• IMMUNE NETWORK
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• 제23권2호
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• pp.15.1-15.17
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• 2023

Innate lymphoid cells (ILCs) are critical immune-response mediators. Although they largely reside in mucosal tissues, the kidney also bears substantial numbers. Nevertheless, kidney ILC biology is poorly understood. BALB/c and C57BL/6 mice are known to display type-2 and type-1 skewed immune responses, respectively, but it is unclear whether this extends to ILCs. We show here that indeed, BALB/c mice have higher total ILCs in the kidney than C57BL/6 mice. This difference was particularly pronounced for ILC2s. We then showed that three factors contributed to the higher ILC2s in the BALB/c kidney. First, BALB/c mice demonstrated higher numbers of ILC precursors in the bone marrow. Second, transcriptome analysis showed that compared to C57BL/6 kidneys, the BALB/c kidneys associated with significantly higher IL-2 responses. Quantitative RT-PCR also showed that compared to C57BL/6 kidneys, the BALB/c kidneys expressed higher levels of IL-2 and other cytokines known to promote ILC2 proliferation and/or survival (IL-7, IL-33, and thymic stromal lymphopoietin). Third, the BALB/c kidney ILC2s may be more sensitive to the environmental signals than C57BL/6 kidney ILC2s since they expressed their transcription factor GATA3 and the IL-2, IL-7, and IL-25 receptors at higher levels. Indeed, they also demonstrated greater responsiveness to IL-2 than C57BL/6 kidney ILC2s, as shown by their greater STAT5 phosphorylation levels after culture with IL-2. Thus, this study demonstrates previously unknown properties of kidney ILC2s. It also shows the impact of mouse strain background on ILC2 behavior, which should be considered when conducting research on immune diseases with experimental mouse models.

• 대한약침학회지
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• 제27권2호
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• pp.131-141
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• 2024

Objectives: Polycystic ovary syndrome (PCOS) is one of the most common disorders and it shows up to 20% prevalence in reproductive-aged women populations, but no cures are available to date. We aimed to investigate the protective effects of Changbudodam-tang (CBD) on cell death signaling pathways, inflammation, and oxidative stress observed in Bone-Marrow derived human mesenchymal stem cell (BM-hMSC) by means of PCOS therapeutics in the future. Methods: BM-hMSCs were applied with cell deaths and injuries. Apoptosis and pyroptosis signals were quenched with their related signaling pathways using quantitative PCR, Western blot, and fluorescence image analysis. Results: Our data clearly displayed hydrogen peroxide- and nigericin-treated cell death signaling pathways via regulations of mitochondrial integrity and interleukin (IL)-1β at the cellular levels (p < 0.01 or 0.001). We further observed that pre-treatment with CBD showed protective effects against oxidative stress by enhancement of antioxidant components at the cellular level, with respect to both protein and mRNA expression levels (p < 0.05, 0.01 or 0.001). The mechanisms of CBD were examined by Western blot analysis, and it showed anti-cell death, anti-inflammatory, and antioxidant effects via normalizations of the Jun N-terminal kinase/mitogen-activated protein kinase kinase 7/c-Jun signaling pathways. Conclusion: This study confirmed the pharmacological properties of CBD by regulation of cellular oxidation and the inflammation-provoked cell death condition of BM-hMSCs, which is mediated by the MKK7/JNK/c-Jun signaling pathway.

• 대한약침학회지
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• 제27권2호
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• pp.162-171
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• 2024

Objectives: Electroacupuncture (EA) has been demonstrated to aid stroke recovery. However, few investigations have focused on identifying the potent molecular targets of EA by comparing EA stimulation between naïve and disease models. Therefore, this study was undertaken to identify the potent molecular therapeutic mechanisms underlying EA stimulation in ischemic stroke through a comparison of mRNA sequencing data obtained from EA-treated naïve control and ischemic stroke mouse models. Methods: Using both naïve control and middle cerebral artery occlusion (MCAO) mouse models, EA stimulation was administered at two acupoints, Baihui (GV20) and Dazhui (GV14), at a frequency of 2 Hz. Comprehensive assessments were conducted, including behavioral evaluations, RNA sequencing to identify differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction (PPI) network analysis, and quantitative real-time PCR. Results: EA stimulation ameliorated the ischemic insult-induced motor dysfunction in mice with ischemic stroke. Comparative analysis between control vs. MCAO, control vs. control + EA, and MCAO vs. MCAO + EA revealed 4,407, 101, and 82 DEGs, respectively. Of these, 30, 7, and 1 were common across the respective groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed upregulated DEGs associated with the regulation of inflammatory immune response in the MCAO vs. MCAO + EA comparison. Conversely, downregulated DEGs in the control vs. control + EA comparison were linked to neuronal development. PPI analysis revealed major clustering related to the regulation of cytokines, such as Cxcl9, Pcp2, Ccl11, and Cxcl13, in the common DEGs of MCAO vs. MCAO + EA, with Esp8l1 identified as the only common downregulated DEG in both EA-treated naïve and ischemic models. Conclusion: These findings underscore the diverse potent mechanisms of EA stimulation between naïve and ischemic stroke mice, albeit with few overlaps. However, the potent mechanisms underlying EA treatment in ischemic stroke models were associated with the regulation of inflammatory processes involving cytokines.

• IMMUNE NETWORK
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• 제21권3호
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• pp.22.1-22.17
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• 2021

Chitinase-3-like-1 (CHI3L1) is known to induce inflammation in the progression of allergic diseases. Previous our studies revealed that 2-({3-[2-(1-cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111; K284), the CHI3L1 inhibiting compound, has the anti-inflammatory effect on neuroinflammation. In this study, we investigated that K284 treatment could inhibit the development of atopic dermatitis (AD). To identify the effect of K284, we used phthalic anhydride (5% PA)-induced AD animal model and in vitro reconstructed human skin model. We analyzed the expression of AD-related cytokine mediators and NF-κB signaling by Western blotting, ELISA and quantitative real-time PCR. Histological analysis showed that K284 treatment suppressed PA-induced epidermal thickening and infiltration of mast cells. K284 treatment also reduced PA-induced release of inflammatory cytokines. In addition, K284 treatment inhibited the expression of NF-κB activity in PA-treated skin tissues and TNF-α and IFN-γ-treated HaCaT cells. Protein-association network analysis indicated that CHI3L1 is associated with lactoferrin (LTF). LTF was elevated in PA-treated skin tissues and TNF-α and IFN-γ-induced HaCaT cells. However, this expression was reduced by K284 treatment. Knockdown of LTF decreased the expression of inflammatory cytokines in TNF-α and IFN-γ-induced HaCaT cells. Moreover, anti-LTF antibody treatment alleviated AD development in PA-induced AD model. Our data demonstrate that CHI3L1 targeting K284 reduces AD-like skin inflammation and K284 could be a promising therapeutic agent for AD by inhibition of LTF expression.

• IMMUNE NETWORK
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• 제22권3호
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• pp.25.1-25.11
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• 2022

IL-34 can promote osteoclast differentiation and activation, which may contribute to steroid-induced osteonecrosis of the femoral head (ONFH). Animal model was constructed in both BALB/c and IL-34 deficient mice to detect the relative expression of inflammation cytokines. Micro-CT was utilized to reveal the internal structure. In vitro differentiated osteoclast was induced by culturing bone marrow-derived macrophages with IL-34 conditioned medium or M-CSF. The relative expression of pro-inflammation cytokines, osteoclast marker genes, and relevant pathways molecules was detected with quantitative real-time RT-PCR, ELISA, and Western blot. Up-regulated IL-34 expression could be detected in the serum of ONFH patients and femoral heads of ONFH mice. IL-34 deficient mice showed the resistance to ONFH induction with the up-regulated trabecular number, trabecular thickness, bone value fraction, and down-regulated trabecular separation. On the other hand, inflammatory cytokines, such as TNF-α, IFN-γ, IL-6, IL-12, IL-2, and IL-17A, showed diminished expression in IL-34 deficient ONFH induced mice. IL-34 alone or works in coordination with M-CSF to promote osteoclastogenesis and activate ERK, STAT3, and non-canonical NF-κB pathways. These data demonstrate that IL-34 can promote the differentiation of osteoclast through ERK, STAT3, and non-canonical NF-κB pathways to aggravate steroid-induced ONFH, and IL-34 can be considered as a treatment target.

• The Korean Journal of Physiology and Pharmacology
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• 제28권1호
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• pp.93-103
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• 2024

Satellite glial cells (SGCs), a major type of glial cell in the autonomic ganglia, closely envelop the cell body and even the synaptic regions of a single neuron with a very narrow gap. This structurally unique organization suggests that autonomic neurons and SGCs may communicate reciprocally. Glial Ca²⁺ signaling is critical for controlling neural activity. Here, for the first time we identified the machinery of store-operated Ca²⁺ entry (SOCE) which is critical for cellular Ca²⁺ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative realtime PCR and immunostaining analyses showed that Orai1 and stromal interaction molecules 1 (STIM1) proteins are the primary components of SOCE machinery in the sympathetic ganglia. When the internal Ca²⁺ stores were depleted in the absence of extracellular Ca²⁺, the number of plasmalemmal Orai1 puncta was increased in neurons and SGCs, suggesting activation of the Ca²⁺ entry channels. Intracellular Ca²⁺ imaging revealed that SOCE was present in SGCs and neurons; however, the magnitude of SOCE was much larger in the SGCs than in the neurons. The SOCE was significantly suppressed by GSK7975A, a selective Orai1 blocker, and Pyr6, a SOCE blocker. Lipopolysaccharide (LPS) upregulated the glial fibrillary acidic protein and Toll-like receptor 4 in the sympathetic ganglia. Importantly, LPS attenuated SOCE via downregulating Orai1 and STIM1 expression. In conclusion, sympathetic SGCs functionally express the SOCE machinery, which is indispensable for intracellular Ca²⁺ signaling. The SOCE is highly susceptible to inflammation, which may affect sympathetic neuronal activity and thereby autonomic output.

• Journal of Plant Biotechnology
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• 제50권
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• pp.127-136
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• 2023

Water scarcity decreases the rate of photosynthesis and, consequently, the yield of banana plants (Musa spp). In this study, transcriptome analysis was performed to identify photosynthesis-related genes in banana plants and determine their expression profiles under water stress conditions. Banana plantlets were in vitro cultured on Murashige and Skoog agar medium with and without 10% polyethylene glycol and marked as BP10 and BK. Chlorophyll contents in the plant shoots were determined spectrophotometrically. Two cDNA libraries generated from BK and BP10 plantlets, respectively, were used as the reference for transcriptome data. Gene ontology (GO) enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) and visualized using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway prediction. Morphological observations indicated that water deficiency caused chlorosis and reduced the shoot chlorophyll content of banana plantlets. GO enrichment identified 52 photosynthesis-related genes that were affected by water stress. KEGG visualization revealed the pathways related to the 52 photosynthesisr-elated genes and their allocations in four GO terms. Four, 12, 15, and 21 genes were related to chlorophyll biosynthesis, the Calvin cycle, the photosynthetic electron transfer chain, and the light-harvesting complex, respectively. Differentially expressed gene (DEG) analysis using DESeq revealed that 45 genes were down-regulated, whereas seven genes were up-regulated. Four of the down-regulated genes were responsible for chlorophyll biosynthesis and appeared to cause the decrease in the banana leaf chlorophyll content. Among the annotated DEGs, MaPNDO, MaPSAL, and MaFEDA were selected and validated using quantitative real-time PCR.

• 한국발생생물학회지:발생과생식
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• 제27권4호
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• pp.185-193
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• 2023

Although increasing evidence of cause-and-effect relationship between BPA exposure and female reproductive disorders have been suggested through many studies, the precise biochemical and molecular mechanism(s) by which BPA interferes with steroidogenesis in the ovarian cells still remain unclear. Therefore, the purpose of this study was to discover the steroidogenic biomarker(s) associated with BPA treatment in human granulosa cell line, KGN. In this study, our results obtained via the analysis of steroidogenesis-related protein expression in KGN cells using quantitative polymerase chain reaction (qPCR) and western blot analyses revealed that the expression levels of steroidogenic acute regulatory (StAR) and aromatase decreased considerably and gradually after BPA treatment in a dose-dependent manner under BPA treatment. Further, remarkable decreases in their expression levels at the cellular levels were also confirmed via immunocytochemistry, and subsequent StAR and aromatase mRNA expression levels showed profiles similar to those observed for their proteins, i.e., both StAR and aromatase mRNA expression levels were significantly decreased under BPA treatment at concentrations ≥0.1 μM. We observed that follicle stimulating hormone upregulated StAR and aromatase protein expression levels; however, this effect was suppressed in the presence of BPA. Regarding the steroidogenic effects of BPA on KGN cells, controversies remain regarding the ultimate outcomes. Nevertheless, we believe that the results here presented imply that KGN cells have a good cellular and steroidogenic machinery for evaluating endocrine disruption. Therefore, StAR and aromatase could be stable and sensitive biomarkers in KGN cells for the cellular screening of the potential risk posed by exogenous and environmental chemicals to female reproductive (endocrine) function.

• Journal of Animal Science and Technology
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• 제66권1호
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• pp.204-218
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• 2024

Elsholtzia fruticosa (EF) is present in tropical regions throughout South Asian countries as well as the Himalayas. Although it has been used as a traditional medicine to treat digestive, respiratory, and inflammatory issues, its effect on preadipocyte differentiation is unknown. In this study, we examined the effects of a methanol extract prepared from EF on the differentiation of 3T3-L1 preadipocytes. Cell differentiation was assessed by microscopic observation and oil-red O staining. The expression of adipogenic and lipogenic genes, including PPARγ and C/EBPα, was measured by western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), to provide insight into adipogenesis and lipogenesis mechanisms. The results indicated that EF promotes the differentiation of 3T3-L1 preadipocytes, with elevated lipid accumulation occurring in a concentration-dependent manner without apparent cytotoxicity. EF enhances the expression of adipogenic and lipogenic genes, including PPARγ, FABP4, adiponectin, and FAS, at the mRNA and protein levels. The effect of EF was more pronounced during the early and middle stages of 3T3-L1 cell differentiation. Treatment with EF decreased C/EBP homologous protein (CHOP) mRNA and protein levels, while increasing C/EBPα and PPARγ expression. Treatment with EF resulted in the upregulation of cyclin E and CDK2 gene expression within 24 h, followed by a decrease at 48 h, demonstrating the early-stage impact of EF. A concomitant increase in cyclin-D1 levels was observed compared with untreated cells, indicating that EF modulates lipogenic and adipogenic genes through intricate mechanisms involving CHOP and cell cycle pathways. In summary, EF induces the differentiation of 3T3-L1 preadipocytes by increasing the expression of adipogenic and lipogenic genes, possibly through CHOP and cell cycle-dependent mechanisms.

• Fisheries and Aquatic Sciences
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• 제26권11호
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• pp.649-659
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• 2023

The present study compared the structure of mixilliped of Fenneropenaeus chinensis between the larval and adult stage and investigated the effect of the structural difference on the immunity of F. chinensis. A fourteen day and a one-month long culture trial were conducted each with postlarvae and adults of F. chinensis in the biofloc, mixed water (50% biofloc:50% clear seawater) and seawater control. Immune-related genes mRNA expressions of postlarvae was analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). And the analysis of adult stage immunity was carried out using phenoloxidase (PO) enzyme activation in haemocyte. In the postlarvae, the final body weights were 51.43 and 58.47 mg for the biofloc water and the control seawater, respectively. On the other hand, the final body weights of the adults were significantly different between biofloc water and seawater. The survival rate showed the opposite trend to the growth rate. Immune related genes mRNA expression levels in the postlarvae in the biofloc water were significantly lower than those in the seawater. While, the adult stage showed significantly higher PO enzyme activations in the biofloc water than in the seawater with the PO enzyme activation increasing proportionally to the biofloc concentration. This result is considerably explained by the observations of setal morphological structures of the third maxilliped: postlarvae have short serrulated setae that compose the small 'net' structure while adults had long and dense plumose setae. It is understood that the morphological difference of the maxilliped structure resulted in the different feeding abilities in the postlarvae and the adult F. chinensis to use bioflocs as food source.