Jammed Microgel-Based Inks for 3D Printing of Complex Structures Transformable via pH/Temperature Variations.

Structure changes mediated by anisotropic volume changes of stimuli-responsive hydrogels are useful for many research fields, yet relatively simple structured objects are mostly used due to limitation in fabrication methods. To fabricate complex 3 dimensional (3D) structures that undergo structure changes in response to external stimuli, jammed microgel-based inks containing precursors of stimuli-responsive hydrogels are developed for extrusion-based 3D printing. Specifically, the jammed microgel-based inks are prepared by absorbing precursors of poly(acrylic acid) or poly(N-isopropylacrylamide) in poly(acrylamide) (PAAm) microgels, and jamming them. The inks exhibit shear-thinning and self-healing properties that allow extrusion of the inks through a nozzle and rapid stabilization after printing. Stimuli-mediated volume changes are observed for the extruded structures when they are post-crosslinked by UV light to form interpenetrating networks of PAAm microgels and stimuli-responsive hydrogels. Using this method, a dumbbell-shaped object that can transform to a biconvex shape, and a gripper that can grasp and lift an object in response to stimuli are 3D-printed. The jammed microgel-based 3D printing strategy is a versatile method useful for variety of applications as diverse types of monomers absorbable in the microgels can be used to fabricate complex 3D objects transformable by external stimuli.

Inhibitory effects of baicalin in the early stage of 3T3-L1 preadipocytes differentiation by down-regulation of PDK1/Akt phosphorylation.

Baicalin is a flavonoid derived from the root of Scutellaria baicalensis and exhibits a broad spectrum of biological activities including anti-adipogenesis. However, the inhibitory role of baicalin in the early stage of 3T3-L1 adipocyte differentiation relevant to the signaling up-stream of peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding proteins (C/EBPs) expression is unclear, and is the subject of the present investigation. We used 3T3-L1 preadipocytes for adipocyte differentiation, Oil Red-O staining for the intracellular lipid accumulation assay, and real-time polymerase chain reaction (RT-PCR) for assaying the expression of major adipocyte transcription factors. We found that baicalin markedly suppressed the Akt phosphorylation in early stage of adipocytes differentiation. In addition, we observed that baicalin and LY294002 (as an inhibitor of Akt phosphorylation) significantly inhibited adipocyte differentiation by down-regulating several adipocyte-specific transcription factors, including PPAR-γ and C/EBPs in 3T3-L1 preadipocytes. Furthermore, we observed that baicalin significantly suppressed the Akt phosphorylation by inhibiting phosphoinositide-dependent kinase 1 (PDK1). These results indicate that the anti-adipogenesis effect of baicalin involves down-regulation of major transcription factors in 3T3-L1 adipocyte differentiation including PPAR-γ, C/EBP-ß, and C/EBP-α through the down-regulation of PDK1/Akt phosphorylation.

Nanotopography-guided migration of T cells.

T cells navigate a wide variety of tissues and organs for immune surveillance and effector functions. Although nanoscale topographical structures of extracellular matrices and stromal/endothelial cell surfaces in local tissues may guide the migration of T cells, there has been little opportunity to study how nanoscale topographical features affect T cell migration. In this study, we systematically investigated mechanisms of nanotopography-guided migration of T cells using nanoscale ridge/groove surfaces. The velocity and directionality of T cells on these nanostructured surfaces were quantitatively assessed with and without confinement, which is a key property of three-dimensional interstitial tissue spaces for leukocyte motility. Depending on the confinement, T cells exhibited different mechanisms for nanotopography-guided migration. Without confinement, actin polymerization-driven leading edge protrusion was guided toward the direction of nanogrooves via integrin-mediated adhesion. In contrast, T cells under confinement appeared to migrate along the direction of nanogrooves purely by mechanical effects, and integrin-mediated adhesion was dispensable. Therefore, surface nanotopography may play a prominent role in generating migratory patterns for T cells. Because the majority of cells in periphery migrate along the topography of extracellular matrices with much lower motility than T cells, nanotopography-guided migration of T cells would be an important strategy to efficiently perform cell-mediated immune responses by increasing chances of encountering other cells within a given amount of time.

Effects of apolipoprotein A5 haplotypes on the ratio of triglyceride to high-density lipoprotein cholesterol and the risk for metabolic syndrome in Koreans.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) around the apolipoprotein A5 gene (APOA5) have pleiotropic effects on the levels of triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C). APOA5 SNPs have also been associated with metabolic syndrome (MS). Here, we constructed haplotypes with SNPs spanning APOA5 and ZNF259, which are approximately 1.3 kb apart, to perform association analyses with the risk for MS and the levels of TG and HDL-C in terms of a TG:HDL-C ratio. METHODS: The effects of three constructed haplotypes (TAA, CGG, and CGA, in the order of rs662799, rs651821, and rs6589566) on the TG:HDL-C ratio and MS were estimated using multiple regression analyses in 2,949 Koreans and in each gender separately (1,082 men and 1,867 women). RESULTS: The haplotypes, CGG and CGA, were associated with the TG:HDL-C ratio and the risk of MS development in both genders. That is, the minor alleles of the rs662799 and rs651821 in APOA5, irrespective of which allele was present at rs6589566, had the marked effects. Interestingly, a C-G-A haplotype at these three SNPs had the most marked effects on the TG:HDL-C ratio and the risk of MS development in women. CONCLUSIONS: We have identified the novel APOA5-ZNF259 haplotype manifesting sex-dependent effects on elevation of the TG:HDL-C ratio as well as the increased risk for MS.

Sho-saiko-to, a traditional herbal medicine, regulates gene expression and biological function by way of microRNAs in primary mouse hepatocytes.

BACKGROUND: Sho-saiko-to (SST) (also known as so-shi-ho-tang or xiao-chai-hu-tang) has been widely prescribed for chronic liver diseases in traditional Oriental medicine. Despite the substantial amount of clinical evidence for SST, its molecular mechanism has not been clearly identified at a genome-wide level. METHODS: By using a microarray, we analyzed the temporal changes of messenger RNA (mRNA) and microRNA expression in primary mouse hepatocytes after SST treatment. The pattern of genes regulated by SST was identified by using time-series microarray analysis. The biological function of genes was measured by pathway analysis. For the identification of the exact targets of the microRNAs, a permutation-based correlation method was implemented in which the temporal expression of mRNAs and microRNAs were integrated. The similarity of the promoter structure between temporally regulated genes was measured by analyzing the transcription factor binding sites in the promoter region. RESULTS: The SST-regulated gene expression had two major patterns: (1) a temporally up-regulated pattern (463 genes) and (2) a temporally down-regulated pattern (177 genes). The integration of the genes and microRNA demonstrated that 155 genes could be the targets of microRNAs from the temporally up-regulated pattern and 19 genes could be the targets of microRNAs from the temporally down-regulated pattern. The temporally up-regulated pattern by SST was associated with signaling pathways such as the cell cycle pathway, whereas the temporally down-regulated pattern included drug metabolism-related pathways and immune-related pathways. All these pathways could be possibly associated with liver regenerative activity of SST. Genes targeted by microRNA were moreover associated with different biological pathways from the genes not targeted by microRNA. An analysis of promoter similarity indicated that co-expressed genes after SST treatment were clustered into subgroups, depending on the temporal expression patterns. CONCLUSIONS: We are the first to identify that SST regulates temporal gene expression by way of microRNA. MicroRNA targets and non-microRNA targets moreover have different biological roles. This functional segregation by microRNA would be critical for the elucidation of the molecular activities of SST.

Development of Cell Culture Platforms for Study of Trabecular Meshwork Cells and Glaucoma Development.

BACKGROUND: Various cell culture platforms that could display native environmental cue-mimicking stimuli were developed, and effects of environmental cues on cell behaviors were studied with the cell culture platforms. Likewise, various cell culture platforms mimicking native trabecular meshwork (TM) composed of juxtacanalicular, corneoscleral and uveal meshwork located in internal scleral sulcus were used to study effects of environmental cues and/or drug treatments on TM cells and glaucoma development. Glaucoma is a disease that could cause blindness, and cause of glaucoma is not clearly identified yet. It appears that aqueous humor (AH) outflow resistance increased by damages on pathway of AH outflow can elevate intraocular pressure (IOP). These overall possibly contribute to development of glaucoma. METHODS: For the study of glaucoma, static and dynamic cell culture platforms were developed. Particularly, the dynamic platforms exploiting AH outflow-mimicking perfusion or increased IOP-mimicking increased pressure were used to study how perfusion or increased pressure could affect TM cells. Overall, potential mechanisms of glaucoma development, TM structures and compositions, TM cell culture platform types and researches on TM cells and glaucoma development with the platforms were described in this review. RESULTS AND CONCLUSION: This will be useful to improve researches on TM cells and develop enhanced therapies targeting glaucoma.

Extracts of Scutellaria baicalensis reduced body weight and blood triglyceride in db/db Mice.

Scutellaria baicalensis has been extensively employed for the clinical treatment of hyperlipidemia, atherosclerosis, hypertension, dysentery, inflammatory diseases, and the common cold. The present study was performed to investigate the anti-obesity and anti-dyslipidemia effect of Scutellaria baicalensis extracts (SBE) in type 2 diabetic db/db mice. Male db/db mice were divided into three groups (n = 5) and orally administrated vehicle (control), SBE 10, and 100 mg/kg body weight/day for 4 weeks everyday. Administration of SBE improves weight gain, hypertriglyceridemia, and hyperinsulinemia in db/db mice. In obese db/db mice, SBE treatment also reduced plasma alanine aminotransferase levels. In the livers of db/db mice, SBE promoted 5' AMP-activated protein kinase activity and restored metabolic process and insulin signaling pathways. Our data demonstrate that SBE exerts potent anti-obesity and anti-hypertriglyceride effects suggesting its useful potential function as adjuvant therapeutic agent for the treatment of weight gain and hypertriglyceridemia.

Comparison of pathways associated with hepatitis B- and C-infected hepatocellular carcinoma using pathway-based class discrimination method.

Molecular signatures causing hepatocellular carcinoma (HCC) from chronic infection of hepatitis B virus (HBV) or hepatitis C virus (HCV) are not clearly known. Using microarray datasets composed of HCV-positive HCC or HBV-positive HCC, pathways that could discriminate tumor tissue from adjacent non-tumor liver tissue were selected by implementing nearest shrunken centroid algorithm. Cancer-related signaling pathways and lipid metabolism-related pathways were predominantly enriched in HCV-positive HCC, whereas functionally diverse pathways including immune-related pathways, cell cycle pathways, and RNA metabolism pathways were mainly enriched in HBV-positive HCC. In addition to differentially involved pathways, signaling pathways such as TGF-ß, MAPK, and p53 pathways were commonly significant in both HCCs, suggesting the presence of common hepatocarcinogenesis process. The pathway clustering also verified segregation of pathways into the functional subgroups in both HCCs. This study indicates the functional distinction and similarity on the pathways implicated in the development of HCV- and/or HBV-positive HCC.

Fibrous hydrogels by electrospinning: Novel platforms for biomedical applications.

Hydrogels, hydrophilic and biocompatible polymeric networks, have been used for numerous biomedical applications because they have exhibited abilities to mimic features of extracellular matrix (ECM). In particular, the hydrogels engineered with electrospinning techniques have shown great performances in biomedical applications. Electrospinning techniques are to generate polymeric micro/nanofibers that can mimic geometries of natural ECM by drawing micro/nanofibers from polymer precursors with electrical forces, followed by structural stabilization of them. By exploiting the electrospinning techniques, the fibrous hydrogels have been fabricated and utilized as 2D/3D cell culture platforms, implantable scaffolds, and wound dressings. In addition, some hydrogels that respond to external stimuli have been used to develop biosensors. For comprehensive understanding, this review covers electrospinning processes, hydrogel precursors used for electrospinning, characteristics of fibrous hydrogels and specific biomedical applications of electrospun fibrous hydrogels and highlight their potential to promote use in biomedical applications.

Network pharmacology-based identification of bioavailable anti-inflammatory agents from Psoralea corylifolia L. in an experimental colitis model.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional oriental medicine, the dried seeds of Psoralea corylifolia L. (PC) have been used to treat various diseases, including gastrointestinal, urinary, orthopedic, diarrheal, ulcer, and inflammatory disorders. AIM OF THE STUDY: Although its various biological properties are well-known, there is no information on the therapeutic effects and bioavailable components of PC against inflammatory bowel disease. Therefore, we focused on the relationship between hydroethanolic extract of PC (EPC) that ameliorates colitis in mice and bioactive constituents of EPC that suppress pro-inflammatory cytokines in macrophages. MATERIALS AND METHODS: We investigated the therapeutic effects of EPC in a dextran sulfate sodium-induced colitis mouse model and identified the orally absorbed components of EPC using UPLC-MS/MS analysis. In addition, we evaluated and validated the mechanism of action of the bioavailable constituents of EPC using network pharmacology analysis. The effects on nitric oxide (NO) and inflammatory cytokines were measured by Griess reagent and enzyme linked immunosorbent assay in lipopolysaccharide (LPS)-induced macrophages. RESULTS: In experimental colitis, EPC improved body weight loss, colon length shortening, and disease activity index. Moreover, EPC reduced the serum levels of pro-inflammatory cytokines and histopathological damage to the colon. Network pharmacological analysis identified 13 phytochemicals that were bioavailable following oral administration of EPC, as well as their potential anti-inflammatory effects. 11 identified EPC constituents markedly reduced the overproduction of NO, tumor necrosis factor-α, and/or interleukin-6 in macrophages induced by LPS. The LPS-induced expression of the nuclear factor kappa-light-chain-enhancer of activated B cells reporter gene was reduced by the 4 EPC constituents. CONCLUSIONS: The results indicate that the protective activity of EPC against colitis is a result of the additive effects of each constituent on the expression of inflammatory cytokines. Therefore, it suggests that 11 bioavailable phytochemicals of EPC could aid in the management of intestinal inflammation, and also provides useful insights into the clinical application of PC for the treatment of inflammatory bowel diseases.

Effects of Alginate Oligosaccharide on Testosterone-Induced Benign Prostatic Hyperplasia in Orchiectomized Rats.

Benign prostatic hyperplasia (BPH) is an age-related disease of the urinary system that affects elderly men. Current treatments for BPH are associated with several adverse effects, thus highlighting the need for alternative agents. Alginate oligosaccharide (AOS), a water-soluble functional oligomer derived from brown algae, inhibits prostate cancer cell proliferation. However, the effects of AOS on BPH and the underlying molecular mechanisms remain unclear. Therefore, here, we aimed to investigate the therapeutic potential of AOS in BPH by using human benign prostatic epithelial cells (BPH-1) and a rat model of testosterone-induced BPH. Treatment with AOS inhibited in vitro and in vivo proliferation of prostatic epithelial cells and the testosterone-induced expression of androgen receptor (AR) and androgen-associated genes, such as those encoding 5α-reductase type 2 and prostate-specific antigen. Oral administration of AOS remarkably reduced the serum levels of dihydrotestosterone (DHT) and testosterone as well as the expression of proliferating cell nuclear antigen, inflammatory cytokines, and enzymes, which showed increased levels in prostatic tissues of rats with testosterone-induced BPH. Taken together, these data demonstrate that AOS suppresses testosterone-induced BPH in rats by downregulating AR and the expression of androgen-associated genes, supporting the hypothesis that AOS might be of potential use for the treatment of BPH.

Hydroethanolic Extract of Fritillariae thunbergii Bulbus Alleviates Dextran Sulfate Sodium-Induced Ulcerative Colitis by Enhancing Intestinal Barrier Integrity.

The incidence of ulcerative colitis (UC), an inflammatory disorder of the gastrointestinal tract, has rapidly increased in Asian countries over several decades. To overcome the limitations of conventional drug therapies, including biologics for UC management, the development of herbal medicine-derived products has received continuous attention. In this study, we evaluated the beneficial effects of a hydroethanolic extract of Fritillariae thunbergii Bulbus (FTB) in a mouse model of DSS-induced UC. The DSS treatment successfully induced severe colonic inflammation and ulceration. However, the severity of colitis was reduced by the oral administration of FTB. Histopathological examination showed that FTB alleviated the infiltration of inflammatory cells (e.g., neutrophils and macrophages), damage to epithelial and goblet cells in the colonic mucosal layer, and fibrotic lesions. Additionally, FTB markedly reduced the gene expression of proinflammatory cytokines and extracellular matrix remodeling. Immunohistochemical analysis showed that FTB alleviated the decrease in occludin and zonula occludens-1 expression induced by DSS. In a Caco-2 monolayer system, FTB treatment improved intestinal barrier permeability in a dose-dependent manner and increased tight junction expression. Overall, FTB has potential as a therapeutic agent through the improvement of tissue damage and inflammation severity through the modulation of intestinal barrier integrity.

Effect of lyophilized gelatin-norbornene cryogel size on calvarial bone regeneration.

Molding processes with molds containing topographical structures have been used for fabrication of hydrogel and cryogel particles. However, they can involve difficulties in separation of fabricated particles with complex shape from the molds or repeated fabrication of the particles although the overall processes do not require much skill and equipment. In this study, molds with etched superhydrophobic patterns have been developed by etching polytetrafluoroethylene (PTFE) blocks in user-defined designs with a femtosecond (FS) laser-based etching system. Lyophilized cryogel particles with various designs and sizes were fabricated by molding precursors with these PTFE molds. Additionally, the clean and easy separation of particles from the molds allowed repeated fabrication of the particles. For an application, relatively 'big' gelatin-norbornene (GelNB) cryogel particles prepared via molding with polydimethylsiloxane (PDMS) molds, swelling in phosphate buffered saline (PBS) and slicing height in half and 'small' GelNB cryogel particles fabricated with the PTFE molds were fabricated. Then, they were used to study scaffold size effect on calvarial bone regeneration. The molds generated with the FS laser-based etching system can be useful for various applications that require the mass production of cryogel particles in various geometries.

Therapeutic effects of Pulsatilla koreana Nakai extract on ovalbumin-induced allergic rhinitis by inhibition of Th2 cell activation and differentiation via the IL-4/STAT6/GATA3 pathway.

Allergic rhinitis (AR), caused by immunoglobulin E (IgE)-mediated inflammation, generally occurs in the upper respiratory tract. T helper type 2 (Th2) cell-mediated cytokines, including interleukin (IL)-4, IL-5, and IL-13, are important factors in AR pathogenesis. Despite various treatment options, the difficulty in alleviating AR and pharmacological side effects necessitate development of new therapies. The root of Pulsatilla koreana Nakai (P. koreana), a pasque flower, has been used as a herbal medicine. However, its effects on AR remain unclear; therefore, we aimed to explore this subject in the current study. The therapeutic effects of P. koreana water extract (PKN) on the pathophysiological functions of the nasal mucosa was examined in ovalbumin (OVA)-induced AR mice. The effect of PKN on Th2 activation and differentiation was evaluated using concanavalin A-induced splenocytes and differentiated Th2 cells from naïve CD4+ T cells. We also investigated the effect of changes in JAK/STAT6/GATA3 signaling on IL-4-induced Th2 cells. In OVA-induced AR mice, PKN administration alleviated allergic nasal symptoms and decreased the total number of immune cells, lymphocytes, neutrophils, and eosinophils in nasal lavage fluid; serum levels of OVA-specific IgE, histamine, and IL-13 were also significantly reduced. PKN also ameliorated OVA-induced nasal mucosal tissue thickening by inhibiting inflammation and goblet cell hyperplasia. PKN treatment significantly inhibited Th2 activity and differentiation through the IL-4/STAT-6/GATA3 pathway in Th2 cells. PKN is an effective AR treatment with the potential to improve patients' daily lives by regulating the allergic inflammatory response induced by Th2 cells.

Prevalence of Metabolic Syndrome according to Sasang Constitutional Medicine in Korean Subjects.

Metabolic syndrome (MS) is a complex disorder defined by a cluster of abdominal obesity, atherogenic dyslipidemia, hyperglycemia, and hypertension; the condition is recognized as a risk factor for diabetes and cardiovascular disease. This study assessed the effects of the Sasang constitution group (SCG) on the risk of MS in Korean subjects. We have analyzed 1,617 outpatients of Korean oriental medicine hospitals who were classified into three SCGs, So-Yang, So-Eum, and Tae-Eum. Significant differences were noted in the prevalence of MS and the frequencies of all MS risk factors among the three SCGs. The odds ratios for MS as determined via multiple logistic regression analysis were 2.004 for So-Yang and 4.521 for Tae-Eum compared with So-Eum. These results indicate that SCG may function as a significant risk factor of MS; comprehensive knowledge of Sasang constitutional medicine may prove helpful in predicting susceptibility and developing preventive care techniques for MS.

Association of the Apolipoprotein A5 Gene -1131T>C Polymorphism with Serum Lipids in Korean Subjects: Impact of Sasang Constitution.

Apolipoprotein A5 (APOA5) was identified as a strong modulator of serum lipids. Moreover, an APOA5 gene -1131T>C polymorphism has been associated with serum lipids, but the results are inconsistent according to ethnic and racial groups. We have genotyped and analyzed 1,619 outpatients of Korean oriental medicine hospitals who were classified into three Sasang constitution groups (SCGs), So-Yang (SY), So-Eum (SE), and Tae-Eum (TE). There were no significant difference in the distribution of the APOA5 -1131T>C genotype among the three SCGs. Subjects with the C allele in SY and TE showed significantly lower serum high-density lipoprotein cholesterol (HDL-C) and higher triglyceride (TG) levels than noncarriers of the C allele. These results show the differences in the prevalence of decreasing serum HDL-C and elevating serum TG levels along with APOA5 -1131T>C polymorphism according to SCG and suggest that SCG may act as a significant risk factor for hypo-HDL-C-emia and hypertriglyceridemia susceptibility.

In Vitro and In Vivo Genotoxicity Assessment of Aristolochia manshuriensis Kom.

Arisolochiae species plants containing aristolochic acids I and II (AA I and AA II) are well known to cause aristolochic acid nephropathy (AAN). Recently, there are various approaches to use AAs-containing herbs after the removal of their toxic factors. However, there is little information about genotoxicity of Arisolochiae manshuriensis Kom. (AMK) per se. To obtain safety information for AMK, its genotoxicity was evaluated in accordance with OECD guideline. To evaluate genotoxicity of AMK, we tested bacterial reverse mutation assay, chromosomal aberration test, and micronucleus test. Here, we also determined the amounts of AA I and II in AMK (2.85 ± 0.08 and 0.50 ± 0.02 mg/g extract, resp.). In bacterial reverse mutation assay, AMK dose-dependently increased revertant colony numbers in TA98, TA100 and TA1537 regardless of metabolic activation. AMK increased the incidence of chromosomal aberration in Chinese hamster ovary-K1 cells, but there was no statistically significant difference. The incidences of micronucleus in bone marrow erythrocyte were significantly increased in mice after oral administration of AMK (5000 mg/kg), comparing with those of vehicle group (P < 0.05). The results of three standard tests suggest that the genotoxicity of AMK is directly related to the AAs contents in AMK.

A Novel Herbal Medicine KIOM-MA Exerts an Anti-Inflammatory Effect in LPS-Stimulated RAW 264.7 Macrophage Cells.

KIOM-MA was recently reported as a novel herbal medicine effective for atopic dermatitis and asthma. In this study, we have demonstrated the inhibitory effect of KIOM-MA on proinflammatory mediator produced in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. KIOM-MA significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as nitric oxide (NO) and prostaglandin E(2) (PGE(2)). Consistent with the inhibitory effect on PGE(2), KIOM-MA suppresses the LPS-induced migration of macrophages and gelatinase activity and the expression of matrix metalloprotease-9 (MMP-9) in a dose-dependent manner. Additionally, KIOM-MA showed a strong suppressive effect on the inflammatory cytokines production such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). We also found that KIOM-MA inhibits the activation of nuclear factor-κB (NF-κB) and represses the activity of extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs). Taken together, we elucidated the mechanism of anti-inflammatory effect of KIOM-MA using RAW 264.7 cells stimulated by LPS.

A time-dependently regulated gene network reveals that Aspergillus protease affects mitochondrial metabolism and airway epithelial cell barrier function via mitochondrial oxidants.

The airway epithelium maintains tight barrier integrity to prevent penetration of pathogens; thus, impairment of the barrier function is an important and common histological feature in asthmatic patients. Proteolytic allergens from fungi, pollen, and house dust mites can disrupt epithelial barrier integrity, but the mechanism remains unclear. Aspergillus oryzae protease (AP)-induced mitochondrial reactive oxygen species (ROS) contribute to the epithelial inflammatory response. However, as mitochondrial ROS affect various cellular functions, such as metabolism, cell death, cell proliferation, and redox homeostasis through signal transduction, it is difficult to understand the detailed action mechanism of AP by measuring changes in a single gene or protein of a specific signaling pathway. Moreover, mitochondrial ROS can directly oxidize DNA to activate transcription, thereby affecting the expression of various genes at the transcriptional level. Therefore, we conducted whole-genome analysis and used a network-based approach to understand the effect of AP and AP-induced mitochondrial ROS in human primary airway epithelial cells and to evaluate the mechanistic basis for AP-mediated epithelial barrier dysfunction. Our results indicate that production of mitochondrial ROS following AP exposure induce mitochondrial dysfunction at an early stage. Over time, changes in genome expression were further expanded without remaining mitochondrial ROS. Specifically, genes involved in the apoptotic functions and intercellular junctions were affected, consequently impairing the cellular barrier integrity. This change was recovered by scavenging mitochondrial ROS at an early point after exposure to AP. In conclusion, our findings indicate that instantly increased mitochondrial ROS at the time of exposure to allergenic proteases consequently induces epithelial barrier dysfunction at a later time point, resulting in pathological changes. These data suggest that antioxidant therapy administered immediately after exposure to proteolytic antigens may be effective in maintaining epithelial barrier function.