scholarly journals HDM-induced Atg5 Mediates TSLP Expressing to Disrupt Epithelial Barrier

2021 ◽  
Author(s):  
Changhui Yu ◽  
Zicong Zhou ◽  
Shixiu Liang ◽  
Zili Zhou ◽  
Jieyi Liu ◽  
...  
Keyword(s):  
Epithelial Barrier ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Mice Model ◽  
Rt Pcr ◽  
Intracellular Process ◽  
Self Protection ◽  
One Way Anova

Abstract Background: Asthma is a complex and heterogeneous disease. Autophagy, process of self-protection in cells, is an intracellular process when cells are being attacked by certain stress. Our team focused upon the disruption of airways epithelial barrier in asthma, and we interested in whether autophagy played a key role in asthma. Methods: 400U HDM was used to treat HBECs and established asthmatic mice model. Western blotting, RT-PCR and immunofluorescence were mainly used to detect autophagy process in vivo and in vitro. One way ANOVA and Mann Whitney test were used for statistic. Results: After treated with HDM, expression of LC3ab increased in vivo and in vitro. Using Rapamycin, 3-MA and Chloroquine to treat HBECs, then we surprisingly found that HDM disrupts epithelial barrier through incomplete autophagy. To find out the connection between asthma and autophagy, we chose known autophagy related genes to determine the association between autophagy and disruption of airway epithelial barrier. Atg5 and atg12 were chosen because these two genes varied upon the time dependent manner. Knocked down the expression of atg5 or atg12 by siRNA, the expression of TSLP, which can induce the disruption of airway epithelial barrier, remarkably reduced. Conclusions: These results demonstrated that HDM induced inflammatory in airway epithelium through autophagy, and then knocked down autophagy related genes alleviated the inflammatory in HBECs.

Reticulon 3-mediated Chk2/p53 activation suppresses hepatocellular carcinogenesis and is blocked by hepatitis B virus

Gut ◽  
2020 ◽  
pp. gutjnl-2020-321386
Author(s):  
Shushu Song ◽  
Yinghong Shi ◽  
Weicheng Wu ◽  
Hao Wu ◽  
Lei Chang ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Dependent Manner ◽  
Mice Model ◽  
Calcium Dependent ◽  
B Virus ◽  
P53 Activation ◽  
Underlying Mechanisms ◽  
Induced Apoptosis

ObjectiveDysfunction of endoplasmic reticulum (ER) proteins is closely related to homeostasis disturbance and malignant transformation of hepatocellular carcinoma (HCC). Reticulons (RTN) are a family of ER-resident proteins critical for maintaining ER function. Nevertheless, the precise roles of RTN in HCC remain largely unclear. The aim of the study is to examine the effect of reticulon family member RTN3 on HCC development and explore the underlying mechanisms.DesignClinical HCC samples were collected to assess the relationship between RTN3 expression and patients’ outcome. HCC cell lines were employed to examine the effects of RTN3 on cellular proliferation, apoptosis and signal transduction in vitro. Nude mice model was used to detect the role of RTN3 in modulating tumour growth in vivo.ResultsWe found that RTN3 was highly expressed in normal hepatocytes but frequently downregulated in HCC. Low RTN3 expression predicted poor outcome in patients with HCC in TP53 gene mutation and HBV infection status-dependent manner. RTN3 restrained HCC growth and induced apoptosis by activating p53. Mechanism studies indicated that RTN3 facilitated p53 Ser392 phosphorylation via Chk2 and enhanced subsequent p53 nuclear localisation. RTN3 interacted with Chk2, recruited it to ER and promoted its activation in an ER calcium-dependent manner. Nevertheless, the tumour suppressive effects of RTN3 were abrogated in HBV-positive cells. HBV surface antigen competed with Chk2 for RTN3 binding and blocked RTN3-mediated Chk2/p53 activation.ConclusionThe findings suggest that RTN3 functions as a novel suppressor of HCC by activating Chk2/p53 pathway and provide more clues to better understand the oncogenic effects of HBV.

scholarly journals The role of secreted Hsp90α in HDM-induced asthmatic airway epithelial barrier dysfunction

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Cuiping Ye ◽  
Chaowen Huang ◽  
Mengchen Zou ◽  
Yahui Hu ◽  
Lishan Luo ◽  
...  
Keyword(s):  
Barrier Function ◽  
Epithelial Barrier ◽  
Airway Epithelial ◽  
Barrier Dysfunction ◽  
Epithelial Barrier Function ◽  
Asthmatic Airway ◽  
Epithelial Barrier Dysfunction

Abstract Background The dysfunction of airway epithelial barrier is closely related to the pathogenesis of asthma. Secreted Hsp90α participates in inflammation and Hsp90 inhibitor protects endothelial dysfunction. In the current study, we aimed to explore the role of secreted Hsp90α in asthmatic airway epithelial barrier function. Methods Male BALB/c mice were sensitized and challenged with HDM to generate asthma model. The 16HBE and Hsp90α-knockdown cells were cultured and treated according to the experiment requirements. Transepithelial Electric Resistance (TEER) and permeability of epithelial layer in vitro, distribution and expression of junction proteins both in vivo and in vitro were used to evaluate the epithelial barrier function. Western Blot was used to evaluate the expression of junction proteins and phosphorylated AKT in cells and lung tissues while ELISA were used to evaluate the Hsp90α expression and cytokines release in the lung homogenate. Results HDM resulted in a dysfunction of airway epithelial barrier both in vivo and in vitro, paralleled with the increased expression and release of Hsp90α. All of which were rescued in Hsp90α-knockdown cells or co-administration of 1G6-D7. Furthermore, either 1G6-D7 or PI3K inhibitor LY294002 suppressed the significant phosphorylation of AKT, which caused by secreted and recombinant Hsp90α, resulting in the restoration of epithelial barrier function. Conclusions Secreted Hsp90α medicates HDM-induced asthmatic airway epithelial barrier dysfunction via PI3K/AKT pathway, indicating that anti-secreted Hsp90α therapy might be a potential treatment to asthma in future.

scholarly journals JAK Inhibitors Suppress Colon Cancer Cachexia-Associated Anorexia and Adipose Wasting in Mice

2020 ◽  
Author(s):  
Gurpreet Arora ◽  
Arun Gupta ◽  
Tong Guo ◽  
Aakash Gandhi ◽  
Aaron Laine ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Colon Adenocarcinoma ◽  
Serum Levels ◽  
Immunoblot Analysis ◽  
Dependent Manner ◽  
Jak Inhibitors ◽  
Rt Pcr ◽  
Stat3 Phosphorylation

ABSTRACTBackgroundCachexia (CX), a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitor factor (LIF) which induces CX. We characterized how LIF promotes CX-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues.MethodsCX was induced in vivo with C26c20 colon adenocarcinoma cells or recombinant LIF administration in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. CX was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These activated adipocytes were processed for lipolysis, immunoblot analysis, and RT-PCR.ResultsTumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing CX-associated adipose wasting and anorexia. We identified two JAK inhibitors that block cytokine-mediated adipocyte lipolysis and IL-6 induction using an in vitro CX lipolysis assay. JAK inhibitors administered to in vivo colon cancer CX mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the CX serum cyto/adipokine signature, 3) a delay in colon cancer CX-associated anorexia and adipose loss, and 4) an improvement in overall survival.ConclusionsJAK inhibitors suppress cytokine-associated adipose loss and anorexia in multiple in vitro and in vivo models of cancer CX.

scholarly journals CircSETD3 (Hsa_circ_0000567) Suppresses Hepatoblastoma Pathogenesis via Targeting the miR-423-3p/Bcl-2-Interacting Mediator of Cell Death Axis

2021 ◽  
Vol 12 ◽  
Author(s):  
Xin Li ◽  
Haojie Wang ◽  
Zhijie Liu ◽  
Alimujiang Abudureyimu
Keyword(s):  
Regulatory Mechanism ◽  
Epithelial Mesenchymal Transition ◽  
Circular Rna ◽  
Cell Counting ◽  
Dependent Manner ◽  
Mice Model ◽  
Gain Of Function ◽  
Mesenchymal Transition

Background: Up until now, the role of circSETD3 (Has_circ_0000567) in regulating cancer development has been reported in several tumors, but the role and regulatory mechanism of circSETD3 in hepatoblastoma (HB) remain unclear.Methods: The qPCR and western blotting were used to determine the mRNA and protein levels in the present study. Stability of circular RNA was detected by RNA digested experiments. The gain-of-function and rescue experiments were used to explore the function and mechanism of circSETD3 in HB. Cell counting kit-8, colony formation, transwell assay, and xenograft mice model were used to detect effects and regulatory mechanism of circSETD3/miR-423-3p/Bim axis on cell aggressive phenotype in vitro and in vivo.Results: Here, we identified that circSETD3 downregulated in both HB clinical tissues and cell lines, compared to that of normal tissues and cells. Further gain-of-function experiments validated that circSETD3 overexpression inhibited cell proliferation, viability, migration, epithelial-mesenchymal transition (EMT) and tumorigenesis, and induced cell apoptosis in HB cells. Next, we validated that miR-423-3p targeted both circSETD3 and 3′ untranslated region (3′UTR) of Bim, and circSETD3 positively regulated Bim in HB cells through sponging miR-423-3p in a competing endogenous RNA (ceRNA)-dependent manner. Furthermore, through conducting reversal experiments, we evidenced that the inhibiting effects of circSETD3 overexpression on HB development were abrogated by upregulating miR-423-3p and downregulating Bim.Conclusion: Taken together, we evidenced that circSETD3 sponged miR-423-3p to upregulate Bim, resulting in the inhibition of HB development.

scholarly journals MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

2013 ◽  
Vol 304 (8) ◽  
pp. L511-L518 ◽  
Author(s):  
Shijing Fang ◽  
Anne L. Crews ◽  
Wei Chen ◽  
Joungjoa Park ◽  
Qi Yin ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Mucin Secretion ◽  
Concentration Dependent Manner

Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.

Targeted Anti-Tumor Research of Mesenchymal Stem Cells Delivered Enzyme-Prodrug Gene CYP450.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3582-3582
Author(s):  
Jishi Wang ◽  
Yuan Yang ◽  
Wei Zhang ◽  
Pengxiang Guo
Keyword(s):  
Nude Mice ◽  
Recombinant Adenovirus ◽  
Annexin V ◽  
Fluorescence Microscope ◽  
Mice Model ◽  
Rt Pcr ◽  
Nude Mice Model ◽  
Adenovirus Vectors

Abstract Abstract 3582 Poster Board III-519 Objective Cytochrome P450(CYP450-CYP1A2 /CYP2B6/CYP2C9) was transfected into human bone marrow-derived mesenchymal stem cells (hBMSCs), and the targed anti-tumor effect of BMSC-CYP450 cooperated with enzyme-prodrug(Dacarbazine (DTIC)/Cyclophosphamide (CPA)) was measured to provide laboratory data base for gene directed enzyme prodrug targeted anti-tumor therapy (GDEPT) which used BMSC as vehicles. Methods We respectively cloned CYP1A2/CYP2B6/CYP2C9 cDNA from human liver and constructed recombinant adenovirus vectors(pAd5CMV-NpA-CYP1A2/ pAd5CMV-NpA-CYP2B6/pAd5CMV-NpA-CYP2C9) which titer was 1×1012 pfu/mL. These hBMSCs were separated, cultured, purified, and detected by morphology, flow cytometry, osteogenic, adipogenic and chondrogenic induction, and RT-PCR(A surface marker for the identification of MSCs-the neural ganglioside GD2 gene). The tropism of BMSCs for cancer cells was detected by Transwell inserts technique. These recombinant vectors were transferred into BMSCs and A375/K562 cells, and the expression of EGFP and CYP1A2/CYP2B6/CYP2C9 was detected by fluorescence microscope, RT-PCR and Western blot respectively. Inverted microscope, MTT and Annexin V-FITC/PI detected the anti-tumor effect of CYP450 recombinant adenovirus vectors combined with chemotherapeutic prodrug DTIC/CPA in vitro. A human melanoma(A375) BALB/c nude mice model and a human myelocytic leukemia(K562) BALB/c nude mice model was constructed and detected by immuno-histochemistry analysis. The CYP1A2 gene tranfected BMSCs were injected into the A375 BALB/c nude mice model in combination with DTIC through caudal vein, while CYP2B6/CYP2C9 gene tranfected BMSCs were injected into K562 BALB/c nude mice model in combination with CPA in the same way. The measurement of tumors size, fluorescence microscope and TUNEL were used to detect anti-tumor effect of BMSCs-CYP1A2 cooperating with DTIC and BMSCs-CYP2B6/CYP2C9 with CPA in vivo. Results We constructed the recombinant adenovirus vectors pAd5CMV-NpA-CYP1A2/pAd5CMV-NpA-CYP2B6/pAd5CMV-NpA-CYP2C9 and pAd5CMV-NpA-EGFP successfully. BMSCs was separated successfully, and it respectively showed that BMSCs can migrate through the polycarbonate filter toward K562 and A375 cells in the lower chamber in vitro. Fluorescence microscope detected the expression of EGFP, while both RT-PCR and Western blot detected high expression of CYP1A2/CYP2B6/CYP2C9 in gene-transfected group cells. Inverted microscope, MTT and Annexin V-FITC/PI confirmed that BMSCs transferred with CYP1A2/CYP2B6/CYP2C9 recombinant adenovirus vectors could activate DTIC/CPA and increase its anti-tumor effect(In the DTIC/CPA concentration(0.05 mmol/L/2.5 mmol/L) which BMSCs was relatively safe, the cell apoptosis was (38.38±2.27)% (P<0.01), (42.69±2.03)% (P<0.01) and (39. 51±1.94)% (P<0.01) in BMSCs-CYP1A2+A375 group, BMSCs-CYP2B6+K562 group and BMSCs-CYP2C9+K562 group respectively. ). A375 and K562 BALB/c nude mice model was constructed successfully. The sizes of the tumor in the nude mice treated with transfected BMSCs and DTIC/CPA were significantly smaller than control case and changed along with concentration(P< 0.01, P< 0.05).BMSCs was congregated to tumor site in fluorescence microscope. Apoptosis of tumor cells was conspicuously more in BMSCs-CYP1A2+A375/BMSCs-CYP2B6+K562/BMSCs-CYP2C9+K562 treatment group than in control group by TUNEL. Conclusion BMSCs had the tropism for cancer cells in vitro and vivo. DTIC can be catalyzed by CYP2E1/CYP1A2, while CPA by CYP2B6/CYP2C9 in vitro and vivo. BMSC-based enzyme prodrug system of CYP2E1/CYP1A2 and DTIC can induce A375 cells apoptosis, while BMSC-based enzyme prodrug system of CYP2B6/CYP2C9 and CPA can induce K562 cells apoptosis in vitro and targetedly in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Paracellular Pathway-Mediated Mycoplasma hyopneumoniae Migration across Porcine Airway Epithelial Barrier under Air-Liquid Interface Conditions

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Haiyan Wang ◽  
Zhenzhen Zhang ◽  
Xing Xie ◽  
Beibei Liu ◽  
Yanna Wei ◽  
...  
Keyword(s):  
Mycoplasma Hyopneumoniae ◽  
Liquid Interface ◽  
Respiratory Pathogen ◽  
Epithelial Barrier ◽  
Airway Epithelial ◽  
Migration Ability ◽  
Content Type ◽  
Air Liquid Interface

ABSTRACT Mycoplasma hyopneumoniae is an important respiratory pathogen of pigs that causes persistent and secondary infections. However, the mechanisms by which this occurs are unclear. In this study, we established air-liquid interface culture systems for pig bronchial epithelial cells (ALI-PBECs) that were comparable to the conditions in the native bronchus in vivo. We used this ALI-PBECs model to study the infection and migration characteristics of M. hyopneumoniae in vitro. Based on the results, we confirmed that M. hyopneumoniae was able to adhere to ALI-PBECs and disrupt mucociliary function. Importantly, M. hyopneumoniae could migrate to the basolateral chamber through the paracellular route but not the transcellular pathway, and this was achieved by reversibly disrupting tight junctions (TJs) and increasing the permeability and damaging the integrity of the epithelial barrier. We examined the migration ability of M. hyopneumoniae using an ALI-PBECs model for the first time. The disruption of the epithelial barrier allowed M. hyopneumoniae to migrate to the basolateral chamber through the paracellular route, which may be related to immune evasion, extrapulmonary dissemination, and persistent infection of M. hyopneumoniae.

scholarly journals Short Isoform Thymic Stromal Lymphopoietin Reduces Inflammation and Aerobic Glycolysis of Asthmatic Airway Epithelium by Antagonizing Long Isoform Thymic Stromal Lymphopoietin

2021 ◽  
Author(s):  
Changhui Yu ◽  
Wufeng Huang ◽  
Zicong Zhou ◽  
Shixiu Liang ◽  
Zili Zhou ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Aerobic Glycolysis ◽  
Thymic Stromal Lymphopoietin ◽  
Epithelial Cell Line ◽  
Receptor Complex ◽  
Airway Epithelial ◽  
Mice Model ◽  
Asthmatic Airway

Abstract Background:Up-regulation of aerobic glycolysis has been reported as a characterization of asthma and facilitates airway inflammation. We has been previously reported that short isoform thymic stromal lymphopoietin (sTSLP) could reduce inflammation in asthmatic airway epithelial. Here we wanted to investigate whether the inhibition of sTSLP on asthma is related to aerobic glycolysis. Methods:Asthmatic model was established in challenging Male BALB/c mice and 16-HBE (human bronchial epithelial) cell line with house dust mite (HDM). Indicators of glycolysis were assessed to measure whether involve in sTSLP regulating airway epithelial inflammation in asthmatic model in vivo and in vitro.Results:sTSLP decreased inflammation of asthmatic airway and aerobic glycolysis in mice. HDM or long isoform thymic stromal lymphopoietin (lTSLP) promoted HIF-1α expression and aerobic glycolysis by miR-223 to target and inhibit VHL (von Hippel-Lindau）expression 16-HBE. Inhibition of aerobic glycolysis restrained HDM- and lTSLP-induced inflammatory cytokines production. sTSLP along had almost no potential to alter aerobic glycolysis of 16-HBE. But sTSLP decreased LDHA (lactate dehydrogenase A) and LD (Lactic acid) levels in BALF, and HIF-1α and LDHA protein levels in airway epithelial of asthma mice model. lTSLP and sTSLP both induced formation of TSLPR and IL-7R receptor complex, and lTSLP obviously facilitated phosphorylation of JAK1, JAK2 and STAT5, while sTSLP induced a little phosphorylation of JAK1 and STAT5. Conclusion: We identified a novel mechanism that lTSLP could promote inflammatory cytokines production by miR-223/VHL/HIF-1α pathway to upregulate aerobic glycolysis in airway epithelial in asthma. This pathway is suppressed by sTSLP through occupying binding site of lTSLP in TSLPR and IL-7R receptor complex.

scholarly journals Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2251
Author(s):  
Jin-A Yu ◽  
Jung-Yun Lee ◽  
Tae Yang Kim ◽  
Hanna Kang ◽  
Su-Young Lee ◽  
...  
Keyword(s):  
Natural Products ◽  
Treated Group ◽  
Dependent Manner ◽  
Red Ginseng ◽  
Mice Model ◽  
Mouse Splenocytes ◽  
Higher Weights ◽  
In Vivo Animal Models

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF (p < 0.01) or GF (p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM (p < 0.01), IgG (p < 0.05), IL-2 (p < 0.01), IL-4 (p < 0.01), IL-6 (p < 0.01), and IFN-γ (p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

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