ The root of dried Celastrus orbiculatus, Azadirachta indica, and Codonopsis lanceolata. Celastrol  is a quinone methide triterpenoid isolated from Celastrus orbiculatus, and showed inhibitory effects against TNF-α-, IKKβ-, or MEKK1-mediated NF-κB activation on the basis of NF-κB luciferase activity 26) could dose-dependently suppress the IKKβ activity, and the mutation of Cys179 in the activation loop of IKKβ abolished its effect, indicating that it targeted amino acid residue Cys179 of IKKβ to inhibit the activation of NF-κB 27), a pentacyclic triterpene, interacted with Cys179 to inhibit the IKKβ activity 27 afforded a new synthetic triterpenoid inhibitor 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid imidazolide (28) Azadirachta indica, nimbolide (29), promoted the apoptosis induced by chemotherapeutic agents in tumor cells Triterpenoids are common type compounds in natural products, so far, some of triterpenoids have been reported from 29 demonstrated that it could modify Cys179 of IKKβ to suppress the IKKβ activity, which was supported by amino acid mutation Cys179Ala 30) from Poria cocos, escin (31), acetyl-11-keto-β-boswellic acid (32) from the genus Boswellia, pristimerin (33) from Menispermum dauricum, oleanolic acid acetate (34) from Vigna angularis, deacetyl ganoderic acid (35) from Ganoderma lucidum, 3-O-acetylrubianol C (36) from Rubia philippinesis, and lancemaside (37) from Codonopsis lanceolata inhibited the IKKβ activity or regulated the phosphorylation of IKKβ 34) exerted an anti-inflammatory effect via inhibiting IKKα/β to reduce the production of embryonic alkaline phosphatase and pro-inflammatory cytokines, including MCP-1, IL-1, IL-8, VCAM-1, and ICAM-1 37), a pentacyclic triterpenoid glucose isolated from Codonopsis lanceolata, inhibited the LPS-mediated inflammatory response in Raw264.7 cells through blocking the IKKβ activation The study on the mechanism of action of Glycyrrhiza inflata is a traditional Chinese medicine to treat inflammatory related diseases, and its major chemical constituents are flavonoids and triterpenoids 38, Figure 12) and licochalcone A (39), isolated from the genus Glycyrrhiza, belong to the chalcone family and possess multiple bioactive activities, such as anti-inflammatory and anti-tumor effects 38) against human T lymphocyte activation α,β-unsaturated ketone moiety in isoliquiritigenin (38) 39) inhibited IKK complex activation and IκB degradation via the covalently binding to amino acid residue Cys179 of IKKβ 40), naringenin (41), and pinocembrin (42), could interact with amino acid residues Thr23, Glu97, Cys99, and Asp166 in the ATP binding domain through hydrogen bonds to suppress the IKKβ activity Figure 15), including berberine (43), vinpocetine (44), cryptopleurin (45), matrine (46), tetrandrine (47), piperlongumine (48), daphnipaxianine A (49), pipernigamides E-G (50-52), tulipiferamide A (53), herbimycin A (54), himalesine (55), and ellipticine (56) 55, Figure 14), isolated from Daphniphyllum himalense, possesses an unprecedented 6/5/6/7/5/6 skeleton, and its proposed biosynthetic pathway was discussed Daphniphyllum himalense, yielded intermediate i, followed by Baeyer-Villager oxidation, ring opening, and esterification to afford 55So far, fourteen natural alkaloids have been reported to possess inhibitory potentials towards IKKβ (54) is an acetomycin antibiotic that possesses a potent Src tyrosine kinase inhibitory activity 54) could reduce the iNOS expression and chemokine production via inactivating NF-κB. Subsequently study demonstrated that herbimycin A (54) could bind to Cys59 in the KD region of IKKβ, resulting in the inhibition of the IKKβ activity, which was supported by the experiment of amino acid mutation (Cys59Ala) 56, Figure 15), an alkaloid from Ochrosia elliptica174. Ellipticine (56) could suppress the IKKβ activity through directly binding to IKKβ, resulting in the inactivation of NF-κB signaling pathway on the basis of kinase and binding experiments, and the result of amino acid mutation suggested that Cys46 in the activation loop of IKKβ played a role in the inhibition of ellipticine (56) 174.Herbimycin A , and its effect in an arthritis animal model 57) on the IKKβ activity, which led to a new era of the development of IKKβ inhibitors. Bingham et al. summarized the structural characteristic of SPC839 (57) and built the aminopyrimidine scaffold to afford a series of aminopyrimidine analogues, such as 58. Although this compound had a better potential than SPC839 (57), its selectivity was not satisfactory. Afterwards, they tried to modify the substituent moiety at the right of 58 through the induction of the electron-withdrawing  or electron-donating (e.g. -OCH3 and -CH3) groups, and found that the electron-withdrawing group was in favor of the IKKβ selectivity  63 and 64 with an IKKβ selectivity 65, whereas its potential had not been improved 1, respectively, and got two representative inhibitors 66 and 67 that possessed a low nanomole level of inhibitory potentials 66 was applied for the investigation on LPS-mediated inflammation in vitro, and showed a remarkable anti-inflammatory effect In 2001, Bhagwat et al. has reported a small molecule with an aminopyrimidine core, SPC839  displayed a potent inhibitory activity against IKKβ (IC50 = 1500 nM) 69-7473 showed a low nanomole potential (IC50 = 40 nM), its performance was not as good as expected at cellular level (IC50 = 5000 nM) 75 afforded from the structure of 74 not only displayed a low nanomole inhibitory effect (IC50 = 8.5 nM), but also possessed reasonable aqueous solubility (0.12 mg/mL in buffer), excellent permeability, and orally bioavailability Figure 18) and optimized this type IKKβ inhibitor through the modification of R1 and R2 substituent groups 1 substituent group, and followed by the modification to produce selective inhibitors 76-8076-78 displayed anti-inflammatory effects against LPS-mediated TNF-α production in vivo, which provided a thought for the development of novel IKKβ inhibitors.In 2002, Murata et al. performed the high-throughput screening based on the library of Bayer compounds, and found that 2-amino-3-cyano-4-aryl-6-(2-hydroxy-phenyl)pyridine analogue (a)quinoxaline  that was a selective IKKβ inhibitor (IKKα IC50/IKKβ IC50 = 13.3) via binding to the allosteric site of IKKβ 81) possessed an excellent pharmacokinetics and in vitro and in vivo anti-inflammatory effects through the release of LPS-mediated inflammatory factors 82-8883 not only possessed a low nanomole potential, but also displayed a good selectivity (IKKα IC50/IKKβ IC50 = 30). Meanwhile, the delightful pharmacokinetic property of 83 led to its perfect anti-inflammatory effect for reducing the release of LPS-mediated TNF-α in vivoIn 2003, Burke and co-workers synthesized 4-(2′-aminoethyl)amino-1,8-dimethylimidazo, and found the advantage of the sulfonamide moiety for the IKKβ inhibitory potency 95 and 96. These two compounds shared the same inhibitory potential against IKKβ and the special selectivity , which was further supported by the molecular stimulation Previously, Christopher's group disclosed 2-amino-3,5-diarylbenzamide inhibitors  in the inhibition of the IKKβ activity, and used it to confirmed the hypothesis 97 and got the more potent inhibitors than the parent 97, such as 98-10098 not only showed the potent inhibitory effect but also possessed a good pharmacokinetics property. Preclinical results indicated the more potent anti-inflammatory (2.5 fold) and anti-tumor (4.3 fold) effects of 100 than the parent 97Recently, Radhakrishnan and co-workers found the hypothesis that IKKβ served as the signaling node to regulate the transcription and translation through the NF-kB and mTOR/p-S6K/p-eIF4EBP axis in tumors 101-103 , and found that 101-103 had a common motif, where the orientation of a primary amide was restricted by an adjacent hydrogen-bonding functionality, therefore, they tried to explore a new template based on the benzamide skeleton for developing IKKβ inhibitors 104-109In 2007, Christopher and colleges analyzed recent IKK inhibitors, e.g. 110 and 111 , Baxter et al. optimized the thiophene core to produce a series of analogs 112-114 showed low nanomole inhibitory activities against IKKβ and good pharmacokinetics 114, a novel class of tricyclic furan derivatives were designed and synthesized by Sugiyama and colleagues, such as 3-[(aminocarbonyl)amino]-benzothieno-furan-2-carboxamide derivatives 115-117b]furan to overcome the low metabolic stability yielded a series of 6-alkoxy derivatives, meanwhile improved oral bioavailabilities of inhibitors. The potent inhibitory activity of these derivatives resulted from an intramolecular non-bonded S…O interaction, except for four hydrogen bond interactions with Glu97, Tyr98, and Cys99, which was further supported by the co-crystal result e]indazole core to afford a library of IKKβ inhibitors, such as 118 and 119, while having unsatisfying inhibitory effects In 2004, through the high throughput screening to find two potential thiophenecarboxamides 120, Figure 24) with a thiazolidine-2,4-dione skeleton and its potential in the inhibition of IKKβ (IC50 = 1500 nM), therefore, they tried to optimize this core via exploring the electron push-pull effect of the right moiety and analyze the SAR 121 and 122 showcased more potent inhibitory effects than the parent (about 4-6 fold) 123 with the cyano group at the right side possessed the sub nanomole level in the inhibition of IKKβ, meanwhile remaining the pharmacokinetics property and anti-inflammatory effect in vitro, which was further explained through the molecular stimulation showing the role of Arg47, Arg55, Trp58, and Leu91 for its potential in vivo experiment, 123 enhanced the mortality of septic shock induced mice , demonstrating its protective potential for septic shock Roh's group found a lead compound  sharing the rhodamine scaffold from druggable and transformative points of view 125-127, and the result of the kinase assessment indicated the selectivity of 127 in the inhibition of the IKKβ inhibitors as well 125-127 did not display satisfactory pharmacokinetic property. Skin's group subsequently optimized its metabolic property through the modification of the right moiety based on the structure of 127, resulting in the production of 128 and 129129 remained a selectivity and inhibitory effect of the rhodanine type inhibitors, while possessed a good metabolic stability  and pharmacokinetic character In 2012, Song et al. employed high-throughput screening to assay the inhibitory effect of the in-house library of compounds, in order to develop the new generation of IKKβ inhibitors In summary, we described the structure and transduction mechanism of IKKβ and its inhibitors from natural products  and chemically synthesized . Furthermore, inhibitory potentials of IKKβ are associated with in various diseases through regulating the NF-κB pathway, including cancer, PD, diabetes, SCI, NAFLD, osteolysis, and arthritis, therefore, it has been considered a potential target. Notably, for inhibitors from chemically synthesized, pyrimidine- and pyrazine-type compounds display low nanomole inhibitory effects, which attributes to the aromatic nitrogen atom in the skeleton of pyrimidine and pyrazine due to its hydrogen bond interaction with Cys99 of IKKβ, but their physical properties (water solubility), bioavailability, and pharmacokinetic parameters remains great improving spaces. Although the clinical trial of IKKβ inhibitors, such as IMD-2560, and SAR-113945, do not show satisfactory results for the safety, efficacy, and selectivity, natural products with more complex and changeable structural skeletons offer endless possibilities for discovering IKKβ inhibitors. Among naturally occurred inhibitors, sesquiterpenoids with an α,β-unsaturated lactone moiety have great potentials because they covalently bind to amino acid residues Cys46 and Cys179 of IKKβ. However, the total synthesis of natural products with multiply chiral centers has always been a difficult problem, therefore, simplification and extraction of effective structures of natural inhibitors may be an efficient means to synthetize new type IKKβ inhibitors in the future.


Compared to pure epoxy, the lowest values of Ɛr and δ were 3.26 and 0.021 with 30 wt.% quartz fibers in the composite, while 40% less weight loss was observed which shows its better thermal stability. The mechanical characteristics encompassing tensile and bending strength were improved by 42.8% and 48.3%. In high-speed communication applications, compared to a pure epoxy composite, adding only a small quantity of quartz fiber can improve the composite material’s dielectric performance, durability, and thermal and mechanical strength.Polymer matrix wave transparent composites are used in a variety of high-speed communication applications. One of the applications of these involves making protective enclosures for antennas of microwave towers, air vehicles, weather radars, and underwater communication devices. Material performance, structural, thermal, and mechanical degradation are matters of concern as advanced wireless communication needs robust materials for radomes that can withstand mechanical and thermal stresses. These polymer composite radomes are installed externally on antennas and are exposed directly to ambient as well as severe conditions. In this research, epoxy resin was reinforced with a small amount of quartz fibers to yield an improved composite radome material compared to a pure epoxy composite with better thermal and mechanical properties. FTIR spectra, SEM morphology, dielectric constant (Ɛ Polymer radomes . Low Ɛr ications . Reinforications . Differeications .r) and dielectric loss factor (δ) of the composite material [r determines the dielectric or polarization of the composite, which is the capacitance ratio of the same size capacitor with dielectrics under vacuum. The δ defines the portion of EM waves that is consumed during its transmission through the material or medium [r = dielectric constant, f = frequency, and tanδ = dielectric loss tangent.Wave transmission performance is mostly assessed by the dielectric constant  for longer durations while retaining their mechanical and thermal stability along with the desired electric performance . Glass fr and low δ, mechanical integrity, and low moisture absorption. A fiber-reinforced composite radome material was fabricated by embedding a small fraction of quartz fibers in epoxy resin. The dielectric constant, dielectric loss, moisture absorption, mechanical properties, and thermal stability were evaluated.Extensive research has been carried out to investigate the properties of fiber-reinforced composites . SelectiThe experimental materials used in the preparation of reinforced composite samples in this research study were epoxy resin and quartz fibers. Their properties are mentioned in r and δ values of the quartz fiber/epoxy composites along with their identification are given in Using the rule of mixture (ROM) for evaluating composite properties , the dieQuartz fiber/epoxy composites were prepared according to the schematic shown in The composite laminates were taken out and then underwent a thermal curing process. The samples prepared for this study were obtained after curing. In Figure.1, “A” refers to epoxy resin, “B” refers to amine curing agent, blue arrow represents cold process (ambient) and red arrow represents hot process (heating).The experimental conditions used for preparation of the quartz fiber/epoxy composites are given in The details of the characterization methodologies used to determine the quartz fiber/epoxy composite properties are given in In this section, the experimental results are presented which include the chemical structure, morphology, electrical, mechanical, and thermal properties.−1 and 2922 cm−1), C-N-C stretching (1174.3 cm−1), C=O (1645 cm−1), symmetric and asymmetric C-O-C stretching (1099 cm−1), C-H deformation (801.2 cm−1), Si-O-Si deformation (467.7 cm−1), and Si-OH compression (891.3 cm−1 and 1005 cm−1) bands. The spectra showed polymerization of the thermoset epoxy resin [xy resin  during cxy resin  due to tr of the material increases the EM wave reflection, with an ultimate reduction in its transmission efficiency due to polarization phenomena of the medium in the external electric field [According to the law of energy conservation , the eleic field .r) and dielectric loss factor (δ) of the pure epoxy composite was 4.1, and for the quartz fiber/epoxy composite EQ-20, it was 3.31, 3.28 for EQ-25, 3.26 for EQ-30, and 3.26 for EQ-35. These measured Ɛr and δ values were different from those estimated from the rule of mixture for composite property estimation [r was 3.26 with quartz factions of 0.30 and 0.35. The results showed that Ɛr varied by 1.5% from 3.31. These decreasing values are due to the interfacial polarization of the composite. The dielectric constant became fixed and was not reduced further even with increasing quartz fiber fraction. Increasing the quartz fiber fraction packed the composite structure and reduced the local charge displacement towards the electric field. The epoxy resin embedded with quartz fibers formed a heterogeneous structure due to their organic–inorganic nature. Upon curing of the epoxy resin, space charge polarization was created with the compact interfacial boundaries and random movement of ions was restricted. Thus, ionic conductivity becomes lower with increasing dielectric dispersion in both the real and imaginary parts of Ɛr. In the composites EQ-30 (0.30) and EQ-35 (0.35), due to the relatively lower resin faction compared to EQ-20 and EQ-25, the curing reaction progressed quickly. Thus the epoxy/amine crosslinking contributed towards the dominancy of dipole relaxation over the ionic conductivity, which ultimately reduced the dielectric constant [r.The dielectric constant  had the highest tensile strength (226.15 MPa). This was a result of the effective wetting of quartz fibers by the epoxy resin and the improved interfacial bonding resulting from the greater contact area between the fibers and resin. These all contributed to increasing the tensile strength.The stress–strain curves obtained during the bend strength test are shown in The weight loss (%) of the pure epoxy and reinforced composites is shown in Apart from the epoxy composite, the quartz fiber/epoxy composites  decomposed differently with a smaller amount of weight loss. The initial decomposition of sample EQ-20 (0.20 quartz fiber) started at 245 °C; for EQ-25 (0.25 quartz fiber), it started at 280 °C; for sample EQ-30 (0.30 quartz fiber), it started at 290 °C; and for sample EQ-35 (0.35 quartz fiber), it started at 308 °C. Significant degradation in these composites was observed within the temperature range of 285 °C and 650 °C. The major weight loss was observed due to the decomposition of the epoxy resin which is a polymeric component. It was seen that increasing the fraction of quartz fibers provided better thermal stability to the composite compared to the pure epoxy composite. This was evident from the decreasing weight loss of 74.25%, 71.3%, 70.3%, and 64.15%, respectively. Increasing the fraction of quartz fibers reduced the epoxy faction in the reinforced composite.Moreover, the lower quantity of epoxy resin was efficiently cured by limiting the molecular chain’s thermal vibration up to some extent. In this manner, heat conduction becomes lower and thus, the quartz fiber/epoxy composite exhibited better thermal stability than the pure epoxy composite. Embedding a small quantity of quartz fibers into the epoxy resin increases the manufacturing cost roughly by 15 to 20% when compared to producing pure epoxy composite, all in pursuit of achieving enhanced properties.In this work, fiber-reinforced composite radome material was prepared, for communication applications, to withstand high working temperature, mechanical, and thermal stability compared to pure epoxy composites. Our research delved into the impact of embedding quartz fibers and determining their optimal quantity required to achieve enhanced electrical, mechanical, and thermal performance in epoxy composites. Quartz fiber/epoxy (EQ) composites were prepared through hand lay-up, compression molding process followed by thermal curing. FTIR analysis indicated the presence of organic and inorganic networks  while SEM morphology showed fair fiber–resin adhesion, interfacial bonding, and compact structure of EQ composites.Compared to the pure epoxy resin embedding a small fraction (0.30) of quartz fibers in epoxy, decreased the dielectric constant by 22% (4.10 to 3.21) and the dielectric loss factor by 40% (0.035 to 0.021). This addition of quartz fibers substantially reduced the polarizability of polarized molecules by decreasing their dielectric constant and transmission loss which is advantageous in radome applications. Embedding quartz fiber up to 30% (wt./wt.) significantly improved the mechanical properties with a 49.72% increase in tensile strength and 52.01% rise in bending strength. Beyond this fiber loading, tensile strength was increased but the rise in the bending strength was not exceptional. Moreover, lower weight losses  refers to the higher thermal stability of EQ composites when compared to the pure epoxy composite. The enhanced electrical, mechanical, and thermal properties can be attributed to the inclusion of quartz fibers, good interfacial properties, fair adhesion compatibility, and a compact and balanced composite structure. In conclusion, adding a small quantity (0.30) of quartz fibers into an epoxy resin yields excellent dielectric characteristics with improved mechanical and thermal properties. Compared to pure epoxy resin composites, quartz fiber/epoxy composites exhibit substantial potential for applications in radomes.


Background: Currently, the prognosis and survival rate for patients bearing non-small cell lung cancer (NSCLC) is still quite poor, mainly due to lack of efficient theranostic paradigms to exert in time diagnostics and therapeutics.Methods: Herein, for NSCLC treatment, we offer a customized theranostic paradigm, termed NIR-IIb fluorescence diagnosis and synergistic surgery/starvation/chemodynamic therapeutics, with a newly designed theranostic nanoplatform PEG/MnCuDCNPs@GOx. The nanoplatform is composed of brightly NIR-II emissive downconversion nanoparticles (DCNPs)-core and Mn/Cu-silica shell loaded with glucose oxidase (GOx) to achieve synergistic starvation and chemodynamic therapy (CDT).Results: It is found that 10% Ce3+ doped in the core and 100% Yb3+ doped in the middle shell greatly improves the NIR-IIb emission up to even 20.3 times as compared to the core-shell DCNPs without Ce3+ doping and middle shell. The bright NIR-IIb emission of the nanoplatform contributes to sensitive margin delineation of early-stage NSCLC (diameter < 1 mm) with a signal-to-background ratio (SBR) of 2.18, and further assists in visualizing drug distribution and guiding surgery/starvation/chemodynamic therapy. Notably, the starvation therapy mediated by GOx-driven oxidation reaction efficiently depletes intratumoral glucose, and supplies H2O2 to boost the CDT mediated by the Mn2+ and Cu2+, which consequently realized a highly effective synergistic treatment for NSCLC.Conclusion: This research demonstrates an efficient treatment paradigm for NSCLC with NIR-IIb fluorescence diganosis and image-guided synergistic surgery/starvation/chemodynamic therapeutics. Nowadays, advanced non-small cell lung cancer (NSCLC) is still devastated for human beings, with a poor prognosis  in vivo imaging with good spatiotemporal resolution, high sensitivity, deep penetration, large SBR and non-invasiveness, which paves the way for sensitive diagnosis of early-stage microscopic tumors e.g., dyes and conjugated polymers) and inorganic ones , and lanthanide-doped downconversion nanoparticles (DCNPs) have been developed via adjusting their composition and morphology via surface modification and/or co-loading of drugs Sensitive diagnosis of early-stage microscopic tumors (diameter < 1 mm) is still quite challenging for conventional imaging modalities, such as visible/NIR-I optical imaging , computed tomography (CT), magnetic resonance imaging (MRI), ultrasound imaging (US), positron emission tomography (PET), and single-photon emission computed tomography (SPET) e.g., surgery, chemo-/targeted and immuno- therapy), present superiority in minimal invasiveness and tumor specificity 2O2) and high concentration of glutathione (GSH) which is an active antioxidant to consume intracellular ROS e.g., Fe2+, Co2+, Mn2+, Cu+) and H2O2 under acid conditions, which exist in tumors rather than normal tissues 2O2 concentration, acidity and metal ion species 2O2 level (≈10×10-6 M), rich-GSH and mild acidity, actions including introduction of continuous H2O2 supply and GSH depletion, and selection of highly reactive metal ion species in mild acidic tumors, contribute to highly efficient CDT X-dynamic therapies such as photodynamic therapy (PDT) and chemodynamic therapy (CDT), emerging as precision alternatives to conventional NSCLC therapies (4:Yb/Er/Ce@NaYbF4@NaYF4), a highly bright NIR-II emissive DCNP core is produced, offering highly sensitive detection and image-guided surgery of NSCLC submillimeter tumors in early stage 2O2, which not only enhances CDT, but also chock off the energy supply, offering combinatory therapeutics of starvation therapy and CDT, and thus greatly boosts the ultimate anticancer treatment outcomes 2+ to deplete GSH with production of Cu+ which further catalyze H2O2 to yield •OH +-catalysis occurs in both weakly acidic and neutral conditions with a Fenton-like reaction rate ten-fold higher than that of the most common CDT agent Fe2+2+ as Fenton-like catalyst reacts with H2O2 with effectively in situ generation of •OH for CDT and O2 for subsequent catalytic starvation reaction 2+Cu2+) silicate shell acts as gate-keeper to minimize side effects and its decomposition leads to the release of GOx, triggering the generation of O2 and •OH. For the synthesized nanoplatform, we systematically evaluated the photophysical properties, the bioimaging and therapeutic performance on NSCLC tumors in vitro and in vivo.Previously, we and others have demonstrated that lanthanide-doped nanoparticles (NPs) can readily integrate “all-in-one” theranostic platforms for image-guided X-dynamic/combinatory therapies with real-time monitoring of the pharmaceutic kinetics and distribution. In this contribution, we formulated multiple functional DCNP nanoplatform to innovate NSCLC treatment paradigm with dual diagnosis of early-stage NSCLC and image-guided synergistic surgery/starvation/chemodynamic therapy. We designed and synthesized PEGylated and glucose oxidase (GOx) loaded DCNPs (PEG/ MnCuDCNPs @GOx), which is composed of the bright NIR-II emissive DCNP core and the Mn/Cu-silica shell, for sensitive diagnosis of early NSCLC, and image-guided synergistic surgery/starvation/chemodynamic therapy Scheme . The rat via customizing the element contents to tune the upconversion (UC) and downconversion (DC) processes. The DCNPs were composed of a Ce3+, Er3+and Yb3+ co-doped NaYF4 core coated by a NaYbF4 middle shell and a NaYF4 passive shell . Similar dopent modulating strategies have been applied to enhance the DC emission at NIR-II window as well by optimizing the Ce3+ concentration in DCNPs cores, achieved a 9-fold and 12.3-fold increase at 1550 nm for Er-RENPs  4:Lu,Yb,Er@NaGdF4:Yb,Ce3+3+ concentration in the DCNPs core, but also highlighted the use of Yb3+-doped middle shell, combination of which has realized an substantial increase of 20.3 folds in the NIR-IIb emission. The bright NIR-IIb fluorescence should be highly beneficial for improving the in vivo imaging fidelity due to the much reduced tissue scattering and interference 4 coating was applied at the outermost layer, which could minimize the quenching effects of surface defects and surrounding solvents when the DCNPs are dispersed in water. Apart from the element doping, the thickness of outermost shell layer also affects luminescence brightness et al. revealed that an outer shell with a thickness of around 5 nm represented the best compromise between efficient UC and bright NIR-II DC emission by studying the quantum yields of the downshifted Er3+ emission around 1520 nm 4 shell thickness around 5 nm to investigate its protection effect on the NIR-IIb luminescence in this nanosystem. By adding different amount of the shell precusor sequencially into the core-shell dispersion, a passivation shell with a thickness of 3.5 to 7.5 nm was grown around the core-shell seed. When the thicknesses of NaYF4 shell varied from 3.5 nm, 4.7 nm to 6.2 nm  result showed the average shell thickness was c.a. 6.2 nm  shells were firstly coated on the surface of DCNPs by a hydrothermal method. Then the obtained DCNPs@mSiO2 further loaded Mn2+/Cu2+ and GOx to form the MnCuDCNPs@GOx by another hydrothermal process and a dipping method, respectively 42+ and Mn(NH3)42+ were persistently generated on the mSiO2 template surface until the mSiO2 shell were completely decomposed, eventually forming MnCuDCNPs. The loading of the Mn2+and Cu2+ further increased the diameter of obtained MnCuDCNPs to c.a. 71.4 nm  (PAH)-assisted layer-by-layer method to offer water dispersity. This was processed by condensation reaction between -COOH group of PEG and -NH2 group on the PAH backbones, which can also enhance the accumulation of the PEG/MnCuDCNPs@GOx at the tumor site via enhanced permeability and retention effect c.a. 77.5 nm as verified by TEM and DLS . Besides, an obvious diffraction peak at 22° shown in DCNPs@mSiO2 and the additional characteristic peaks of MnCuDCNPs confirmed the presence of amorphous silica, the copper silicate and manganese silicate (CuMn6SiO12), respectively 2+ (2+ (641 eV), Mn3+ (642 eV), and Mn4+ (644 eV)  B. Above 2 adsorption/desorption isotherms and pore-size distribution diagram of MnCuDCNPs. The corresponding Brunauer-Emmett-Teller (BET) surface area was calculated to be 314.68 m2g-1, the pore volume is around 0.54 cm3g-1, and the average pore size is nearly 5.01 nm, respectively, which indicate the mesoporous nature of the silica layer. The surface area and mesoporous structure is large enough for substance transfer and active molecular loading 2g-1, 0.19 cm3g-1, 4.33 nm after the coating of GOx . The MnCuDCNPs@GOx shows additional obvious peaks at 1082, 950, and 806 cm-1, belonging to the stretching vibration bands of Si-OH and Si-O-Si on the coated mSiO2 shell -1 which corresponds to the C=O vibration in GOx are discovered on the spectra of MnCuDCNPs@GOx, indicating the formation of MnCuDCNPs@GOx composites The absorption spectra of GOx, MnCuDCNPs and MnCuDCNPs@GOx were conducted to further confirm the successful loading of GOx. The GOx presented a special peak at 277 nm corresponding to the oxidized flavin group 2O2 and gluconic acid 2O2 generation and pH change. Figure 2O2 were produced and the pH value decreased when the sample was incubated with higher concentration of glucose. These results confirm that the high enzymatic activity of GOx was hardly affected after being loaded onto the mesoporous silica shell. To investigate stability of the interaction between the absorbed GOx and the MnCuDCNPs, we used the bicinchoninic acid (BCA) kit to colorimetricly detect and quantify the desorbed protein concentration. As shown in the As GOx can catalyze glucose to produce H2O2 at pH 7.4, implying the generation of •OH. After 25 min, the re-addition of H2O2 into the system leads to the continuous decline of the intensity, indicative of the dependence of •OH generation on H2O2 concentration. When the pH decreases from 7.4 to 5.5, the MB degradation efficiency was obviously elevated due to higher production of •OH and ultimately led to a 50% MB degradation  to capture free radicals, as the short-lived free radicals could be generated to comparative long-lived radical-DMPO adducts. ESR spectrum shows that the addition of PEG/MnCuDCNPs@GOx into H2O2 and GSH aqueous solution induced the production of plenty of •OH  2 generation to monitor the decomposition of H2O2 by PEG/MnCuDCNPs@GOx. As expected, the O2 production was obviously increased after addition of PEG/MnCuDCNPs@GOx into H2O2 solution -labeled cell imaging experiment and intracellular ROS level assay to investigate the cellular uptake of PEG/MnCuDCNPs@GOx and intracellular •OH production, respectively. The A549 lung tumor cells exhibit the clearly green fluorescence after co-incubation with FITC-labeled PEG/MnCuDCNPs@GOx for 2 h, indicating the efficient intracellular uptake of PEG/MnCuDCNPs@GOx . The 2′,-1) of glucose were added to the DMEM medium to simulate the glucose-supplying tumor environment, and then PEG/MnCuDCNPs@GOx (0 - 200 mg L-1) was added and co-incubated with cancer cells for 24 h. Figure -1 of PEG/MnCuDCNPs@GOx and 1000 mg L-1 of glucose, the A549 NSCLC cell survival was suppressed to c.a. 14%. Moreover, when compared with DCNPs@SiO2, PEG/MnCuDCNPs@GOx could exhibit far stronger potency towards A459 NSCLC cells at a fixed glucose concentration of 500 mg L-1  PEG/MnCuDCNPs@GOx could exert CDT to ablate cancer cell due to the Mn2+Cu2+-mediated Fenton-like reactions in the acid TME, whereas the CDT efficacy alone is unsatisfactory; ii) with both the loading of GOx and addition of proper amount of glucose, the therapeutic efficiency of the sample could be dramatically enhanced. The latter phenomenon could result from two aspects. For one thing, the GOx could deplete intratumoral glucose and O2 to kill cancer cells by starvation therapy and produce H2O2 to boost the CDT efficacy; for another, the PEG/MnCuDCNPs@GOx not only initiates Fenton-like reactions to ablate cancer cells, but also catalyze H2O2 decomposition, releasing O2 to afford sustainable glucose depletion with the aid of GOx. We also used the Glucose detection kit (O-toluidine method) to perform the glucose depletion assay in vitro. As the concentration of PEG/MnCuDCNPs@GOx increased, the content of glucose gradually decreased  through tail vein injection. As Figure c.a. 1 mm3 was successfully established, which was confirmed by the bioluminescence experiment and HE staining , ultrabright NIR-IIb emission of 20.3-fold increase was achieved under 980 nm laser excitation. By virtue of the high detection sensitivity, the bright NIR-II fluorescence of PEG/MnCuDCNPs@GOx successfully delineated the xenograft NSCLC tumor with the volume as small as c.a. 1 mm3 with a SBR of 2.18, and assisted in the intraoperative NIR-II imaging of mice bearing early-stage NSCLC tumors. Furthermore, the DCNPs were decorated with mesoporous silica to load GOx and Mn2+Cu2+ to fulfill therapeutic functions. The GOx-driven oxidation reaction effectively boosted the Mn2+Cu2+-mediated Fenton-like reaction for chemodynamic therapy, and cut off the energy supply to starve the tumor cells. Conversely, the MnCuDCNPs could also catalyze H2O2 decomposition to produce O2 to support the starvation therapy. Benefiting from the mutually-supported combinatory therapeutics, the cell survival was suppressed to only 13.98% when cocultured with the PEG/MnCuDCNPs@GOx for 24 h. The tumor growth of the mice transplanted with subcutaneous tumors was more effectively suppressed by PEG/MnCuDCNPs@GOx, compared with groups treated only with GOx or MnCuDCNPs. Simply put, this research demonstrated a novel synergistic theranostic nanoplatform of NSCLC, which might open up a new avenue for the diagnosis and treatment of NSCLC.To the best of our knowledge, we firstly demonstrated that NIR-IIb fluorescence diagnosis and image-guided synergistic surgery/starvation/chemodynamic therapeutics is efficient for NSCLC treatment. For the theranostic nanoplatform, PEG/MnCuDCNPs@GOx, by adjusting the doping concentration of the CeSupplementary materials and methods, figures.Click here for additional data file.


Thus, our studies have identified the β-catenin pathway as a key regulator of pro-inflammatory response during RSV infection of human lung epithelial cells. This pathway was induced during RSV infection via a non-canonical Wnt-independent mechanism involving paracrine/autocrine action of extracellular HBD3 activating cell surface Wnt receptor complex by directly interacting with the LRP5 receptor.Respiratory Syncytial Virus (RSV) is a non-segmented negative-sense RNA virus belonging to the paramyxovirus family. RSV infects the respiratory tract to cause pneumonia and bronchiolitis in infants, elderly, and immunocompromised patients. Effective clinical therapeutic options and vaccines to combat RSV infection are still lacking. Therefore, to develop effective therapeutic interventions, it is imperative to understand virus-host interactions during RSV infection. Cytoplasmic stabilization of β-catenin protein results in activation of canonical Wingless (Wnt)/β-catenin signaling pathway that culminates in transcriptional activation of various genes regulated by T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. This pathway is involved in various biological and physiological functions. Our study shows RSV infection of human lung epithelial A549 cells triggering β-catenin protein stabilization and induction of β-catenin mediated transcriptional activity. Functionally, the activated β-catenin pathway promoted a pro-inflammatory response during RSV infection of lung epithelial cells. Studies with β-catenin inhibitors and A549 cells lacking optimal β-catenin activity demonstrated a significant loss of pro-inflammatory chemokine interleukin-8 (IL-8) release from RSV-infected cells. Mechanistically, our studies revealed a role of extracellular human beta defensin-3 (HBD3) in interacting with cell surface Wnt receptor LDL receptor-related protein-5 (LRP5) to activate the non-canonical Wnt independent β-catenin pathway during RSV infection. We showed gene expression and release of HBD3 from RSV-infected cells and silencing of HBD3 expression resulted in reduced stabilization of β-catenin protein during RSV infection. Furthermore, we observed the binding of extracellular HBD3 with cell surface localized LRP5 protein, and our  Respiratory Syncytial Virus (RSV) is a respiratory RNA virus causing pneumonia and bronchiolitis in infants, the elderly, and immunocompromised patients . It is eHuman lung epithelial cells play an important role in RSV infection since these cells are the major target of RSV during the early phases of respiratory tract infection .ProductiCanonical Wingless (Wnt)/β-catenin pathway is a well-established signaling cascade regulating the expression of Wnt target genes involved in various biological and physiological functions . Wnt is Our study has demonstrated β-catenin activation by RSV in infected human lung epithelial A549 cells. Furthermore, we show β-catenin mediated transcriptional activity promoting pro-inflammatory response in RSV-infected A549 cells. Interestingly, in contrast to canonical Wnt ligand-dependent β-catenin pathway activation by extracellular Wnt ligands, we have uncovered a yet unknown non-canonical Wnt ligand-independent β-catenin pathway activation during RSV infection. We show extracellular human beta defensin-3 (HBD3) released from RSV-infected cells stabilizing β-catenin protein in infected cells. Furthermore, HBD3 interacted with Wnt receptor LRP5 to stabilize β-catenin protein for its activation of the β-catenin pathway in RSV-infected cells. Thus, in our current study, we have identified extracellular HBD3 as an “alarmin” molecule stimulating Wnt-independent β-catenin pathway in RSV-infected cells for triggering a pro-inflammatory response.Wnt/ β-catenin canonical pathway is activated following the activation of the Fzd/LRP receptor complex by extracellular Wnt ligand . This evFirst, we investigated whether RSV infection triggers β-catenin protein stabilization since this event results in β-catenin-mediated transcriptional activation of Wnt-responsive genes. To evaluate the status of β-catenin protein, we infected human lung epithelial A549 cells with RSV. At various post-infection time-periods, the level of β-catenin protein was assessed by western blotting with anti-β-catenin antibody. RSV infection triggered β-catenin protein stabilization since enhanced levels of β-catenin protein were detected in RSV infected cells ,B.We next examined whether the accumulation of β-catenin protein in RSV-infected cells resulted in the activation of TCF/LEF transcription factors. For these studies, we transfected A549 cells with TOP-Flash luciferase reporter plasmid. TOP-Flash luciferase reporter contains a promoter for binding (and activation) of activated TCF/LEF transcriptional factors upstream of the luciferase gene . TherefoCXCL8 gene; Compared to macrophages, human lung epithelial cells like A549 cells do not robustly produce pro-inflammatory cytokines like TNF, IL-6, and IL-1β. Instead, RSV-infected A549 cells trigger efficient production of pro-inflammatory chemokine IL-8  or β-catenin inhibitors (iCRT3 and iCRT14) during RSV infection. Medium supernatant was collected from infected cells to analyze IL-8 production by ELISA. β-catenin activity is essential for an optimal pro-inflammatory response during RSV infection, since significant loss of IL-8 production was observed in cells treated with β-catenin inhibitors ,B. ApproSince blocking β-catenin activity resulted in dampened pro-inflammatory response during RSV infection ,B, we fuThe typical strategy for employing CRISPR/Cas9 to disrupt a gene has been to target near the 5′ end and isolate a mutation that should lead to a frameshift and premature termination codon and no protein would be expected . InsteadTo validate the loss of β-catenin activity in Δ-catenin cells, we transfected FL-catenin and Δ-catenin cells with TOP-Flash luciferase reporter plasmid. Transfected cells were then treated with lithium chloride (LiCl), a potent inducer of β-catenin-mediated transcriptional activity . LuciferNext, we investigated whether β-catenin activity is required for a pro-inflammatory response during RSV infection. For these studies, we infected FL-catenin and Δ-catenin cells with RSV. As expected, RSV triggered a pro-inflammatory response in FL-catenin cells since high levels of IL-8 was produced from infected FL-catenin cells . In contTo identify non-canonical extracellular ligands for activation of the β-catenin pathway via a Wnt-independent mechanism, we focused our attention on human beta-defensins or HBDs. HBDs (primarily HBD1-3) are cationic anti-microbial peptides derived from mucosal epithelial cells . HBDs arWe first evaluated the ability of purified HBD3 to activate the β-catenin pathway by stabilizing cytoplasmic β-catenin protein. For these studies, we treated A549 cells with purified HBD3 (10 μg/ml) for 16 h. Previous studies showed that treating A549 cells with more than 20 μg/ml of purified HBD3 protein triggers cytotoxicity . TherefoSimilar to RSV-infected cells , HBD3 alNext, we investigated whether HBD3 plays any role in modulating β-catenin protein stability during RSV infection. We focused on β-catenin protein stability since stabilization of β-catenin protein results in activation of β-catenin mediated transcriptional activity, including expression/production of pro-inflammatory mediator IL-8. For these studies, we silenced HBD3 expression in A549 cells by siRNA. Efficient silencing was evident from western blot analysis showing reduced HBD3 protein levels in cells transfected with HBD3-specific siRNA compared to cells transfected with control scrambled siRNA ,B. HBD3 We have identified HBD3 as a ligand involved in β-catenin activation during RSV infection. Therefore, we next investigated the mechanism by which HBD3 may confer its β-catenin activation function. Nineteen different Wnt ligands engage with cell surface receptor complexes comprising of 10 different frizzled receptors (Fzd) and LRP5/6 to activate β-catenin by triggering its stabilization . In the To identify whether HBD3 can interact with Wnt receptors, we first evaluated the plausible interaction of HBD3 with LRP5. We chose LRP5 since, unlike human Fzd receptors, which consist of 10 structurally related proteins, Wnt can trigger signaling by interacting with LRP5 in complex with one of the Fzd receptors. We initially examined the possible interaction of extracellular HBD3 with cell surface localized LRP5 by using purified biotinylated-HBD3 protein (biot-HBD3). The interaction of extracellular HBD3 with cell surface localized LRP5 was studied by incubating chilled  A549 cells over-expressing FLAG-tagged LRP5 protein with biot-HBD3. After incubation, cell lysate precipitated with avidin-agarose, was subjected to western blotting with anti-FLAG antibody to detect FLAG-LRP5. HBD3 interacted with cell surface LRP5, since immune-blotting of avidin-precipitated lysate with anti-FLAG antibody detected FLAG-LRP5 protein . In contin vitro interaction assay, we incubated purified non-biotinylated HBD3 (control) and biotinylated-HBD3 proteins with avidin-agarose. Subsequently, avidin-agarose beads were incubated with recombinant purified truncated LRP5 protein. Following incubation, the bound proteins were subjected to western blotting with an anti-LRP5 antibody. Our studies revealed direct interaction of HBD3 with LRP5 since we detected the HBD3-LRP5 complex bound to the agarose beads , four EGF-like domains , and three LDLR type A domains . Four β-se beads . Interesin silico modeling studies to obtain detailed information about the LRP5-HBD3 interaction.Since HBD3 directly interacted with LRP5, we next performed a series of Wnt ligands interact promiscuously with 10 FZD receptors and two LRPs (LRP5 and LRP6), and each Wnt ligand has the potential to activate several pairs of LRP and FZD receptors . PreviouLRP5 belongs to a single-pass transmembrane family of proteins. The extracellular portion of LRP5 consists of four β-propeller domains  connected by epidermal growth factor (EGF)-like domains , and three LDLR (low-density lipoprotein receptor) type A (LA) domains preceding the transmembrane helix . Each paFZD8 receptors are class F G protein-coupled receptors (GPCRs) and are known to activate the canonical β-catenin pathway . FZD8 reTo examine the stability of the interactions among the LRP5-HBD3 and LRP5-FZD8-HBD3 complexes, we performed MD simulations of the complexes, each for 500 ns. Overall, the binary and ternary complexes were observed to be stable throughout the simulation time. During the first 50–100 ns of the simulations, the complexes underwent notable conformational changes , finetuning interactions between the interfaces, and then stabilized through the rest of the simulation time .To evaluate the critical H-bonds involved in formation and stabilization of the complexes, all three interface hydrogen bond interactions  were analyzed ,B. SeverIn the case of the HBD3-FZD8 interaction interface, K30 of HBD3 formed H-bonds with sidechain ε nitrogen of H124 . SimilarTo evaluate the critical interactions involved in stabilizing the complex, residue contacts at all three interfaces of complexes  were analyzed. We applied a distance-based cutoff (4 Å) to evaluate all non-bonded interactions involved in the interacting surfaces. As several residue interactions were observed among the interacting proteins, only interactions that were observed in more than 80% of the simulation time were discussed here. From the contact analysis of the LRP5-HBD3 interface, the residues G673, K675, E676, H711, E714, F715, W780, R805, H847, F849, W863, F888, M890, P972, L973, H974, G975 and P1010 of LRP5 were observed to be in contact with the HBD3 residues, including M1, R2, H4, Y5, L13, I24, I25, L28, Y31, Y32 and V35. The N-terminal residue M1 of HBD3 forms contact with the PE3 β-propeller domain in the cleft formed in between the PE3 and PE4 β-propeller domains. In addition, the α helix in HBD3, formed by residues 24–36, mainly interacted at the surface of the PE3 domain .HBD3 was sandwiched between LRP5 and FZD8. HBD3 residues, such as F8, L11, F12, L15, P17, V18, K30, R34, G37, R39, and C40, formed contacts with the FZD8 receptor residues, including G18, G20, Y21, Q22, I51, Q52, C53, S54, P55, P93, L94, Q97, and Y98. Interestingly, HBD3 appears to engage in a significantly greater number of contacts with FDZ8 than with LPR5 . In our We performed the solvent-accessible surface area calculation (SASA) to estimate the extent to which the proteins’ surfaces were buried  during the formation of LPR5-HBD3 and LPR5-HBD3-FZD3 complexes. The SASA values represent the average calculated through the entire 500 ns MD simulations. The fraction of BSA for each protein is significantly different, ranging from 5 to 30% . The PE3There is an urgent need to develop antiviral therapeutics and vaccines for Respiratory Syncytial Virus (RSV) since it is a leading cause of mortality and morbidity among infants, the elderly, and immunocompromised patients . To achiThe canonical Wnt/β-catenin pathway is a key signal transduction pathway involved in a wide spectrum of biological mechanisms , includiLung injury during RSV infection is associated with an influx of inflammatory immune cells into the airway . NeutropIn addition to unfolding the β-catenin pathway as an IL-8 inducer during RSV infection, we have also identified a new mechanism triggering β-catenin activation during viral infection. We have identified human beta-defensin 3 (HBD3) as an extracellular ligand involved in activation β-catenin by virtue of interacting with the well-established Wnt receptor LRP5 which is part of the β-catenin activating Fzd-LRP5/6 receptor complex. Defensins are anti-microbial cationic peptides regulating pathogen burden, immunity, and host defense . Apart fin vitro interaction studies with purified HBD3 and truncated LRP5 proteins. Our in-silico studies also provided a structural basis for the interactions between LRP5 and HBD3. Importantly, we obtained the potential binding orientations and critical residues at the interacting surfaces of the proteins using protein–protein docking. Subsequently, we evaluated the stability of the complex using 0.5 μs long MD simulations  were purchased from American Type Culture Collection (ATCC). Cells were cultured in complete Dulbecco’s modified Eagle medium  containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100ug/ml streptomycin unless otherwise stated , 2013. HCells were infected with RSV and mKate2-RSV at the multiplicity of infection (MOI) of 1 or 3. Virus adsorption was performed for 1.5 h (at 37°C) in OPTI-MEM medium (Gibco). Following adsorption, cells were washed with Dulbecco’s phosphate-buffered saline  and infection was continued in the presence or absence of a complete medium. In some experiments, cells were pre-treated with either vehicle (DMSO) or Wnt/β-catenin pathway inhibitors  and infected with RSV in the absence and presence of these inhibitors. For human beta-defensins 3 (HBD3) treatment studies, A549 cells were treated with 10ug/ml of purified HBD3 (Peprotech) or vehicle (PBS with 0.1% BSA). Cells were also treated with either lithium chloride  or vehicle (water) for 24 h. A549 cells were transfected with either empty-FLAG plasmid or FLAG-tagged LRP5  by using Lipofectamine 2000 (Life Technologies).A549 cells expressing either full-length β-catenin protein (FL-catenin) or truncated β-catenin protein (Δ-catenin) were generated by Synthego Corporation using CRISPR-Cas9 technology with the sgRNA sequence of GAGTGGTAAAGGCAATCCTG located in exon 3. Individual clones were isolated by limiting dilution from the cell pool provided by Synthego. To identify those that would likely have diminished activity without being a null, the mutation(s) carried by each clone was determined by PCR amplification of the region surrounding the sgRNA target site using F primer 5′ATCCCCCTGCTTTCCTCTCT3′ and R primer 5′ACATAGCAGCTCGTACCCTC3′. Clones that carried deletions were sequenced to determine the mutation.Cells  were co-transfected with M50 Super 8× TOP-Flash firefly luciferase reporter plasmid (Addgene) and a plasmid encoding Renilla luciferase for 16 h. Transfected cells were either infected with RSV or treated with purified HBD3 protein. Luciferase plasmid-transfected FL-catenin and Δ-catenin cells were also treated with LiCl. Following infection/treatment, the Dual-Luciferase® reporter assay system (Promega) was used for luciferase analysis. Luciferase activity was determined using a microplate luminometer (Promega).Scrambled control siRNA and HBD3 siRNA were purchased from Santa Cruz Biotechnology. A549 cells were transfected with these siRNAs (60 pmol) by using Lipofectamine 2000 (Life Technologies). HBD3 silencing was confirmed by western blotting with an HBD3 antibody.Human IL-8 ELISA kit was purchased from Invitrogen. HBD3 ELISA kit was obtained from MyBioSource. IL-8 and HBD3 levels in the medium supernatant were determined following the manufacturer’s instructions. ELISA values of the experimental group  represent values obtained following subtraction of background signal from the control group .A549 cells were lysed using 1%-Triton X-100 (pH 7.4), EDTA-free protease inhibitor cocktail (Roche Diagnostics) in PBS. Cell lysates were subjected to SDS-PAGE and separated proteins were transferred onto 0.2 μm nitrocellulose membrane  and blotted with specific antibodies. β-catenin and FLAG antibodies were purchased from Sigma-Aldrich. LRP5 antibody was obtained from Cell signaling. β-actin antibody was purchased from Bethyl Laboratories. HBD3-8A antibody was deposited to the DSHB by Starner, T. (DSHB Hybridoma Product hBD-3-8A). An anti-RFP antibody was purchased from Invitrogen. Western blots were quantified using ChemiDoc™ XRS + software Image Lab 5.1 (BioRad).Total RNA was extracted using TRIzol reagent (Life Technologies) following supplier’s instructions. Isolated RNA was treated with RNase-free DNase I (Thermoscientific) and cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR was performed using 2X Taq Red master mix (Apex) in a final reaction of 25 μl. The amplification cycle was as follows: an initial denaturing step (95°C for 3 min) was followed by 34 cycles of denaturing (95°C for 30 s), annealing (55.7°C for 30 s–), and extending (72°C for 1 min), followed by 5 min at 72°C for elongation. Following amplification, the PCR products were analyzed on 2% agarose gels and bands were visualized by ChemiDoc XRS (BioRad). The PCR product bands were quantified using ChemiDoc™ XRS + software Image Lab 5.1 (BioRad). Housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The primers used to detect the indicated genes are listed below:Human GAPDH forward, (5′ GATCATCAGCAATGCCTCCT-3′) and human GAPDH reverse, (5′ TGTGGTCATGAGTCCTTCCA-3′).Human HBD3 forward, (5′ TCCAGGTCATGGAGGAATCAT-3′) and human HBD3 reverse, (5′ CGAGCACTTGCCGATCTGT-3′).online.com). The purified truncated LRP5 protein consisted of E3 and E4 regions (aa769–aa1016) of the LRP5 extracellular domain. For the interaction studies, biotinylated-HBD3 and non-biotinylated HBD3 (control) were incubated (16 h at 4°C) with avidin–agarose beads. Following washing, the agarose beads were further incubated (16 h at 4°C) with purified truncated LRP5 protein. Washed beads were boiled with SDS sample buffer, followed by SDS-PAGE and western blotting with LRP5 antibody.The EZ-link® TFPA-PEG3-Biotin kit  was used to biotinylate purified HBD3 protein (Peprotech). Biotinylation was performed in the dark as per the manufacturer’s instructions. For cell surface interaction studies, A549 cells were transfected with either an empty-FLAG vector or LRP5-FLAG for 16 h. After 16 h, cells were cooled to 4°C for 2 h, and the chilled cells were subsequently incubated with biotinylated-HBD3 for 4 h at 4°C. Cells were lysed with PBS containing 1% TritonX-100 and protease inhibitors. Cell lysates were incubated with NeutrAvidin-agarose beads (Thermo Fisher Scientific) for 16 h at 4°C. After washing the agarose beads with wash buffer (10 mM Tris–HCL with protease inhibitor), the proteins bound to avidin–agarose beads were subjected to western blotting with anti-FLAG antibody. For cell-free interaction studies, we biotinylated purified HBD3 protein and purchased purified truncated LRP5 protein with Fc tag , and Student’s t-test was utilized to determine significance.All data were analyzed using GraphPad Prism software (6.0). For ELISA and luciferase assay, a significance test was carried out using Student’s https://alphafold.ebi.ac.uk/entry/P81534; https://saves.mbi.ucla.edu/; To examine the potential interactions of HBD3 with LRP5 and Frizzled-8 receptor (FZD8), we assembled the binary (HBD3-LRP5), and ternary (HBD3-LRP5-FZD8) complexes using protein–protein docking of the three proteins. Multiple experimental structures are available for FZD8, whereas the structures for LRP5 and HBD3 are yet to be resolved. For FZD8, we used the X-ray crystal structure of the protein in complex with a surrogate Wnt agonist  docking of HBD3 with LPR5, and (2) docking of the LRP5-HBD3 complex with FZD8. For docking of HBD3 with LRP5, the following residues of the PE3 (β propeller) domain of LRP5  were selected as the potential interaction site . For HBD+ and 174 Cl− ions. The simulations were performed under periodic boundary conditions and with the Particle Mesh Ewald (PME) method for calculating the long-range electrostatic interactions  and R01GM137022 (SN).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To elucidate the role of Mucin1 (MUC1) in the trophoblast function (glucose uptake and apoptosis) of gestational diabetes mellitus (GDM) women through the Wnt/β-catenin pathway.Glucose uptake was analyzed by plasma GLUT1 and GLUT4 levels with ELISA and measured by the expression of GLUT4 and INSR with immunofluorescence and Western blotting. Apoptosis was measured by the expression of Bcl-2 and Caspase3 by Western blotting and flow cytometry. Wnt/β-catenin signaling measured by Western blotting. In vitro studies were performed using HTR-8/SVneo cells that were cultured and treated with high glucose (HG), sh-MUC1 and FH535 .MUC1 was highly expressed in the placental trophoblasts of GDM, and the Wnt/β-catenin pathway was activated, along with dysfunction of glucose uptake and apoptosis. MUC1 knockdown resulted in increased invasiveness and decreased apoptosis in trophoblast cells. The initial linkage between MUC1, the Wnt/β-catenin pathway, and glucose uptake was confirmed by using an HG-exposed HTR-8/SVneo cell model with MUC1 knockdown. MUC1 knockdown inhibited the Wnt/β-catenin signaling pathway and reversed glucose uptake dysfunction and apoptosis in HG-induced HTR-8/SVneo cells. Meanwhile, inhibition of Wnt/β-catenin signaling could also reverse the dysfunction of glucose uptake and apoptosis.In summary, the increased level of MUC1 in GDM could abnormally activate the Wnt/β-catenin signaling pathway, leading to trophoblast dysfunction, which may impair glucose uptake and induce apoptosis in placental tissues of GDM women.The online version contains supplementary material available at 10.1186/s40659-023-00460-3. Gestational diabetes mellitus (GDM), defined as abnormal glucose metabolism and insulin resistance during pregnancy, has been increasingly prevalent and estimated to affect one in every six pregnancies worldwide in recent decades . The occIn GDM-complicated pregnancies, placentas exhibit dysfunction of trophoblast cells compared to normal pregnancies , 9. The The mucin (MUC) family includes MUC1-MUC22, and it provides a protective effect for epithelial cells of various organs through secretion and binding mode  20]. As. As20]. β-catenin, as the crucial factor in the Wnt/β-catenin signaling pathway, has a special binding site with MUC1. A previous study demonstrated that MUC1 could stimulate the Wnt/β-catenin pathway and its downstream genes  , 29. In In this study, we demonstrated that the maternal plasma MUC1 levels in the third trimester were significantly elevated in GDM patients (GDM group) compared to healthy subjects (Con group) with ELISA -induced trophoblast cells (HTR-8/SVneo cells) were used to mimic GDM conditions in vitro. HTR-8/SVneo cells were exposed to 30 mM glucose, and we found that trophoblast cell viability was significantly decreased in the HG group but increased in the MUC1 knockdown group. In addition, MUC1 knockdown reversed the cell viability of trophoblast cells exposed to HG Fig. A. The exTo understand the impact of high glucose and MUC1 on Wnt/β-catenin signaling, we compared the expression levels of several members of Wnt/β-catenin signaling by Western blotting. The results revealed that the Wnt/β-catenin pathway was also activated in HG-induced HTR8/SVneo cells, which showed a similar trend to human GDM placentas, while the Wnt/β-catenin pathway was inhibited in MUC1 knockdown HTR8/SVneo cells. The activation and inhibition of Wnt/β-catenin signaling was represented by the corresponding expression levels of β-catenin and p-β-catenin in the HG group and sh-MUC1 group  guidelines. To ensure homogeneity between the samples, the GDM pregnancy had no other related diseases, such as preeclampsia or pregestational diabetes. In the normal group, the OGTT assays were normal and without other complications. The clinical characteristics of the pregnant women enrolled in this study are presented in Supplementary file 1: Table The site of the placenta was located approximately 2 cm from the umbilical insertion site. Blood on the placenta was immediately washed 3 times with phosphate-buffered saline (PBS) and then placed in liquid nitrogen or 4% paraformaldehyde for further studies. In addition, peripheral venous blood was collected from pregnant women in the control group and GDM group on the first day of the last hospital admission  before delivery using anticoagulant tubes, 2 ml peripheral venous blood was collected and then centrifuged , and then the supernatant  was collected and stored at -80 °C for further study.Whole blood samples were collected from normal and GDM patients. The substances to be tested in the sera were measured by UV spectrophotometry using detection kits according to the manufacturer’s instructions . The kit details are provided: Human MUC1 ELISA kit , Human GLUT4 ELISA kit , and Human GLUT1 ELISA kit .−ΔΔCt method.Total RNA was extracted from human placentas and HTR8 cell lines using a TRIzol kit . First-strand cDNA was synthesized to a final volume of 20 µl using a SuperScript RIII first-strand kit . Following reverse transcription, PCR amplification of the cDNA was performed using the corresponding specific primers  overnight at 4 °C. The sections were stained with fluorescent secondary antibodies (anti-rabbit IgG/FITC or goat anti-mouse IgG/FITC) . The nuclei were stained with DAPI (Invitrogen). The sections were photographed using a fluorescence microscope . A minimum of 6 random images from 3 samples were analyzed per group.Proteins were isolated from the placenta of normal and GDM and HTR-8/SVneo cells using RIPA  buffer containing protease and phosphatase inhibitors. Protein concentrations were quantified by BCA assay . Extracted proteins were separated by 10% SDS‒PAGE and transferred to polyvinylidene difluoride membranes . The membrane was blocked with 5% nonfat milk, incubated overnight at 4 °C with primary antibody in TBST buffer and shaken gently. After incubation with the secondary antibody, i.e., either horseradish peroxidase goat anti-rabbit IgG  or horseradish peroxidase goat anti-mouse IgG , the samples were treated with SuperSignal™ West Femto chemiluminescent substrate  and then photographed by the Gel Doc™ XR + System . ImageJ software was used to capture the chemiluminescent signals and analyze the data. The following primary antibodies were used: MUC1 , GLUT4 , INSR , Bcl-2 , Caspase-3 , β-Catenin , p-β-Catenin , GSK-3β , p-GSK-3β , Wnt3a , TCF4 , c-Myc , CyclinD1 , and β-actin . All experiments were performed with at least three replicates.2. HTR-8/SVneo cells were treated for 72 hours with 30 mM D-glucose as the high glucose (HG) group, and normal medium (5.5 mM D-glucose) was used as a control group. HTR-8/SVneo cells (2 × 105 cells/mL) were seeded into 6-well plates and incubated for 24 h until the cells reached 60–70% confluence. Then, the cells were transfected with negative control (sh-NC group) and MUC1-shRNA (sh-MUC1 group) plasmids purchased from Shanghai GeneChem Co., Ltd. . The target sequence for MUC1 was as follows: shMUC1: 5’-CCGGCCGGGATACCTACCATCCTATCTCGAGATAGGATGGTAGGTATCCCGGTTTTTG-3’, and the negative control shRNA (sh-NC) sequence was 5’-TTCTCCGAACGTGTCACGT-3’. The vector map and sequence result of the sh-MUC1 plasmid are presented in Supplementary file 1: Results S1-2. The transfection assay was performed according to the manufacturer’s protocol for Lipofectamine 2000. FH535 was purchased from EMD Millipore .The human trophoblast cell line HTR8/SVneo was purchased from Zhongqiao Xinzhou Biotechnology Co., Ltd. . HTR-8/SVneo cells were cultured in DMEM supplemented with 10% fetal bovine serum  and 1% penicillin‒streptomycin solution (HyClone) at 37°C with 5% CO6) were collected by centrifugation at 1,000 rpm for 5 min and resuspended in 1 mL of cold PBS. The cells were then stained with an Annexin V-FITC apoptosis kit  and detected using a FACScan flow cytometer . The acquired data were analyzed using FCS-Express software version 3.0 (De Novo). Three independent experiments were performed.After transfection and treatment, HTR-8/SVneo cells (1 × 106) were collected and washed with cold PBS and then centrifuged, and the supernatant was discarded. Then, 1 ml of -20 °C 70% ethanol was added, gently mixed and fixed overnight at -20 °C. After overnight incubation at -20 °C and washing with PBS, the cells were stained with PI and subjected to flow cytometry. Then, the distribution of cells in the G1, S, and G2/M phases of the cell cycle was determined. Three independent experiments were performed.Cell cycle analysis was also detected by flow cytometry. After transfection and treatment, HTR-8/SVneo cells .A24h was the area of the wound measured 24 h after the scratch was performed.A4 cells/ml) and treated in the absence or presence of 50 mM D-glucose for 12, 24, 48 and 72 h. Briefly, 10 µl of CCK8 reagent  was added to 96-well plates and incubated continually for 2 h at 37 °C. The absorbance values were measured at 450 nm using a Bio-Rad Model 450 microplate reader .Cell viability was measured using a cell counting kit-8 . Cells from the Con, sh-NC and sh-MUC1 groups were cultured in 96-well plates . Statistical analysis was performed using the SPSS 23.0 statistical package program. A comparison of the means between two groups was performed using the Mann‒Whitney U or Student’s t test to estimate the differences. Multiple group comparisons of the means were performed by one-way analysis of variance (ANOVA). Blinded outcome assessment was implemented. All values are presented as the mean ± SD (standard deviation). All statistical data (including n number for the data) are presented in Supplementary file 1: Table S3. P < 0.05 was considered statistically significant.Below is the link to the electronic supplementary material.Supplementary file 1


Atopic dermatitis (AD) is an inflammatory, heterogeneous, chronic skin disorder characterized by recurrent eczematous lesions and intense pruritus, and the pathophysiology mechanism of AD is known for immune dysregulation and inflammatory responses. Wuguchong (maggot) has been widely used in the wound field and found with pharmacological properties of the anti-inflammatory and immunomodulatory function. Recently, some polysaccharides were proven to have beneficial effects on AD skin lesions in mice and humans. However, the effect of the polysaccharide extracted from Wuguchong (PEW) on AD remains to be investigated. In the present study, we examined the anti-inflammatory and immunomodulatory effects of PEW on AD and explored the potential mechanisms. Balb/c mice were orally administrated with PEW to evaluate the therapeutic effect of PEW on 2,4-dinitrochlorobenzene (DNCB)-induced AD. Oral PEW administration significantly ameliorated the lesions and symptoms in AD mice, such as the ear thickness and ear swelling degree, epidermal and dermal thickness, and the infiltration of mast cells. In addition, PEW treatment decreased the levels of serum IgE and histamine, the frequencies of Th1 and Th17 cells, as well as the mRNA expression levels of Th1 and Th17 cytokines and nuclear transcript factors . Furthermore, the activation of the NF-κB pathway and the phosphorylation of MAPKs  were significantly suppressed by PEW treatment. Taken together, our study suggests that PEW exerts anti-inflammatory and immunomodulatory effects through inhibition of Th1 and Th17 responses and downregulation of NF-κB and MAPK pathways, PEW would be developed as a promising immune therapy for AD. Atopic dermatitis (AD), also known as eczema and atopic eczema, is a chronic, heterogeneous, inflammatory skin disorder characterized by intense itching and recurrent eczematous lesions and has wide-ranging clinical phenotypes and courses . The pre+ T cells are the dominant cellular infiltration in AD and are differentiated into Th1, Th2, Th17, Th22, and regulatory T cell subsets  pathway plays important roles in immunity and inflammation, deregulated NF-κB activation contributes to the pathogenic processes of various inflammatory diseases including AD . The canchanisms . In addichanisms . Phosphochanisms . And thechanisms .via binding their high-affinity surface receptors to IgE , the larvae of nd field . Pharmacnd field . Howevernd field . So, mornd field . Besidesnd field . As prevThe PEW was isolated from the dried bodies of Wuguchong according to water extraction and alcohol precipitation methods and characterized by GPC (gel permeation chromatogram) and HPLC (high-performance liquid chromatography). The molecular weight of PEW is 32.9 kDa, and its monosaccharide composition includes rhamnose, glucose, arabinose, mannose, xylose, galactose, galacturonic acid, and glucuronic acid as previously reported .The present study was approved by the Ethical Committee on Animal Research of Dalian Medical University (approved number: AEE19075). Balb/c female mice  were bred in an animal facility under standard laboratory conditions in the SPF-level Experimental Animal Center of Dalian Medical University .n = 9–10/group) as follows:After 1 week of acclimation, all mice were shaved about 2.5 cm × 2.5 cm on their dorsal skin on day 0 and then randomly divided into 5 groups  + physiological saline.Group 4: DNCB + PEW (2000 mg/kg.d) + physiological saline.Group 5: DNCB + dexamethasone  + physiological saline.The mice were orally administered with physiological saline, PEW (1000 mg/kg.d), PEW (2000 mg/kg.d), and dexamethasone (DEX) (1 mg/kg.d) from day 4 to day 14. PEW and dexamethasone were separately dissolved in 0.9% physiological saline to get 100 mg/mL PEW solution, 200 mg/mL PEW solution, and 0.1 mg/mL dexamethasone solution, respectively. Mice were administered by oral gavage daily at a dose volume of 0.01 mL/g using a plastic syringe (1 mL) equipped with a straight, stainless-steel, bulb-tipped gavage needle .DNCB  was applied to the shaved dorsal skin and the back of the right ear of Balb/c mice to induce AD-like symptoms, based on the previously described procedure with minor modification . On day The ear thickness was measured on day 0 and 24 h after each DNCB application with a thickness gauge . Both ears of each mouse were punched with a 7 mm diameter skin puncher after the mice were sacrificed, the ear swelling degree was measured as the right ear weight minus the left ear weight.The dorsal skin tissues of mice were cut and fixed in 4% paraformaldehyde after being sacrificed. The fixed tissues were embedded in paraffin and then sliced into 4 µm sections. Hematoxylin and eosin (H&E) and toluidine blue (TB) were stained on the tissue sections for histological analysis. Epidermal thickness and dermal thickness were analyzed in H&E-stained sections at a magnification of ×100. Thickness was measured in five randomly selected fields from each sample. Toluidine blue (TB) stain for the measurement of mast cell infiltration and mast cell was counted in 5 random high-power fields at a magnification of ×400.Blood samples were collected immediately after the mice were sacrificed. The serum samples were obtained from blood using a centrifuge  and stored at −80°C until use. The level of serum IgE and serum histamine were measured by mouse IgE ELISA kit  and mouse histamine kit  according to the manufacturer’s instructions, respectively.6) were cultured with the cell stimulation cocktail (plus protein transport inhibitors)  for 6 h in a 5% CO2 incubator at 37°C. Then, the cells were collected and strained with FITC-labeled rat anti-mouse CD4 mAb  for 20 min at 4°C in the dark. The cells were fixed and permeabilized after being thoroughly resuspended in BD Cytofix/Cytoperm solution  for 20 min at 4°C and then washed with BD Perm/Wash™ buffer  two times. Next, the permeabilized cells were stained with PE-labeled rat anti-mouse IFN-γ mAb , PE-Cy7-labeled rat anti-mouse IL-4 mAb , PerCp-Cy5.5-labeled rat anti-mouse IL-17A mAb  and APC-labeled rat anti-mouse IL-22 mAb  for 20 min at 4°C in the dark. Th1 cells were defined as CD4+ IFN-γ+, Th2 cells were defined as CD4+ IL-4+, Th17 cells were defined as CD4+ IL-17A+, and Th22 cells were defined as CD4+ IL-22+. Data was harvested with a BD FACSVerse Flow Cytometer  and analyzed using FlowJo software .Splenocytes were collected as single-cell suspension after the mice were sacrificed. The prepared splenocytes  according to the manufacturer’s protocol. 10 μg of total RNA was used to synthesize the first-strand complementary DNA (cDNA) with the All-in-One First-Strand cDNA Synthesis kit . The relative mRNA expression was quantified using RT-qPCR with Top Green qPCR SuperMix  performed on an ABI Prism 7,500 device . Levels of target genes were normalized with respect to the expression of GAPDH, and the primer sequences used in this study are listed as follows .Total proteins were extracted from mice’s dorsal skin tissues using the Whole Cell Lysis Assay Kit , followed by the protein concentrations determined with the BCA assay kit . The denatured protein was separated by 10% SDS-PAGE and then transferred onto PVDF membranes . After being blocked with 5% BSA for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies: NF-κB (p65), p-IκBα (Ser32), p-p38 (Thr180/Tyr182), p38, p-JNK (Thr183/Tyr185), JNK, p-ERK (Thr202/Tyr204), ERK  and GAPDH . Then, the HRP Goat Anti-Rabit IgG (H + L) secondary antibody  was added and incubated for 1 h at room temperature. The protein bands were visualized with the ECL FemtoLight kit  and analyzed with the ImageJ software .T-test and one-way analysis of variance (ANOVA). The data were expressed as the mean ± standard deviation (SD), and p < 0.05 was considered statistically significant.All statistical analyses were performed using Prism 8.0 , and differences across groups were analyzed using unpaired Student’s To explore the therapeutic properties of PEW on AD, a Balb/c AD model was established by applying DNCB on the dorsal skin and the back of the right ear , 2A. As p < 0.001) (p < 0.001) [In < 0.001) . In addi< 0.001) .p < 0.001), whereas the PEW-treated groups (1000 mg/kg.d and 2000 mg/kg.d) displayed significantly lower levels than the AD model group (p < 0.01)  .+ Th cells in DNCB-induced AD mice by detecting the intracellular expression of IFN-γ, IL-4, IL-17A, and IL-22 using Flow cytometry assay. In the present study, the percentage of Th1, Th2, Th17, and Th22 cells of the AD model group were all significantly increased compared to the normal control group. Two PEW-treated groups showed significantly lower percentages of Th1 and Th17 cell subsets than the AD model group , Th2 (GATA-3 and IL-4), Th17 (ROR-rt and IL-17A), and Th22 (Ahr and IL-22) in different groups of mouse skin tissue were measured by RT-qPCR. Consistent with previous results, all nuclear transcription factors and cytokines in the AD model group were significantly increased compared with the normal control group, and the mRNA expression of Th1 (T-bet and IFN-γ) and Th17 (ROR-rt and IL-17A) nuclear transcription factors and cytokines were significantly suppressed in PEW-treated groups compared to the AD model group  pathways were reported to be activated in DNCB-induced AD-like skin lesions in mice. In the present study, the effect of PEW on the activation of NF-κB and the phosphorylation of IκB-α and MAPKs  signaling pathways were measured by Western Blotting analysis with mice dorsal skin tissue. As shown in + Th cell subsets. The results demonstrated that PEW markedly alleviates DNCB-induced AD-like symptoms mainly via inhibition of Th1 and Th17 responses and downregulation of NF-κB and MAPK signaling pathways.Wuguchong (maggot) has been widely used in the wound field since antiquity, including diabetic foot ulcers, chronic wounds, pressure ulcers, venous ulcers, etc., due to its pharmacological properties of antibacterial activity, anti-inflammatory activity, proangiogenic function, and neurogenesis . BesidesEssential features of AD are intense itching and eczematous lesions, with a relapsing or chronic disease course . Histolo+ cells , including the p38, ERK, and JNK subfamilies, are the other classic inflammation-related pathways. Cross-talking with NF-κB signaling, activation of MAPKs contributes to the induction of several genes which together mediate the inflammatory response . Phosphovia immunologic, metabolic, and neuroendocrine pathways (+ T cell dysfunction (via the regulation of gut microbiota (+ T cell differentiation, also, it is possible that PEW ameliorates AD via modulating effects of diets. PEW may play its role in AD by influencing the interaction between diets, gut microbes, and immunity. Further studies are warranted to clarify whether PEW can alleviate AD inflammation by regulating intestinal microbiota, mediating diets’ influence, or preventing obesity.Furthermore, recent evidence indicates that obesity can also influence the immune system and is associated with atopic dermatitis . A high-pathways . Neonatafunction . A recenfunction . In our crobiota , we hypoOur current study indicates that PEW significantly inhibits Th1 and Th17 responses in DNCB-induced AD mice, which may be used as a promising immune therapy targeting Th1 and Th17 pathways. However, AD immunoendotypes are in part associated with race or ethnic group . For insIn conclusion, our findings indicate that PEW treatment can alleviate skin inflammation in mice with DNCB-induced AD. PEW significantly inhibited the generation of Th1 and Th17 cells, and profoundly reduced the expression of Th1 and Th17 cytokines and nuclear transcription factors including IFN-γ, T-bet, IL-17, and ROR-rt. Furthermore, we demonstrated the underlying mechanisms of action involved in suppressing inflammation as well as the Th1 and Th17 responses are through the inactivation of NF-κB and MAPK pathways. The potential mechanisms related to regulating gut microbiota and preventing obesity remain to be explored. This study verifies the therapeutic effect of PEW on AD and provides a scientific rationale for this bioactive ingredient as a promising immune therapy for AD. We will purify PEW for further studies in the future.