The Potential of Campanula takesimana Callus Extract to Enhance Skin Barrier Function

Atopic dermatitis (AD) is a prevalent inflammatory skin disease characterized by epidermal barrier dysfunction and Th2-skewed inflammation. Campanula takesimana (C. takesimana), a Korean endemic plant grown on Ulleng Island, has long been associated with a traditional alternative medicine for asthma, tonsillitis, and sore throat. In this study, we reported the effect of C. takesimana callus extract on upregulating epidermal barrier-related proteins dysregulated by Th2 cytokines. C. takesimana callus extract induced the expression of skin barrier proteins, such as filaggrin, claudin-1, and zonula occludens-1, in both human primary keratinocytes and Th2-induced AD-like skin-equivalent models. Additionally, RNA sequencing analysis demonstrated that C. takesimana callus extract partially restored Th2 cytokine-induced dysregulation of the epidermal development and lipid metabolic pathways. Considering the advantages of callus as a sustainable eco-friendly source of bioactive substances, and its effect on skin barrier proteins and lipid metabolic pathways, C. takesimana callus extracts can possibly be utilized to improve the integrity of the skin barrier.


Introduction
A typical pathological feature of atopic dermatitis (AD) is skin barrier dysfunction.Filaggrin (FLG), a filament-associated protein that binds to keratin fibers in epithelial cells, plays an essential role in stratum corneum formation and barrier function [1][2][3][4].During the final stage of epidermal development, FLG is broken down into free amino acids and derivatives, serving as a source of natural moisturizing factors (NMFs) that control stratum corneum hydration and pH [5][6][7][8].Tight junctions in the granular layer support the formation of the skin barrier and regulate transdermal water loss [9].Transmembrane proteins such as claudin-1 (CLDN-1) and zonula occludens-1 (ZO-1), which constitute tight junctions, have been reported to be reduced in the skin of patients with AD [9][10][11].Th2 cytokines, such as IL-4 and IL-13, downregulate the expression of FLG and tight junction proteins, leading to the disruption of skin barrier function [2, 12,13].Defects in skin barrier proteins contribute to dry and inflamed skin, providing a site for allergen penetration and sensitization [14][15][16][17].Additionally, damaged epithelial cells secrete chemokines and pro-inflammatory cytokines, such as interleukin IL-1, thymic stromal lymphopoietin, IL-25, and IL-33, further promoting Th2-skewed inflammation [18,19].Therefore, effectively restoring the Th2-induced dysregulation of skin barrier proteins can improve skin barrier function in AD patients.
Campanula takesimana (C.takesimana), also known as Korean bellflower or Seomchorongkkot, is a Korean endemic herb that grows on Ulleng Island.Ulleng Island is a volcanic island located in the East Sea of Korea, characterized by various plant communities.This island exhibits unique vegetation and plant distribution influenced by environmental characteristics [20].Currently, there are 550 species of native plants, including C. takesimana.In Korean folklore, C. takesimana has been associated with an effective herbal remedy for Th2-mediated diseases, such as bronchitis and asthma [21].Furthermore, the root of C. takesimana has been used in herbal medicine to treat asthma, tonsillitis, and sore throat [20].However, the mechanisms by which C. takesimana regulates Th2 inflammatory diseases are not well understood.
A callus is defined as a mass of dedifferentiated cells which possesses totipotency [22].Calluses exhibit high plasticity for cell differentiation, which is a central characteristic.The induction of a callus in vitro has significant benefits compared to using the plant itself as a source of ingredients with biological activity, as it does not harm the environment and can be grown in vitro.
In this study, we induced calluses from C. takesimana and validated their efficacy on the expression of barrier proteins in primary keratinocytes and 3D skin equivalents.The extract from a C. takesimana callus successfully restored the Th2-induced dysregulation of the skin barrier-associated proteins and lipid metabolic pathways.These findings suggest that C. takesimana, a native herb of Ulleng Island, may be useful in enhancing epidermal barrier function.

Induction of Calluses and the HPLC Analysis of the Callus Extract
We induced callus formation from C. takesimana leaves using callus culture technology.The sterilized and cut C. takesimana leaves were placed in gel media supplemented with various combinations of auxin and plant growth regulators (PGRs) (Figure 1a-c).After 4 weeks, undifferentiated calluses had formed (Figure 1d,e), with several calluses turning brown by 8 weeks (Figure 1f).We selected the culture conditions that promoted the highest growth rate with minimal browning of the callus and propagated it in the chosen culture medium supplemented with 1 mg/L of 2,4-dichlorophenoxyacetic acid, an auxin-based growth regulator.Calluses grown in the selected culture medium exhibited no browning (Figure 1g).Subsequently, we transferred the calluses from the gel medium to liquid medium supplemented with 1 mg/L of 2,4-dichlorophenoxyacetic acid and cultured them in 10 L-capacity bioreactors for mass culture of the calluses (Figure 1h,i).The calluses were harvested and dried to extract bioactive compounds.
Next, we analyzed the C. takesimana callus extract using high-performance liquid chromatography (HPLC) and compared it to those in the C. takesimana plant extract.The analysis of both the plant extract and the callus extract revealed differences in their components, as observed in the chromatograms.In the callus extract, guanine, adenine, uridine, adenosine, and guanosine were detected, whereas these peaks did not appear in the plant extract (Figure S1a,b), indicating the higher proliferating activity of the callus compared to the plant itself.

Decreased Expression of FLG, ZO-1, and CLDN-1 by Th2 Cytokines Was Reversed by the C. takesimana Callus Extract
To explore the function of the C. takesimana callus extract on the skin, we assessed its impact on IL-4/IL-13-induced alterations in normal human epidermal keratinocytes (NHEKs).Various concentrations of the C. takesimana callus extract were added to the NHEK culture medium for 24 h for the viability and proliferation assay.Within the concentration range of 0-4 mg/mL, the callus extract significantly increased keratinocyte viability (Figure S2a) and proliferation (Figure S2b) at a concentration of 0.4 mg/mL.Based on the results, we selected concentrations of 0.04 mg/mL and 0.4 mg/mL for further experiments.Next, we analyzed the C. takesimana callus extract using high-performance liquid chromatography (HPLC) and compared it to those in the C. takesimana plant extract.The analysis of both the plant extract and the callus extract revealed differences in their components, as observed in the chromatograms.In the callus extract, guanine, adenine, uridine, adenosine, and guanosine were detected, whereas these peaks did not appear in the plant extract (Figure S1a,b), indicating the higher proliferating activity of the callus compared to the plant itself.

Decreased Expression of FLG, ZO-1, and CLDN-1 by Th2 Cytokines Was Reversed by the C. takesimana Callus Extract
To explore the function of the C. takesimana callus extract on the skin, we assessed its impact on IL-4/IL-13-induced alterations in normal human epidermal keratinocytes (NHEKs).Various concentrations of the C. takesimana callus extract were added to the NHEK culture medium for 24 h for the viability and proliferation assay.Within the concentration range of 0-4 mg/mL, the callus extract significantly increased keratinocyte viability (Figure S2a) and proliferation (Figure S2b) at a concentration of 0.4 mg/mL.Based on the results, we selected concentrations of 0.04 mg/mL and 0.4 mg/mL for further experiments.
Barrier-associated proteins such as FLG and tight junction proteins, including ZO-1 and CLDN-1, are known to be downregulated in the skin of individuals with AD due to enhanced Th2 inflammation [23][24][25].To mimic Th2-induced barrier disruption, we treated NHEKs with Th2 cytokines, namely IL-4 and IL-13.The mRNA expression of FLG increased in calcium chloride-treated differentiated keratinocytes, but was significantly decreased by IL-4 and IL-13 treatment (Figure S3a,b).Next, we investigated whether treatment with the C. takesimana plant extract or C. takesimana callus extract on differentiated keratinocytes could regulate the expression of epidermal barrier-associated proteins.Although the C. takesimana plant extract had no effect on FLG expression (Figure S4a,b), the Barrier-associated proteins such as FLG and tight junction proteins, including ZO-1 and CLDN-1, are known to be downregulated in the skin of individuals with AD due to enhanced Th2 inflammation [23][24][25].To mimic Th2-induced barrier disruption, we treated NHEKs with Th2 cytokines, namely IL-4 and IL-13.The mRNA expression of FLG increased in calcium chloride-treated differentiated keratinocytes, but was significantly decreased by IL-4 and IL-13 treatment (Figure S3a,b).Next, we investigated whether treatment with the C. takesimana plant extract or C. takesimana callus extract on differentiated keratinocytes could regulate the expression of epidermal barrier-associated proteins.Although the C. takesimana plant extract had no effect on FLG expression (Figure S4a,b), the C. takesimana callus extract induced FLG expression in a concentration-dependent manner in differentiated keratinocytes, both in the absence of Th2 cytokines (Figure 2a, left panel) and in their presence (Figure 2a, right panel).
When we treated differentiated NHEKs with the C. takesimana callus extract, the mRNA expression of the tight junction protein ZO-1 increased at 400 µg/mL (Figure 2b, left panel).However, in the presence of IL-4 and IL-13, the callus extract did not significantly alter ZO-1 expression (Figure 2b, right panel).On the other hand, CLDN-1 expression was unaffected by the callus extract in differentiated primary keratinocytes without Th2 cytokines (Figure 2c, left panel), but it was significantly elevated by the callus extract in the presence of IL-4 and IL-13 (Figure 2c, right panel).
To validate the impact of the callus extract on epidermal development, we used skin equivalents cultured with IL-4 and IL-13.IL-4 and IL-13 significantly downregulated FLG, ZO-1, and CLDN-1 expression in the epidermal layer of the skin equivalents, and the C. takesimana callus extract successfully restored the expression of these skin barrier-related proteins (Figure 2d).These findings collectively demonstrated that the callus extract from C. takesimana exhibits a beneficial effect on upregulating epidermal barrier proteins and tight junction proteins that are downregulated in AD-affected skin.When we treated differentiated NHEKs with the C. takesimana callus extract, the mRNA expression of the tight junction protein ZO-1 increased at 400 µg/mL (Figure 2b, left panel).However, in the presence of IL-4 and IL-13, the callus extract did not significantly alter ZO-1 expression (Figure 2b, right panel).On the other hand, CLDN-1 expression was unaffected by the callus extract in differentiated primary keratinocytes without Th2 cytokines (Figure 2c, left panel), but it was significantly elevated by the callus extract in the presence of IL-4 and IL-13 (Figure 2c, right panel).
To validate the impact of the callus extract on epidermal development, we used skin equivalents cultured with IL-4 and IL-13.IL-4 and IL-13 significantly downregulated FLG, ZO-1, and CLDN-1 expression in the epidermal layer of the skin equivalents, and the C. takesimana callus extract successfully restored the expression of these skin barrier-related proteins (Figure 2d).These findings collectively demonstrated that the callus extract from C. takesimana exhibits a beneficial effect on upregulating epidermal barrier proteins and tight junction proteins that are downregulated in AD-affected skin.

The C. takesimana Callus Extract Restores Lipid Biosynthesis and the Epidermal Development Pathway
We analyzed the whole genes that are downregulated or upregulated by Th2 cytokines and restored with the C. takesimana callus extract in differentiated NHEKs via RNA sequencing analysis.A total of 609 differentially expressed genes (DEGs) were identified as downregulated by Th2 cytokines and upregulated by the C. takesimana callus extract.On the other hand, 148 DEGs were identified as upregulated by Th2 cytokines and

The C. takesimana Callus Extract Restores Lipid Biosynthesis and the Epidermal Development Pathway
We analyzed the whole genes that are downregulated or upregulated by Th2 cytokines and restored with the C. takesimana callus extract in differentiated NHEKs via RNA sequencing analysis.A total of 609 differentially expressed genes (DEGs) were identified as downregulated by Th2 cytokines and upregulated by the C. takesimana callus extract.On the other hand, 148 DEGs were identified as upregulated by Th2 cytokines and downregulated by the C. takesimana callus extract.The top 30 upregulated and downregulated DEGs by the C. takesimana callus extract are demonstrated in Table 1.Gene ontology (GO) analysis demonstrated that several pathways were restored with the C. takesimana callus extract, which was suppressed by Th2 cytokines.These pathways include the cholesterol biosynthetic process, sterol biosynthetic process, sphingolipid metabolic process, epidermis development, and response to endoplasmic reticulum stress (Figure 3a).Regarding the GO pathways that were increased by Th2 cytokines and decreased by the C. takesimana callus extract, they include the positive regulation of translation in response to stress, nucleosome disassembly, and regulation of mitochondrial membrane potential, among others (Figure 3b).
role in maintaining the barrier function of the skin, was markedly restored with the C. takesimana callus extract (Figure 4c).Several genes involved in the regulation of transcription, DNA templated, were highly upregulated in the presence of IL-4 and IL-13.However, their relative expression levels were significantly reduced by the C. takesimana callus extract (Figure 4d).Likewise, genes involved in the regulation of mitochondrial membrane potentials, such as superoxide dismutase 1 (SOD1) and peroxiredoxin 3 (PRDX3), displayed a similar trend (Figure 4e).The mRNA expression of (f) HMGCR, (g) ACER1, (h) SGPP2, and (i) TGM3 was analyzed via qRT-PCR.All data were analyzed via the one-way ANOVA analysis followed by Tukey's multiple comparisons test and shown as the mean ± SD (n = 4 per group; ** p < 0.01 and **** p < 0.0001).
To validate the RNA sequencing analysis, we confirmed the expression of HMGCR, ACER1, SGPP2, and TGM3, which are involved in the cholesterol biosynthesis pathway, sphingolipid metabolic processes, and skin development, through qRT-PCR.Consistent with the RNA sequencing results, these genes were downregulated in Th2 cytokinetreated keratinocytes and restored in callus extract-treated keratinocytes (Figure 4f-i).

Discussion
Previous studies have provided evidence for the antioxidative and anti-inflammatory effects of C. takesimana [20,21].The ethyl acetate fraction of the C. takesimana extract exhibited free radical scavenging activity comparable to that of ascorbic acid [20].Moreover, the extract of C. takesimana leaves has been found to inhibit the production of prostaglandin E2 induced by lipopolysaccharides [26].These previous findings suggest the potential therapeutic value of C. takesimana in modulating inflammatory responses.In our study, we found another beneficial effect of C. takesimana in that the callus of C. takesimana increases the expression of barrier-related proteins and potentially restores lipid metabolism and the epidermal development process.
In this study, we induced calluses from the leaves of C. takesimana and obtained an extract using hot water.Since C. takesimana has been used as an herbal medicine to improve bronchitis and asthma in Korea, we attempted to investigate whether C. takesimana can restore barrier-related proteins affected by Th2 cytokines.We observed that the C. takesimana callus extract not only increased the expression of FLG, ZO-1, and CLDN-1 in the keratinocytes but also in the 3D skin equivalents.Interestingly, these effects of C. takesimana on FLG expression were only observed in the callus extract.When the callus extract and the plant extract were treated with the differentiating NHEKs, the plant extract did not induce FLG expression and did not restore the reduced FLG expression caused by Th2 cytokines.
Th2-skewed immune response is a key pathomechanism in AD.Increased Th2 cytokines, including of IL-4 and IL-13, lead to a reduction in skin barrier-related proteins, such as FLG, and impair skin barrier function.Several studies have shown that a lack of FLG interferes with epidermal maturation function, as well as alters skin lipid composition and organization.In addition, FLG mutations cause reduced NMF, which increases skin pH and accelerates barrier dysfunction [4][5][6].Furthermore, tight junction proteins expressed in the stratum granulosum are another core component in the formation of the epidermal barrier.Tight junctions and the stratum corneum have a synergistic effect on the formation of a strong skin barrier, and reduced expression of tight junction proteins and decreased skin barrier function have been well reported in AD [27,28].Thus, restoring skin barrier-related proteins is important in controlling the disease activity of AD.Since our study showed that the extract of C. takesimana callus successfully restored Th2-induced dysregulation of FLG and tight junction proteins, we speculate that it could improve the skin barrier function of patients with AD.
In addition to epidermal differentiation and barrier formation, RNA sequencing analysis revealed that lipid metabolism-related pathways, including the cholesterol biosynthetic process, sterol biosynthetic process, and sphingolipid metabolic process, were initially suppressed by Th2 cytokines but partially restored with the C. takesimana callus extract.In AD, there is a reduction in ceramides, one of the main lipids that surround the outer layer of corneocytes, within the stratum corneum [29,30].Th2 cytokines can regulate the expression of ceramide metabolic enzymes and modulate ceramide levels [31,32].The diminished synthesis of ceramides, which are important components of the skin barrier lipids, leads to impaired formation of the lamellar body, corneocyte lipid envelope, and an overall reduction in epidermal lipids, resulting in skin barrier dysfunction.In our study, IL-4 and IL-13 reduced the expression of genes associated with the lipid metabolic pathways, and the C. takesimana callus extract restored the expression of these genes.These results indicate that the C. takesimana callus extract can not only restore barrier protein expression but also restore the ceramide metabolic pathway.
Plant cells possess high plasticity for cell differentiation.In response to various stresses, such as wounding or pathogen infection, plants can generate unorganized cell masses known as calluses.A callus is defined as a mass of dedifferentiated cells or somatic embryos with a single-cell origin, meaning the callus is similar to plant cells in that it possesses totipotency.The induction of a callus in vitro has significant benefits compared to using the plant itself as a source of ingredients with biological activity, as it does not destroy the environment and can be grown in vitro.Additionally, the callus compared to the plant itself could be more biologically beneficial, as demonstrated in our study.
Taken together, the C. takesimana callus extract could help in regulating skin barrier proteins and lipid metabolism pathways, ultimately strengthening the skin barrier function.Considering the advantages of calluses as a sustainable and eco-friendly source of active materials, C. takesimana callus extracts could be possibly utilized for improving skin barrier function.

Induction of the Callus and Optimization of Culture Medium
To induce the callus, the C. takesimana leaves were sterilized sequentially with 70% ethanol for 30 s and 0.3% sodium hypochlorite for 20 min, washed with distilled water three times, and then cut into 0.5-1 cm portions.The leaves were cultured in a medium supplemented with various combinations of auxin and plant growth regulator (PGR) under dark conditions at 25 ± 2 • C.After 8 weeks, the color, morphology, and differentiation of the callus were compared, and the best combination of auxin and PGR for culture was selected.The C. takesimana callus was then mass-cultured in the selected culture medium.

Preparing Test Samples from C. takesimana Callus and Leaf
The callus was dried at 60 • C for 2 days to remove water and then powdered.The dried plant material and callus of C. takesimana were extracted under reflux by adding 25 times the volume of water and heating at 100 • C for 1 h.Each extract was centrifuged at 13,000 rpm for 10 min and filtered through a 0.45 µm membrane filter (PTFE, Advantec, Tokyo, Japan) to obtain each extract stock solution.

HPLC Analysis of the Samples
To compare the chromatographic data of the water extracts from the callus and leaf, instrumental analysis using HPLC (1260 Infinity II system, Agilent Technologies, Santa Clara, CA, USA) was carried out.All reagents using their mobile phase were of HPLC grade.Water and acetonitrile of Samchun Pure Chemicals (Republic of Korea) and trifluoroacetic acid of Alfa Aesar (Schiltigheim, France) were used.The analytical column was Shim-pack GIS C18 (5 µm, 4.6 × 250 mm; Shimadzu, Kyoto, Japan), and the eluent was a mixture of mobile phase A (water containing 0.1% trifluoroacetic acid) and B (acetonitrile containing 0.1% trifluoroacetic acid).The elution was performed under the following conditions: The elution was performed with mobile phase A at 100% (mobile phase B at 0%) for 5 min, followed by an increase in mobile phase B at a rate of 1% per minute.The flow rate was 1.0 mL/min (with an injection volume of 20 µL), and the data were collected at UV 255 nm.All samples were filtered through a 0.45 µm syringe filter (PTFE, Advantec) before injection.

Cell Culture
NHEKs were cultured in dermal cell basal medium (ATCC, Manassas, VA, USA) supplemented with bovine pituitary extract and recombinant growth factors (ATCC).Cultures were maintained in a 5% CO 2 , 37 • C incubator.For differentiation, cells were seeded and grown to 90% confluency and then cultured in the keratinocyte medium containing 1.5 mM CaCl 2 for 5 days.Th2 cytokines, 10 ng/mL of IL-4 and IL-13 (Peprotech, NJ, USA), were treated with keratinocytes to induce the atopic dermatitis-like changes in gene expression in the presence or absence of the C. takesimana callus extract.

The Cell Viability and Proliferation Assay
Cell viability was measured using the EZ-Cytox Plus assay kit (Dogenbio, Seoul, Republic of Korea).Cells were seeded on 48-well plates at a density of 1.0 × 10 4 cells/well, and the callus extract was treated at different concentrations for 24 h.The viability and proliferation assay was performed according to the manufacturer's protocol.In brief, cells treated with the callus extract were exposed to 10 µL of EZ-Cytox reagent, followed by a 4 h incubation period.Subsequently, absorbance was measured at 480 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA).Cell viability was calculated relative to the absorbance of the control group, and the cell proliferation rate was determined by dividing the absorbance by the protein quantity, normalized to the values of the control group.

RNA Isolation and Quantitative Real-Time PCR
The mRNA expression level was analyzed via quantitative real-time PCR using SYBR (Bioneer, Daejeon, Republic of Korea).Total RNA was isolated using TRIzol (Invitrogen, Waltham, MA, USA), and cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and oligo dT primers (Cosmogenetech, Seoul, Republic of Korea).The PCR conditions were as follows: initial denaturation for 15 min at 95 • C; followed by 40 cycles of 95 • C for 15 s, 60 • C for 45 s, and 72 • C for 30 s, for primer annealing and extension.Relative mRNA levels were calculated via normalization to the reference gene, GAPDH.

RNA Sequencing Data Analysis
RNA sequencing analysis was performed using differentiated NHEKs, which were treated with 10 ng/mL of IL-4 and 10 ng/mL of IL-13 with or without 0.4 mg/mL of callus extract for 5 days.RNA sequencing analyses were performed at Theragen Bio Institute (Suwon, Republic of Korea).The libraries were prepared for 150 bp paired-end sequencing using the TruSeq RNA sample prep kit (Illumina, San Diego, CA, USA).A total of 1 µg of RNA molecules was purified and fragmented, and then synthesized as single-stranded cDNAs via random hexamer priming.Using this as a template to synthesize the second strand, a double-stranded cDNA was prepared.cDNA libraries were amplified with PCR after a sequential process of end repair, A-tailing, and adapter ligation.The quality of these cDNA libraries was evaluated with the Agilent 2100 BioAnalyzer (Agilent, USA), and was quantified with the KAPA library quantification kit (Kapa Biosystems, Wilmington, MA, USA) in accordance with the manufacturer's library quantification protocol.Cluster amplification of denatured templates was followed by paired-end (2 × 150 bp) sequencing using Illumina Novaseq6000 (Illumina).
FastQC (v0.12.0) was used to examine the quality of raw RNA-seq data, and the adapter sequences were removed using Trimmomatic (v0.40) [33].Then, clean reads were aligned to the human reference genome (hg38) using STAR (v2.7.10a) [34], and RSEM (v1.3.3)[35] was used to quantify the gene expression levels.EBSeq [36] was used to calculate the differentially expressed genes (DEGs) based on the expected counts.The genes with a false discovery rate (FDR) of 0.05 or less and the absolute value of log2 fold change of 0.5 or higher were considered DEGs.
Gene set enrichment analysis (GSEA) was subsequently carried out on the DEGs to illustrate the functions of the C. takesimana callus extract and its biological pathways using Enrichr [37].The gene ontology (GO) terms with a p-value less than 0.05 were considered statistically significant.The top 10 terms were analyzed and displayed using dot plots.The size of the dot represented the count of genes, and the color represents the p-value.
Heatmaps were generated to illustrate the effect of the C. takesimana callus extract by comparing the subsets of DEGs, which were selected based on having adjusted p-values of 0.05 or less and a log2 fold change value of 1.0 or higher.The relative intensities of the identified bioactive compounds were visualized using red color for the higher concentration and blue color for the lower concentration.

Producing a 3D-Reconstructed Human Skin Equivalent
The 3D-reconstructed human skin equivalents were made in triplicate, in accordance with the previous report [38].Briefly, primary dermal fibroblast sheets were created by fibroblasts secreting their own extracellular matrix for 4 weeks.Primary keratinocytes were grown on a dermal sheet to form an epidermal layer.A dermal sheet and a dermalepidermal equivalent were stacked to generate a full-thickness skin layer, and the skin equivalents were incubated at the air-liquid interface for 14 days.Ascorbic acid was added to the culture medium during the whole culture period to induce extracellular matrix secretion.The skin equivalents were grown in the culture medium with 100 ng/mL of IL-4 and IL-13 cytokines in the presence or absence of the callus extracts over a period of the air-liquid interface to make an in vitro atopic dermatitis-like 3D-reconstructed human skin equivalent model.

Figure 1 .
Figure 1.Callus induction from Campanula takesimana (C.takesimana).(a,b) Sterilization of leaf tissue.(c) Cutting the leaf tissue for plating.(d,e) Plating the leaf tissue on callus induction media.(f) Callus formation out of the leaf tissue.(g) Selecting a callus with the highest growth rate with no browning.(h) Suspension culture.(i) Bioreactor culture for mass production.

Figure 1 .
Figure 1.Callus induction from Campanula takesimana (C.takesimana).(a,b) Sterilization of leaf tissue.(c) Cutting the leaf tissue for plating.(d,e) Plating the leaf tissue on callus induction media.(f) Callus formation out of the leaf tissue.(g) Selecting a callus with the highest growth rate with no browning.(h) Suspension culture.(i) Bioreactor culture for mass production.

Figure 2 .
Figure 2. C. takesimana callus extract induces FLG, ZO-1, and CLDN-1 expression in NHEKs and skin equivalents.(a-c) A total of 40 µg/mL or 400 µg/mL of C. takesimana callus extract in the absence (left panel) or presence (right panel) of 10 ng/mL of IL-4/IL-13.The mRNA expression for (a) FLG, (b) ZO-1, and (c) CLDN-1 was measured via qRT-PCR.(d) Skin equivalents were treated with C. takesimana callus extract with or without 100 ng/mL of IL-4/IL-13.The expression of FLG, ZO-1, and CLDN-1 was detected via immunofluorescence staining.Hoechst was used for nuclear staining.Scale bar = 50 µm.All data were analyzed using the one-way ANOVA followed by Tukey's multiple comparisons test and shown as the mean ± SD (n = 4 per group; * p < 0.05; n.s.not significant).

Figure 2 .
Figure 2. C. takesimana callus extract induces FLG, ZO-1, and CLDN-1 expression in NHEKs and skin equivalents.(a-c) A total of 40 µg/mL or 400 µg/mL of C. takesimana callus extract in the absence (left panel) or presence (right panel) of 10 ng/mL of IL-4/IL-13.The mRNA expression for (a) FLG, (b) ZO-1, and (c) CLDN-1 was measured via qRT-PCR.(d) Skin equivalents were treated with C. takesimana callus extract with or without 100 ng/mL of IL-4/IL-13.The expression of FLG, ZO-1, and CLDN-1 was detected via immunofluorescence staining.Hoechst was used for nuclear staining.Scale bar = 50 µm.All data were analyzed using the one-way ANOVA followed by Tukey's multiple comparisons test and shown as the mean ± SD (n = 4 per group; * p < 0.05; n.s.not significant).

Figure 3 .
Figure 3. Gene set enrichment analysis comparing DEGs in control NHEKs, IL−4/IL−13-treated NHEKs, and NHEKs treated with both IL−4/IL−13 and C. takesimana callus extract.(a) The top 10 gene ontology terms enriched in genes downregulated by Th2 cytokines and upregulated by the C. takesimana callus extract.(b) The top 10 gene ontology terms enriched in genes upregulated by Th2 cytokines and downregulated by the C. takesimana callus extract.

Figure 3 .
Figure 3. Gene set enrichment analysis comparing DEGs in control NHEKs, IL−4/IL−13-treated NHEKs, and NHEKs treated with both IL−4/IL−13 and C. takesimana callus extract.(a) The top 10 gene ontology terms enriched in genes downregulated by Th2 cytokines and upregulated by the C. takesimana callus extract.(b) The top 10 gene ontology terms enriched in genes upregulated by Th2 cytokines and downregulated by the C. takesimana callus extract.

Table 1 .
The top 30 upregulated and downregulated DEGs.FDR, false discovery rate; and FC, fold change.The functions of the genes were described with reference to http://www.ncbi.nlm.nih.gov/gene (accessed on 21 November 2023).