Aurantio-obtusin Alleviates Dry Eye Disease by Targeting NF-κB/NLRP3 Signaling in Rodent Models.

Dry eye disease (DED) is a common inflammatory ocular surface disorder, seriously affecting the quality of life of patients. Aurantio-obtusin (AO) is a bioactive anthraquinone compound isolated from Semen Cassiae which has multiple pharmacological activities. Nonetheless, the specific function of AO in DED is unclarified. In this study, a rodent DED model was established by benzalkonium chloride (BAC) induction, followed by topical administration of AO. The results showed that topical application of AO increased tear production, mitigated ocular surface disruption and maintained the number of goblet cells in BAC-induced DED rats (p˂0.05). ELISA revealed that AO treatment significantly (p˂0.001) reduced the production of proinflammatory cytokines and chemokines in the conjunctiva and cornea of BAC-induced DED rats. Immunohistochemical staining and western blotting showed that AO treatment suppressed the expression levels of NLR family pyrin domain containing 3 (NLRP3) inflammasome-related proteins, and inhibited activation of nuclear factor kappa B (NF-κB) signaling pathway in rat conjunctiva and cornea (p˂0.001). In conclusion, AO treatment alleviates BAC-induced DED in rats by inhibiting NF-κB/NLRP3 inflammasome signaling pathway.

Gene expression profiles and bioinformatics analysis in lung samples from ovalbumin-induced asthmatic mice.

BACKGROUND: Asthma is characterized by chronic inflammation and airway remodeling. However, limited study is conducted on the gene expression profiles of ovalbumin (OVA) induced asthma in mice. Here, we explored the gene expression profiles in lung tissues from mice with OVA-induced asthma using microarray and bioinformatics analysis. METHODS: For establishment of OVA-induced asthma model, mice first received intraperitoneal sensitization with OVA on day 0, 7 and 14, followed by atomizing inhalation of OVA 3 times a week for 8 weeks. The lung tissues were collected and subjected to microarray analysis, bioinformatics analysis and expression validation. RESULTS: Microarray data of lung tissues suggested that 3754 lncRNAs and 2976 mRNAs were differentially expressed in lung tissues between control and asthmatic mice, including 1647 up-regulated and 2106 down-regulated lncRNAs, and 1201 up-regulated and 1766 down-regulated mRNAs. GO analysis displayed that the up-regulated genes were enriched in inflammatory response, leukocyte migration involved in inflammatory response, and Notch signaling pathway. KEGG pathway analysis indicated that the enriched pathway terms of the up-regulated gene included Toll-like receptor signaling pathway and Th17 cell differentiation signaling pathway. Additionally, based on the previously published literatures on asthma and inflammation, we screened out down-regulated genes, such as Smg7, Sumo2, and Stat5a, and up-regulated genes, such as Myl9, Fos and Tlr4. According to the mRNA-lncRNA co-expression network, we selected lncRNAs associated with above genes, including the down-regulated lncRNAs of NONMMUT032848, NONMMUT008873, NONMMUT009478, and NONMMUT006807, and the up-regulated lncRNAs of NONMMUT052633, NONMMUT05340 and NONMMUT042325. The expression changes of the above genes were validated in lung tissues by real-time quantitaive PCR and Western blot. CONCLUSIONS: Overall, we performed gene microarray on lung samples from OVA-induced asthmatic mice and summarized core mRNAs and their related lncRNAs. This study may provide evidence for further research on the therapeutic targets of asthma.

Salidroside Mitigates Airway Inflammation in Asthmatic Mice via the AMPK/Akt/GSK3ß Signaling Pathway.

INTRODUCTION: This study aimed to explore the effects and mechanisms of salidroside (SAL) in airway inflammation in asthmatic mice. METHODS: Mice were sensitized with ovalbumin (OVA) to establish an asthma model. They were divided into the control group, OVA group, SAL low-dose group (SAL-L), SAL high-dose group (SAL-H), and dexamethasone (DXM) group. The airway reactivity of the mice was measured, and the total cells, neutrophils, eosinophils, and lymphocytes were counted, respectively. The levels of IL-4, IL-5, IL-13, and IFN-γ in bronchoalveolar lavage fluid (BALF) were detected by ELISA. Immunohistochemistry was used to detect the expression levels of p-AMPK, p-Akt, and p-GSK3ß. Western blot was used to detect cytokine levels in lung tissue and p-AMPK, p-Akt, and p-GSK3ß levels in LPS-induced 16HBE cells. RESULTS: The airway hyperresponsiveness of asthmatic mice in the SAL-H group decreased (p < 0.05), and the total number of cells, neutrophils, eosinophils, and lymphocytes decreased significantly (p < 0.05). In addition, the airways of mice showed airway inflammatory infiltration and goblet cell proliferation, and the corresponding cellular inflammatory factors IL-4, IL-5, and IL-13 were significantly decreased. However, the expression of IFN-γ in BALF and lung tissues was increased (p < 0.05). Moreover, after the mice were treated with SAL, the phosphorylation level of AMPK was significantly increased, which further reduced the phosphorylation levels of Akt and GSK3ß (p < 0.05). Both SAL and AMPK inhibitors exerted effects on LPS-induced 16HBE cells, consistent with in vivo results. CONCLUSION: SAL can inhibit bronchial hyperresponsiveness and reduce tracheal inflammation by increasing AMPK phosphorylation and inhibiting Akt and GSK3ß signaling pathways.

DEK-targeting aptamer DTA-64 attenuates bronchial EMT-mediated airway remodelling by suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathway in asthma.

This study is to investigate the inhibitory effects and mechanisms of DEK-targeting aptamer (DTA-64) on epithelial mesenchymaltransition (EMT)-mediated airway remodelling in mice and human bronchial epithelial cell line BEAS-2B. In the ovalbumin (OVA)-induced asthmatic mice, DTA-64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA-64 reduced collagen deposition, transforming growth factor 1 (TGF-ß1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α-SMA), as well as weekend matrix metalloproteinases (MMP-2 and MMP-9) and NF-κB p65 activity. In the in vitro experiments, we used TGF-ß1 to induce EMT in the human epithelial cell line BEAS-2B. DEK overexpression (ovDEK) or silencing (shDEK) up-regulated or down-regulated TGF-ß1 expression, respectively, on the contrary, TGF-ß1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF-ß1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF-ß1-mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA-64 against EMT of asthmatic mice and BEAS-2B might partially be achieved through suppressing TGF-ß1/Smad, MAPK and PI3K signalling pathways. DTA-64 may be a new therapeutic option for the management of airway remodelling in asthma patients.

Cryptotanshinone attenuates allergic airway inflammation through negative regulation of NF-κB and p38 MAPK.

This study is to determine the role and mechanism of cryptotanshinone (CTS) in allergic airway inflammation. Asthma induced by OVA was established in BALB/c mice. We found increased airway hyperresponsiveness (AHR), increased inflammatory cell infiltration, elevated levels of TNF-α, interleukin-1ß (IL-1ß), IL-4, IL-5, IL-6 and IL-13, decreased interferon gamma (IFN-γ) in lung tissue, increased content of total immunoglobulin E (IgE), OVA specific IgE, Eotaxin, ICAM-1, VCAM-1, nuclear factor-kappaB (NF-κB) and phosphorylation of p38 MAPK in lung tissue. However, the administration of CTS significantly decreased AHR in asthmatic mice, reduced inflammation around the bronchioles and inflammatory cells around airway, regulated cytokine production, reduced the total IgE and OVA-specific IgE levels, and inhibited NF-κB activation and p38 MAPK phosphorylation. In vitro experiments in 16 HBE cells revealed that CTS attenuated CAM-1 and IL-6 expression. These results indicate that CTS alleviates allergic airway inflammation by modulating p38 MAPK phosphorylation and NF-κB activation.

Imperatorin alleviates ROS-mediated airway remodeling by targeting the Nrf2/HO-1 signaling pathway.

In this study, we investigated the role and mechanism of imperatorin (IMP) in chronic inflammation and airway remodeling. The levels of TNF-α, IL-1ß, IL-6, IL-8, VEGF, α-SMA, and ROS were detected by ELISA, immunohistochemistry (IHC), immunofluorescence, and Western blot. In addition, we evaluated the effect of IMP on MAPK, PI3K/Akt, NF-κB, and Nrf2/HO-1 signaling pathways. IMP treatment obviously attenuated the production of inflammatory cytokines and inflammatory cells in bronchoalveolar lavage fluid of OVA-induced airway remodeling model. Meanwhile, it significantly inhibited inflammatory cell infiltration, goblet cell hyperplasia, collagen deposition, VEGF production, α-SMA, and ROS expression. Our study has shown that IMP could regulate the signaling pathways including MAPK, PI3K/Akt, NF-κB, and Nrf2/HO-1 to release the inflammatory responses. IMP might attenuate airway remodeling by the down-regulation of Nrf2/HO-1/ROS/PI3K/Akt, Nrf2/HO-1/ROS/MAPK, and Nrf2/HO-1/ROS/NF-κB signaling pathways.

Recombinant pyrin domain protein attenuates allergic inflammation by suppressing NF-κB pathway in asthmatic mice.

Pyrin domain (PYD), a subclass of protein motif known as the death fold, is frequently involved in inflammation and immune responses. PYD modulates nuclear factor-kappa B (NF-κB) signalling pathway upon various stimuli. Herein, a novel recombinant pyrin domain protein (RPYD) was generated. Its role and mechanism in inflammatory response in an ovalbumin (OVA) induced asthma model was investigated. After OVA challenge, there was inflammatory cell infiltration in the lung, as well as airway hyper-responsiveness (AHR) to inhaled methacholine. In addition, eosinophils increased in the bronchoalveolar lavage fluids, alone with the elevated levels of Th-2 type cytokines [interleukin (IL)-4, IL-5 and IL-13], eotaxin, and adhesion molecules. However, the transnasal administration of RPYD before the OVA challenge significantly inhibited these asthmatic reactions. Moreover, RPYD markedly suppressed NF-κB translocation, reduced phosphorylation of p38 MAPK, and thus attenuated the expression of intercellular adhesion molecule 1 and IL-6 in the BEAS-2B cells stimulated by proinflammatory cytokines in vitro. These findings indicate that RPYD can protect asthma host from OVA-induced airway inflammation and AHR via down-regulation of NF-κB and p38 MAPK activities. RPYD may be used as a potential medicine for the treatment of asthma in clinic.

Protective Effect and Mechanism of Alprostadil in Acute Respiratory Distress Syndrome Induced by Oleic Acid in Rats.

BACKGROUND This study investigated the role and mechanism of alprostadil in acute respiratory distress syndrome (ARDS) induced by oleic acid (OA) in rats. MATERIAL AND METHODS Sprague-Dawley rats were randomly divided into control, OA model, and OA + Alprostadil (2.5, 5, and 10 µg/kg, respectively) groups. The ARDS model was induced by femoral vein injection of OA, and alprostadil was administrated immediately. Lung injury was evaluated by lung wet-dry weight ratio (W/D) and histological analyses. Expressions of ACE, inflammatory mediators, apoptotic-related proteins, and proteins in the MAPKs and NF-κB signaling pathways were determined by Western blot or immunohistochemical staining. RESULTS Compared with the control group, the OA model group had significantly increased W/D, lung injury score, and collagen deposition at 3 h after OA injection. However, alprostadil (10 µg/kg) treatment significantly reduced OA-induced elevation of these indicators. Additionally, OA-induced expression of TNF-α and IL-1ß were suppressed by alprostadil. The OA-induced activation of nuclear factor (NF) κB p65 was also reduced by alprostadil. Furthermore, we found that Alprostadil had an inhibitory effect on the phosphorylation of JNK, ERK1/2, and p38 MAPKs. Alprostadil inhibited Bax but increased Bcl-2, indicating a suppressive role in apoptosis. Remarkably increased expression of ACE in the OA model group was observed, which was decreased by alprostadil. CONCLUSIONS Alprostadil has a protective effect on ARDS induced by OA in rats, possibly through inhibiting apoptosis, suppressing the activation of MAPKs and NF-κB signaling pathways, and decreasing ACE protein expression. Therefore, the use of alprostadil in clinical ARDS treatment is promising.

Cornuside inhibits mast cell-mediated allergic response by down-regulating MAPK and NF-κB signaling pathways.

AIMS: The present study is to investigate the effect of cornuside on mast cell-mediated allergic response, as well as its possible mechanisms of action. METHODS: To test the anti-allergic effects of cornuside in vivo, local extravasation was induced by local injection of anti-dinitrophenyl immunoglobulin E (IgE) followed by intravenous antigenic challenge in passive cutaneous anaphylaxis model rats. Mast cell viability was determined using MTT assay. Histamine content from rat peritoneal mast cells was measured by the radioenzymatic method. To investigate the mechanisms by which cornuside affects the reduction of histamine release, the levels of calcium uptake were measured. To examine whether cornuside affects the expression of pro-inflammatory cytokines, Western blotting and ELISA were carried out. RESULTS: Oral administration of cornuside inhibited passive cutaneous anaphylaxis in rats. Presence of cornuside attenuated IgE-induced histamine release from rat peritoneal mast cells. The inhibitory effect of cornuside on histamine release was mediated by the modulation of intracellular calcium. In addition, cornuside decreased phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated production and secretion of pro-inflammatory cytokines such as TNF-α and IL-6 in human mast cells. The inhibitory effect of cornuside on pro-inflammatory cytokines was dependent on nuclear factor-κB and p38 mitogen-activated protein kinase. CONCLUSIONS: The present study provides evidence that cornuside inhibits mast cell-derived inflammatory allergic reactions by blocking histamine release and pro-inflammatory cytokine expression. Furthermore, in vivo and in vitro anti-allergic effects of cornuside suggest a possible therapeutic application of this agent in inflammatory allergic diseases.

Salidroside alleviates cholestasis-induced liver fibrosis by inhibiting hepatic stellate cells via activation of the PI3K/AKT/GSK-3ß signaling pathway and regulating intestinal flora distribution.

Salidroside (SAL), a phenylpropanoid bioactive compound, has various pharmacological properties, including antioxidant, anti-inflammatory, and hepatoprotective effects. However, the pharmacological effects and mechanisms of action of SAL on cholestatic liver injury are unclear. This study investigated the mechanism and effects of salidroside (SAL) on intestinal flora distribution and hepatic stellate cell (HSC) activation in cholestatic hepatic fibrosis. Bile duct ligation was used to cause cholestasis BALB/c mice. The therapeutic efficacy of SAL in liver fibrosis was assessed via serum/tissue biochemical analyses and liver tissue hematoxylin and eosin and Masson staining. Inflammation and oxidative stress were analyzed using enzyme-linked immunosorbent assay and western blotting. HSC were activated in vitro using lipopolysaccharide, and the effects of SAL on HSC migration and inflammatory factor expression were detected via scratch, transwell, and western blotting assays. The effects of SAL on the PI3K/AKT/GSK-3ß pathway in vivo and in vitro were detected using western blotting. 16sRNA sequencing was used to detect the effect of SAL on the diversity of the intestinal flora. Ileal histopathology and western blotting were used to detect the protective effect of SAL on the intestinal mucosal barrier. SAL reduces liver inflammation and oxidative stress and protects against liver fibrosis with cholestasis. It inhibits HSC activation and activates the PI3K/AKT/GSK-3ß pathway in vitro and in vivo. Additionally, SAL restores the abundance of intestinal flora, which contributes to the repair of the intestinal mucosal barrier, inhibits endotoxin translocation, and indirectly inhibits HSC activation, reversing the course of cholestatic liver fibrosis. SAL inhibits HSC activation through the PI3K/AKT/GSK-3ß pathway and improves intestinal flora distribution, thereby protecting and reversing the progression of hepatic fibrosis.

IL-4 activates ULK1/Atg9a/Rab9 in asthma, NLRP3 inflammasomes, and Golgi fragmentation by increasing autophagy flux and mitochondrial oxidative stress.

During asthma, there is an intensification of pulmonary epithelial inflammation, mitochondrial oxidative stress, and Golgi apparatus fragmentation. However, the underlying mechanism remains largely unknown. Therefore, this study investigated the roles of ULK1, Atg9a, and Rab9 in epithelial inflammation, mitochondrial oxidative stress, and Golgi apparatus fragmentation. We found that ULK1 gene knockout reduced the infiltration of inflammatory cells, restored the imbalance of the Th1/Th2 ratio, and inhibited the formation of inflammatory bodies in the lung tissue of house dust mite-induced asthma mice. Moreover, we demonstrated that Atg9a interacted with ULK1 at S467. ULK1 phosphorylated Atg9a at S14. Treatment with ULK1 activator (LYN-1604) and ULK1 inhibitor (ULK-101) respectively promoted and inhibited inflammasome activation, indicating that the activation of inflammasome induced by house dust mite in asthma mice is dependent on ULK1. For validation of the in vivo results, we then used a lentivirus containing ULK1 wild type and ULK1-S467A genes to infect Beas-2b-ULK1-knockout cells and establish a stable cell line. The results suggest that the ULK1 S467 site is crucial for IL-4-induced inflammation and oxidative stress. Experimental verification confirmed that Atg9a was the superior signaling pathway of Rab9. Interestingly, we found for the first time that Rab9 played a very important role in inflammation-induced fragmentation of the Golgi apparatus. Inhibiting the activation of the ULK1/Atg9a/Rab9 signaling pathways can inhibit Golgi apparatus fragmentation and mitochondrial oxidative stress in asthma while reducing the production of NLRP3-mediated pulmonary epithelial inflammation.

More potential uses of specific perforator flaps in the calf - A cadaveric study on the subdermal vascular structure of the lower leg.

BACKGROUND: The perforator flap has garnered significant interest since its inception due to its advantage of not needing a vascular network at the deep fascial level. Perforator flaps are commonly utilized in different flap transplant surgeries, and the thigh flap is presently the most widely used perforator flap. Is it possible for the calf to replace the thigh as a more suitable site for harvesting materials? Currently, there is a lack of relevant anatomical research. This study aims to address this question from an anatomical and imaging perspective. METHODS: This study used cadavers to observe the branches and courses of perforators on the calf and the distribution of skin branches using microdissection techniques, digital X-ray photography, and micro-computed tomography techniques. RESULTS: The perforators had three main branches: the vertical cutaneous branch, the oblique cutaneous branch, and the superficial fascial branch. The superficial fascial branch traveled in the superficial fascia and connected with the nearby perforators. The vertical and oblique cutaneous branches entered the subdermal layer and connected with each other to create the subdermal vascular network. CONCLUSIONS: We observed an intact calf cutaneous branch chain between the cutaneous nerve and the perforator of the infrapopliteal main artery at the superficial vein site. Utilizing this anatomical structure, the calfskin branch has the potential to serve as a substitute for thigh skin flap transplantation and may be applied to perforator flap transplantation in more locations.

Effect of FTY-720 on Pulmonary Fibrosis in Mice via the TGF-ß1 Signaling Pathway and Autophagy.

We investigated whether FTY-720 might have an effect on bleomycin-induced pulmonary fibrosis through inhibiting TGF-ß1 pathway, and up-regulating autophagy. The pulmonary fibrosis was induced by bleomycin. FTY-720 (1 mg/kg) drug was intraperitoneally injected into mice. Histological changes and inflammatory factors were observed, and EMT and autophagy protein markers were studied by immunohistochemistry and immunofluorescence. The effects of bleomycin on MLE-12 cells were detected by MTT assay and flow cytometry, and the related molecular mechanisms were studied by Western Blot. FTY-720 considerably attenuated bleomycin-induced disorganization of alveolar tissue, extracellular collagen deposition, and α-SMA and E-cadherin levels in mice. The levels of IL-1ß, TNF-α, and IL-6 cytokines were attenuated in bronchoalveolar lavage fluid, as well as protein content and leukocyte count. COL1A1 and MMP9 protein expressions in lung tissue were significantly reduced. Additionally, FTY-720 treatment effectively inhibited the expressions of key proteins in TGF-ß1/TAK1/P38MAPK pathway and regulated autophagy proteins. Similar results were additionally found in cellular assays with mouse alveolar epithelial cells. Our study provides proof for a new mechanism for FTY-720 to suppress pulmonary fibrosis. FTY-720 is also a target for treating pulmonary fibrosis.

JTE-013 Alleviates Pulmonary Fibrosis by Affecting the RhoA/YAP Pathway and Mitochondrial Fusion/Fission.

Pulmonary fibrosis may be due to the proliferation of fibroblasts and the aggregation of extracellular matrix, resulting in the stimulation of inflammation damage, destroying lung tissue structure, seriously affecting the patient's respiratory function, and even leading to death. We investigated the role and mechanism of JTE-013 in attenuating bleomycin (BLM)-induced pulmonary fibrosis. BLM-induced pulmonary fibrosis was established in mice. Type 2 alveolar epithelial cells (MLE-12) were stimulated with sphingosine monophosphate (S1P) in vitro. JTE-013, an S1PR2 (sphingosine 1-phosphate receptor 2) antagonist, and Verteporfin were administered in vivo and in vitro. IL-4, IL-5, TNF-α, and IFN-γ were measured by ELISA. IL-4 and IFN-γ positive cells were detected by flow cytometry. Inhibition of S1PR2 with JTE-013 significantly ameliorated BLM-induced pathological changes and inflammatory cytokine levels. JTE-013 also significantly reduced the expression of RHOA/YAP pathway proteins and mitochondrial fission protein Drp1, apoptosis, and the colocalization of α-SMA with YAP, Drp1, and Tom20, as detected by immunohistochemistry, immunofluorescence staining, TUNEL, and Western blot. In vitro, S1PR2 and YAP knockdown downregulated RHOA/YAP pathway protein expression, Drp1 phosphorylation, and Drp1 translocation, promoted YAP phosphorylation and phenotypic transformation of MFN2, and inhibited the up-regulation of mitochondrial membrane potential, reactive oxygen species production, and cell apoptosis (7.13% vs. 18.14%), protecting the integrity of the mitochondrial dynamics. JTE-013 also inhibited the expression of fibrosis markers α-SMA, MMP-9, and COL1A1, and alleviated the symptoms of pulmonary fibrosis. Conclusively, JTE-013 has great anti-pulmonary fibrosis potential by regulating RHOA/YAP and mitochondrial fusion/fission.

DEK deficiency suppresses mitophagy to protect against house dust mite-induced asthma.

DEK protein is highly expressed in asthma. However, the mechanism of DEK on mitophagy in asthma has not been fully understood. This study aims to investigate the role and mechanism of DEK in asthmatic airway inflammation and in regulating PINK1-Parkin-mediated mitophagy, NLRP3 inflammasome activation, and apoptosis. PINK1-Parkin mitophagy, NLRP3 inflammasome, and apoptosis were examined after gene silencing or treatment with specific inhibitors (MitoTEMPO, MCC950, and Ac-DEVD-CHO) in house dust mite (HDM) or recombinant DEK (rmDEK)-induced WT and DEK-/- asthmatic mice and BEAS-2B cells. The regulatory role of DEK on ATAD3A was detected using ChIP-sequence and co-immunoprecipitation. rmDEK promoted eosinophil recruitment, and co-localization of TOM20 and LC3B, MFN1 and mitochondria, LC3B and VDAC, and ROS generation, reduced protein level of MnSOD in HDM induced-asthmatic mice. Moreover, rmDEK also increased DRP1 expression, PINK1-Parkin-mediated mitophagy, NLRP3 inflammasome activation, and apoptosis. These effects were partially reversed in DEK-/- mice. In BEAS-2B cells, siDEK diminished the Parkin, LC3B, and DRP1 translocation to mitochondria, mtROS, TOM20, and mtDNA. ChIP-sequence analysis showed that DEK was enriched on the ATAD3A promoter and could positively regulate ATAD3A expression. Additionally, ATAD3A was highly expressed in HDM-induced asthma models and interacted with DRP1, and siATAD3A could down-regulate DRP1 and mtDNA-mediated mitochondrial oxidative damage. Conclusively, DEK deficiency alleviates airway inflammation in asthma by down-regulating PINK1-Parkin mitophagy, NLRP3 inflammasome activation, and apoptosis. The mechanism may be through the DEK/ATAD3A/DRP1 signaling axis. Our findings may provide new potential therapeutic targets for asthma treatment.

Analysis of Differentially Expressed MicroRNAs in OVA-induced Airway Remodeling Model Mice.

MicroRNAs (miRNAs) can participate in airway remodeling by regulating immune molecule expression. Here, we aimed to identify the differential miRNAs involved in airway remodeling. Airway remodeling was induced by ovalbumin in female BALB/C mice. The differentially expressed miRNAs were screened with microarray. GO (Gene Ontology) and KEGG enrichment analysis was performed. The miRNA target gene network and miRNA target pathway network were constructed. Verification with real-time PCR and Western blot was performed. We identified 63 differentially expressed miRNAs (50 up-regulated and 13 down-regulated) in the lungs of ovalbumin-induced airway remodeling mice. Real-time PCR confirmed that 3 miRNAs (mmu-miR-1931, mmu-miR-712-5p, and mmu-miR-770-5p) were significantly up-regulated, and 4 miRNAs (mmu-miR-128-3p, mmu-miR-182-5p, mmu-miR-130b-3p, and mmu-miR-20b-5p) were significantly down-regulated. The miRNA target gene network analysis identified key mRNAs in the airway remodeling, such as Tnrc6b (trinucleotide repeat containing adaptor 6B), Sesn3 (sestrin 3), Baz2a (bromodomain adjacent to zinc finger domain 2a), and Cux1 (cut like homeobox 1). The miRNA target pathway network showed that the signal pathways such as MAPK (mitogen-activated protein kinase), PI3K/Akt (phosphoinositide 3-Kinase/protein kinase B), p53 (protein 53), and mTOR (mammalian target of rapamycin) were closely related to airway remodeling in asthma. Collectively, differential miRNAs involved in airway remodeling (such as mmu-miR-1931, mmu-miR-712-5p, mmu-miR-770-5p, mmu-miR-128-3p mmu-miR-182-5p, and mmu-miR-130b-3p) as well as their target genes (such as Tnrc6b, Sesn3, Baz2a, and Cux1) and pathways (such as MAPK, PI3K/Akt, p53, mTOR pathways) have been identified. Our findings may help to further understand the pathogenesis of airway remodeling.

Panax notoginseng saponin R1 attenuates allergic rhinitis through AMPK/Drp1 mediated mitochondrial fission.

We investigated whether Panax notoginseng saponin (PNS-R1) attenuates allergic rhinitis (AR) through AMPK/Drp1-mediated mitochondrial fission. AR model was established in mice by Ovalbumin (OVA). In vitro, human nasal epithelial cells (HNEpCs) were stimulated using recombinant human interleukin 13 (IL-13). PNS-R1 was administrated in vivo and in vitro. Then, HE staining of nasal tissue, ELISA detection of immunoglobulin E (IgE) and proinflammatory cytokine levels in serum and nasal lavage fluid, flow cytometry analysis of Th1/Th2 ratio and apoptosis, TUNEL staining, Western blot, detection of reactive oxygen species (ROS) and mitochondrial ROS, immunofluorescence analysis of Tom20 and mitochondrial fission protein Drp1 co-localization, and mitochondrial membrane potential detection, were performed. PNS-R1 attenuated allergic symptoms in AR mice, decreased OVA-specific IgE, IL-4, IL-6, IL-8, IL-13, and TNF-α levels, and restored the Th1/Th2 imbalance. Meanwhile, we found that PNS-R1 treatment significantly reduced apoptosis, ROS production, and co-localization of Tom20 and Drp1 in the nasal epithelium of AR mice. In vitro, we found that PNS-R1 upregulated mitochondrial membrane potential and reduced ROS and mitochondrial ROS production as well as Cleaved-caspase-3/9, Bax, Cyt-c, Apaf-1 expression and mitochondrial fission. Mechanistically, we found that PNS-R1 downregulated Drp1 phosphorylation (Ser 616) and Drp1 translocation in an AMPK-dependent manner, promoted MFN2 expression, and reduced TXNIP, NLRP3, Caspase-1, and IL-1ß expression. PNS-R1 may protect mitochondrial integrity by inhibiting AMPK/Drp1 and TXNIP/NLRP3 signaling pathway, thereby alleviating AR symptoms in mice. PNS-R1 may have great potential as a therapeutic agent for AR.

Effects of G-Rh2 on mast cell-mediated anaphylaxis via AKT-Nrf2/NF-κB and MAPK-Nrf2/NF-κB pathways.

Background: The effect of ginsenoside Rh2 (G-Rh2) on mast cell-mediated anaphylaxis remains unclear. Herein, we investigated the effects of G-Rh2 on OVA-induced asthmatic mice and on mast cell-mediated anaphylaxis. Methods: Asthma model was established for evaluating airway changes and ear allergy. RPMCs and RBL-2H3 were used for in vitro experiments. Calcium uptake, histamine release and degranulation were detected. ELISA and Western blot measured cytokine and protein levels, respectively. Results: G-Rh2 inhibited OVA-induced airway remodeling, the production of TNF-α, IL-4, IL-8, IL-1ß and the degranulation of mast cells of asthmatic mice. G-Rh2 inhibited the activation of Syk and Lyn in lung tissue of OVA-induced asthmatic mice. G-Rh2 inhibited serum IgE production in OVA induced asthmatic mice. Furthermore, G-Rh2 reduced the ear allergy in IgE-sensitized mice. G-Rh2 decreased the ear thickness. In vitro experiments G-Rh2 significantly reduced calcium uptake and inhibited histamine release and degranulation in RPMCs. In addition, G-Rh2 reduced the production of IL-1ß, TNF-α, IL-8, and IL-4 in IgE-sensitized RBL-2H3 cells. Interestingly, G-Rh2 was involved in the FcεRI pathway activation of mast cells and the transduction of the Lyn/Syk signaling pathway. G-Rh2 inhibited PI3K activity in a dose-dependent manner. By blocking the antigen-induced phosphorylation of Lyn, Syk, LAT, PLCγ2, PI3K ERK1/2 and Raf-1 expression, G-Rh2 inhibited the NF-κB, AKT-Nrf2, and p38MAPK-Nrf2 pathways. However, G-Rh2 up-regulated Keap-1 expression. Meanwhile, G-Rh2 reduced the levels of p-AKT, p38MAPK and Nrf2 in RBL-2H3 sensitized IgE cells and inhibited NF-κB signaling pathway activation by activating the AKT-Nrf2 and p38MAPK-Nrf2 pathways. Conclusion: G-Rh2 inhibits mast cell-induced allergic inflammation, which might be mediated by the AKT-Nrf2/NF-κB and p38MAPK-Nrf2/NF-κB signaling pathways.

MicroRNA-182-5p Attenuates Asthmatic Airway Inflammation by Targeting NOX4.

Bronchial asthma is characterized by chronic airway inflammation, airway hyperresponsiveness, and airway remodeling. MicroRNA (miRNA) has recently been implicated in the pathogenesis of asthma. However, the mechanisms of different miRNAs in asthma are complicated, and the mechanism of miRNA-182-5p in asthma is still unclear. Here, we aim to explore the mechanism of miRNA182-5p in asthma-related airway inflammation. Ovalbumin (OVA)-induced asthma model was established. MiRNA Microarray Analysis was performed to analyze the differentially expressed miRNAs in the asthma model. We found that the expression of miRNA-182-5p was significantly decreased in OVA-induced asthma. In vitro, IL-13 stimulation of BEAS-2B cells resulted in a significant up-regulation of NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4), accompanied by mitochondrial damage-induced apoptosis, NLRP3 (NOD-like receptor family pyrin domain-containing 3)/IL-1ß activation, and reduced miRNA-182-5p. In contrast, overexpression of miRNA-182-5p significantly inhibited epithelial cell apoptosis and NLRP3/IL-1ß activation. In addition, we found that miRNA-182-5p could bind to the 3' untranscripted region of NOX4 mRNA and inhibit epithelial cell inflammation by reducing oxidative stress and mitochondrial damage. In vivo, miRNA-182-5p agomir treatment significantly reduced the percentage of eosinophils in bronchoalveolar lavage fluid, and down-regulated Th2 inflammatory factors, including IL-4, IL-5, and OVA induced IL-13. Meanwhile, miRNA-182-5p agomir reduced the peribronchial inflammatory cell infiltration, goblet cell proliferation and collagen deposition. In summary, targeting miRNA-182-5p may provide a new strategy for the treatment of asthma.

[DEK-targeting aptamer DTA-64 inhibits epithelial-mesenchymal transition of airway epithelial cells in bronchial asthmatic mice via blocking TGF-ß1 signaling pathway].

Objective To investigate the inhibitory effect of DEK targeting aptamer 64 (DTA-64) on airway inflammation and epithelial to mesenchymal transition (EMT) induced by ovalbumin (OVA) in asthmatic mice. Methods Thirty-two female BALB/c mice (8 weeks old) were randomly divided into PBS group, OVA model group, DTA-64 group (1 µg/mouse), and control aptamer group, with 8 in each. HE staining of lung tissues was used to detect inflammatory cell infiltration around the airways; immunohistochemical staining was used to detect DEK expression around the airways; ELISA was used to detect serum IgE, and Th2-type cytokines IL-4, IL-5, IL-13 and Th1-type cytokine IFN-γ in bronchoalveolar lavage fluid (BALF); Western blot was applied to detect the EMT-related proteins α-SMA, Snail+Slug, vimentin, and E-cadherin, and TGF-ß1/Smad, MAPK, PI3K, AKT, as well as mTOR in lung; and flow cytometry was used to observe the α-SMA expression in the lung single cell suspensions. Results DEK protein was highly expressed in the lung tissues of OVA group mice and decreased in the DTA-64 group mice; DTA-64 reduced the infiltration of eosinophils and neutrophils around the airways, down-regulated serum OVA-specific IgE and IL-4, IL-5, IL-13 in BALF, and up-regulated IFN-γ; DTA-64 also reduced the expressions of vimentin, α-SMA, Snail+Slug in the lung tissue, and up-regulated epithelial marker E-cadherin. DTA-64 inhibited the expressions of TGF-ß1 and its downstream canonical pathways Smad2/3 and Smad4, as well as the phosphorylation of non-canonical TGF-ß1 pathways ERK1/2, p38 MAPK, JNK and PI3K/AKT/mTOR. Conclusion DTA-64 may inhibit the airway inflammation and EMT induced by OVA in asthmatic mice via blocking TGF-ß1/Smad, MAPK and PI3K signaling pathways, thereby alleviating airway remodeling in asthma.