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1.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1865(6): 158660, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32058034

RESUMO

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that promotes low-density lipoprotein receptor (LDLR) degradation and thereby regulating plasma levels of LDL cholesterol. Previous studies have revealed the role of the C-terminal domain (CTD) of PCSK9 in its secretion, however, how CTD regulates PCSK9 secretion is not completely understood. Additionally, SEC24A, the cargo adaptor protein of the coat protein complex II, has been implicated in the secretion of mouse PCSK9. Here, we investigated how CTD and SEC24 regulated PCSK9 secretion in humans. We found that mutant PCSK91-528, in which amino acids from 529 to the end (amino acid 692) were deleted, was maturated and secreted from cells as effectively as the wild-type protein. On the other hand, lacking amino acids 454 to 692 in mutant PCSK91-453 significantly reduced its maturation and secretion, but to a lesser extent when compared to mutants PCSK91-446, PCSK91-445 and PCSK91-444, that all markedly impaired PCSK9 maturation. However, mutant PCSK91-444 virtually eliminated PCSK9 secretion while PCSK91-446 and PCSK91-445 could still be adequately detected in culture medium. Interestingly, mutation of Pro445 to other amino acid residues considerably impaired the secretion of mutant PCSK91-445 but not the full-length protein. We also found that natural variants in CTD including S462P, S465L, E482G, R495Q and A522T impaired PCSK9 secretion. Further, the knockdown of SEC24A, SEC24B, SEC24C but not SEC24D reduced secretion of the full-length PCSK9 but not mutant PCSK91-446. Therefore, SEC24A, SEC24B, and SEC24C facilitate endogenous PCSK9 secretion from cultured human hepatocytes, that are most likely mediated by the CTD of PCSK9. Our studies also indicate that the CTD of PCSK9 may allosterically and independently modulate the stability of the hinge region. Collectively, these data revealed that the CTD of PCSK9 and the hinge region play a critical role in PCSK9 maturation and secretion.

2.
Dig Dis Sci ; 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31897898

RESUMO

BACKGROUND: Emerged as important regulators in cancer progression, circular RNAs have been tested to participate in diverse biological processes. Former studies have suggested that circular RNA_LARP4 (circLARP4) exerts indispensable function on the development of different cancers such as gastric cancer and ovarian cancer. Nonetheless, the specific role of circLARP4 has not been discovered in ESCC. AIMS: The aim of this study is to explore the biological function and regulatory mechanism of circLARP4 in ESCC. METHODS: CircLARP4, miR-1323, and PTEN expression levels were quantified by RT-qPCR. CCK-8, EdU, caspase-3 activity, wound healing, transwell, and western blot assays were chosen to assess ESCC cell growth. Luciferase reporter, RIP, and RNA pull-down assays were performed to examine the interaction between miR-1323 and circLARP4 (or PTEN). RESULTS: CircLARP4 expression was observably downregulated in ESCC cell lines, and overexpressed circLARP4 restrained cell proliferation and migration whereas boosted cell apoptosis in ESCC. Molecular mechanism experiments revealed that circLARP4 could act as a sponge for miR-1323 and negatively modulated miR-1323 expression in ESCC. Interestingly, the repression of miR-1323 was correlated with inhibitive cell proliferation, migration, and promotive apoptosis. Besides, miR-1323 bound with PTEN, and PTEN expression was negatively regulated by miR-1323 whereas positively regulated by circLARP4 in ESCC. Moreover, rescue assays testified that miR-1323 overexpression or PTEN deficiency could countervail the function of circLARP4 overexpression on ESCC progression. More importantly, circLARP4 played an inhibitory role in PI3K/AKT pathway. CONCLUSIONS: CircLARP4 sponges miR-1323 and hampers tumorigenesis of ESCC through modulating PTEN/PI3K/AKT pathway.

4.
J Cell Physiol ; 235(4): 3657-3668, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31583713

RESUMO

Pancreatic ductal adenocarcinoma (PDA) responds poorly to treatment. Efforts have been exerted to prolong the survival time of PDA, but the 5-year survival rates remain disappointing. Understanding the molecular mechanisms of PDA development is significant. MEK/ERK pathway signaling has been proven to be important in PDA. lncRNA-mRNA networks have become a vital part of molecular mechanisms in the MEK/ERK pathway. Herein, weighted gene coexpression network analysis was used to investigate the coexpressed lncRNA-mRNA networks in the MEK/ERK pathway based on GSE45765. Differently expressed long noncoding RNA (lncRNA) and messenger RNA (mRNA) were found and 10 modules were identified based on coexpression profiles. Gene ontology and Kyoto Encyclopedia of Genes and Genomes were then performed to analyze the coexpressed lncRNA and mRNA in different modules. PDA cells and tissues were used to validate the analysis results. Finally, we found that NONHSAT185150.1 and B4GALT6 were negatively correlated with MEK1/2. By analyzing GSE45765, the genome-wide profiles of lncRNA-mRNA network after MEK1/2 was established, which might aid the development of drug-targeting MEK1/2 and the investigation of diagnostic markers.

5.
Mol Psychiatry ; 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477794

RESUMO

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3' untranslated regions (3'UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3'UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.

6.
Liver Int ; 39(11): 2102-2111, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31379118

RESUMO

BACKGROUND: Drug-induced liver injury (DILI) is the most common reason for a drug to be withdrawn from the market. Apart from stopping the offending drug, no regimens are available for treating idiosyncratic DILI in clinical practice. METHODS: We carried out a randomized, double-blind, multidoses, active drug controlled, multicentre phase II trial to assess the safety and efficacy of the study drug, magnesium isoglycyrrhizinate (MgIG), as compared to tiopronin, a standard therapy for DILI in China. The primary outcome was the proportion of alanine aminotransferase (ALT) normalization at week 4 after study drug administration. Logistic regression was used to examine the odds of ALT normalization between low dose (Group A) and high dose (Group B) vs active control (Group C). RESULTS: One hundred and seventy-four eligible subjects were randomized and enrolled into three groups: 59 in group A, 56 in group B and 59 in group C. It was shown that group A and group B lowered ALT level even at early stage of study drug administration; when compared with Group C (61.02%), the proportions of ALT normalization at week 4 were significantly greater in Group A (84.75%, P = .0029) and Group B (85.71%, P = .0037) respectively. The results from the univariate logistic model showed that the odds of ALT normalized among subjects in Group A were about 3.6 times greater (OR = 3.55, 95% CI: 1.47-8.57, P = .0049) than subjects in Group C. Similar effect was observed among subjects in Group B (OR = 3.83, 95% CI: 1.54-9.55, P = .0039). CONCLUSIONS: This trial provided preliminary evidence that MgIG is an effective and safe treatment for patients with acute DILI.

7.
Int Immunopharmacol ; 75: 105774, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31351363

RESUMO

BACKGROUND: The purpose of the present study was to evaluate the protective effect of Magnesium Isoglycyrrhizinate (MI) on Epirubicin (EPI)-induced hepatic encephalopathy (HE) and explore its underlying mechanism. METHODS: Mice were divided randomly into groups for treatments as follows: control group, EPI group (Model group), EPI + MI (25, 50 mg/kg) group. Morris water maze test were conducted to evaluate the spatial learning and memory ability. The serum and hippocampus levels of oxidative stress or inflammation were uncovered with the detection of superoxide dismutase (SOD), malondialdehyde (MDA), and pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α). RESULTS: As a result, treatment with MI effectively ameliorated the EPI-induced decline in the ability of spatial learning and memory. MI also significantly relieved the severity of oxidative stress or inflammation in serum and hippocampus, which was accompanied with regulating liver functional parameters. Western blot data demonstrated that administration of MI could regulate the redox-related expressions of Txnip, Trx, Nrf2, HO-1, p-IκB-α, p-NF-κB, Caspase-3, Caspase-9, Bax and Bcl-2 in EPI-stimulated hepatic encephalopathy (HE). And the potency of MI treatments on Nrf2, NF-κB expression was also confirmed with immunohistochemical analysis. CONCLUSIONS: Taken together, the protective effect of Magnesium Isoglycyrrhizinate on EPI-induced hepatic encephalopathy might be mediated via the Txnip/Nrf2/NF-κB signaling pathway.


Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Epirubicina/efeitos adversos , Encefalopatia Hepática/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Saponinas/uso terapêutico , Triterpenos/uso terapêutico , Animais , Proteínas de Transporte/imunologia , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/imunologia , Citocinas/sangue , Citocinas/imunologia , Encefalopatia Hepática/induzido quimicamente , Encefalopatia Hepática/imunologia , Hipocampo/efeitos dos fármacos , Hipocampo/imunologia , Masculino , Malondialdeído/sangue , Malondialdeído/imunologia , Memória/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/imunologia , NF-kappa B/imunologia , Fármacos Neuroprotetores/farmacologia , Saponinas/farmacologia , Aprendizagem Espacial/efeitos dos fármacos , Superóxido Dismutase/sangue , Superóxido Dismutase/imunologia , Tiorredoxinas/imunologia , Triterpenos/farmacologia
8.
Biochem Pharmacol ; 166: 139-152, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31085161

RESUMO

High fructose intake is a risk of glomerular podocyte dysfunction. Podocyte apoptosis has emerged as a major cause of podocyte loss, exacerbating proteinuria. Magnesium isoglycyrrhizinate (MgIG) is usually used as a hepatoprotective agent in clinic. Liver and kidney injury often occurs in human diseases. Recent report shows that MgIG improves kidney function. In this study, we found that MgIG significantly alleviated kidney dysfunction, proteinuria and podocyte injury in fructose-fed rats. It also restored fructose-induced podocyte apoptosis in rat glomeruli and cultured differentiated podocytes. Of note, high-expression of miR-193a, downregulation of Wilms' tumor protein (WT1) and RelA, as well as upregulation of C-Maf inducing protein (C-mip) were observed in these animal and cell models. The data from the transfection of miR-193a mimic, miR-193a inhibitor, WT1 siRNA or LV5-WT1 in cultured differentiated podocytes showed that fructose increased miR-193a to down-regulate WT1, and subsequently activated C-mip to suppress RelA, causing podocyte apoptosis. These disturbances were significantly attenuated by MgIG. Taken together, these results provide the first evidence that MgIG restrains fructose-induced podocyte apoptosis at least partly through inhibiting miR-193a to upregulate WT1, supporting the application of MgIG with a novel mechanism-of-action against podocyte apoptosis associated with fructose-induced kidney dysfunction.

9.
J Lipid Res ; 60(3): 516-527, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30617148

RESUMO

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes LDL receptor (LDLR) degradation, increasing plasma levels of LDL cholesterol and the risk of cardiovascular disease. We have previously shown that, in addition to the epidermal growth factor precursor homology repeat-A of LDLR, at least three ligand-binding repeats (LRs) of LDLR are required for PCSK9-promoted LDLR degradation. However, how exactly the LRs contribute to PCSK9's action on the receptor is not completely understood. Here, we found that substitution of Asp at position 172 in the linker between the LR4 and LR5 of full-length LDLR with Asn (D172N) reduced PCSK9 binding at pH 7.4 (mimic cell surface), but not at pH 6.0 (mimic endosomal environment). On the other hand, mutation of Asp at position 203 in the LR5 of full-length LDLR to Asn (D203N) significantly reduced PCSK9 binding at both pH 7.4 and pH 6.0. D203N also significantly reduced the ability of LDLR to mediate cellular LDL uptake, whereas D172N had no detectable effect. These findings indicate that amino acid residues in the LRs of LDLR play an important role in PCSK9 binding to the receptor.

10.
Acta Pharmacol Sin ; 40(7): 879-894, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30568253

RESUMO

Increasing evidence has demonstrated that excessive fructose intake induces liver fibrosis. Epithelial-mesenchymal transition (EMT) driven by transforming growth factor-ß1 (TGF-ß1)/mothers against decapentaplegic homolog (Smad) signaling activation promotes the occurrence and development of liver fibrosis. Magnesium isoglycyrrhizinate is clinically used as a hepatoprotective agent to treat liver fibrosis, but its underlying molecular mechanism has not been identified. Using a rat model, we found that high fructose intake reduced microRNA (miR)-375-3p expression and activated the janus-activating kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) cascade and TGF-ß1/Smad signaling, which is consistent with the EMT and liver fibrosis. To further verify these observations, BRL-3A cells and/or primary rat hepatocytes were exposed to high fructose and/or transfected with a miR-375-3p mimic or inhibitor or treated with a JAK2 inhibitor, and we found that the low expression of miR-375-3p could induce the JAK2/STAT3 pathway to activate TGF-ß1/Smad signaling and promote the EMT. Magnesium isoglycyrrhizinate was found to ameliorate high fructose-induced EMT and liver fibrosis in rats. More importantly, magnesium isoglycyrrhizinate increased miR-375-3p expression to suppress the JAK2/STAT3 pathway and TGF-ß1/Smad signaling in these animal and cell models. This study provides evidence showing that magnesium isoglycyrrhizinate attenuates liver fibrosis associated with a high fructose diet.


Assuntos
Cirrose Hepática/tratamento farmacológico , MicroRNAs/metabolismo , Saponinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Triterpenos/uso terapêutico , Animais , Linhagem Celular , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Frutose , Janus Quinase 2/metabolismo , Cirrose Hepática/induzido quimicamente , Masculino , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Saponinas/farmacologia , Proteínas Smad Reguladas por Receptor/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Triterpenos/farmacologia
11.
Cell Physiol Biochem ; 51(5): 2324-2340, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30537738

RESUMO

BACKGROUND/AIMS: Circular RNAs (circRNAs) are key regulators in the development and progression of human cancers, however its role in non-small cell lung cancer (NSCLC) tumorigenesis is not well understood. The aim of this study is to identify the expression level of circPVT1 in NSCLC and further investigated its functional relevance with NSCLC progression both in vitro and in vivo. METHODS: Quantative real-time PCR was used for the measurement of circPVT1 in NSCLC specimens and cell lines. Fluorescence in situ hybridization analysis (FISH) assay was used for the identification of sublocation of circPVT1 in NSCLC cells. Bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation (RIP) were performed to verify the binding of c-Fos at circPVT1 promoter region, and the direct interaction between circPVT1 and miR-125b. Gain- or loss-function assays were performed to evaluate the effects of circPVT1 on cell proliferation and invasion. Western blot and immunohistochemistry assays were performed to detect the protein levels involved in E2F2 pathway. RESULTS: We found that circPVT1 was upregulated in NSCLC specimens and cells. The transcription factor c-Fos binded to the promoter region of circPVT1, resulting in the overexpression of circPVT1 in NSCLC. Knockdown of circPVT1 suppressed NSCLC cell proliferation, migration and invasion, and increased apoptosis. In addition, circPVT1 mediated NSCLC progression via the regulation of E2F2 signaling pathway. More importantly, circPVT1 was predominantly abundant in the cytoplasm of NSCLC cells, and circPVT1 could serve as a competing endogenous RNA to regulate E2F2 expression and tumorigenesis in a miR-125b-dependent manner, which is further verified by using an in vivo xenograft model. CONCLUSION: circPVT1 promotes NSCLC cell growth and invasion, and may serve as a promising therapeutic target for NSCLC patients. Therefore, silence of circPVT1 could be a future direction to develop a novel treatment strategy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Fator de Transcrição E2F2/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , RNA/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Fator de Transcrição E2F2/genética , Éxons , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA Longo não Codificante/genética , Transdução de Sinais
13.
Tissue Cell ; 53: 53-60, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30060827

RESUMO

Eukaryotic elongation factors 2 (eEF2) plays an essential role in the GTP-dependent translocation of the ribosome along mRNA. Previous studies have shown that eEF2 is overexpressed in various tumors. However, little is known about the role of eEF2 in ovarian cancer. The aim of the present study is to examine the effect of eEF2 on ovarian cancer proliferation. We first measured eEF2 protein expression by western blot using six fresh ovarian cancer tissues from G1 to G3. The results showed that eEF2 expression gradually increased from G1 to G3. Additionally, eEF2 expression correlated significantly with grade (P = 0.045), FIGO stage (P = 0.035) and Ki67 (P < 0.05). Additionally, there was a significant positive association between eEF2 expression and Ki67 (r = 0.855). Cox's proportional hazards model also showed that eEF2 (P = 0.004) and Ki67 (P < 0.001) were an independent prognostic factor of overall survival in ovarian cancer patients. In vitro, after the release of ovarian cancer cell line (HO8910) from serum starvation, the expression of eEF2, cyclinD1 and PCNA was up-regulated. Moreover, silencing eEF2 in HO8910 cells decreased cell proliferation. Finally, we hypothesize that eEF2 may be activated in a positive feedback cycle through inactivation of eEF2K via the PI3K/Akt/mTOR pathway. These data provide novel insights into developing experimental therapies for ovarian cancer.


Assuntos
Proliferação de Células , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Fator 2 de Elongação de Peptídeos/metabolismo , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , Prognóstico
14.
Data Brief ; 18: 69-75, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29896493

RESUMO

The data presented herein are related to the research article entitled "Magnesium isoglycyrrhizinate blocks fructose-induced hepatic NF-κB/NLRP3 inflammasome activation and lipid metabolism disorder" (Zhao et al., 2017) [1]. This article describes the effects of magnesium isoglycyrrhizinate on 24-h food or water intake in fructose-fed rats at 15-week. In addition, this article expands the effect of magnesium isoglycyrrhizinate on the animal body weight change during 1-17 week. The field dataset is made publicly available to enable critical or extended analyzes.

15.
Biomed Pharmacother ; 97: 1000-1010, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29136767

RESUMO

To take full advantage of combination therapy of Docetaxel (DTX) and Magnesium isoglycyrrhizinate (MGIG), the pharmacokinetic- pharmacodynamic- toxicodynamics (PK-PD-TD) interaction of DTX and MGIG in non-small cell lung tumor-bearing mice was investigated in the present study. A model, an integrated semi-mechanistic PK-PD-TD, was established for elucidating the exposure-effect-toxicity relationship between DTX and MGIG. A tumor growth and a transit compartmental system were applied to imitate the growth and death of tumor cell. An indirect model with precursor-dependence was induced to clarify the temporal relationship between liver injury and drug exposure. No PK interaction between DTX and MGIG in plasma, liver and tumor was observed. In PD and TD results, MGIG had no antitumoral activity on non-small cell lung carcinoma, while it showed a strong hepatoprotection on DTX-induced liver injury. The PK-PD parameters of anti-tumor effect were related with the tumor growth characteristics, the kinetics of the tumor death and drug potency. In the PK-TD model, it was estimated about the elevation rate of ALT after DTX challenge in hepatocytes as well as plasma. MGIG reduced the DTX-induced ALT release rate from hepatocyte efficiently. Based on parameters estimated via PK-PD-TD correlation, the model successfully predicted the tumor growth kinetics and hepatoprotection at different dose regimes. Therefore, this prospective model might provide an alternative approache to the optimization of new experiment design.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Saponinas/farmacologia , Taxoides/farmacologia , Triterpenos/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Docetaxel , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Saponinas/administração & dosagem , Saponinas/toxicidade , Taxoides/administração & dosagem , Taxoides/toxicidade , Triterpenos/administração & dosagem , Triterpenos/toxicidade , Ensaios Antitumorais Modelo de Xenoenxerto
16.
FASEB J ; 31(12): 5543-5556, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28821630

RESUMO

Interleukin 17A (IL-17A) and complement (C') activation have each been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). We have reported that IL-17A induces epithelial injury via TGF-ß in murine bronchiolitis obliterans; that TGF-ß and the C' cascade present signaling interactions in mediating epithelial injury; and that the blockade of C' receptors mitigates lung fibrosis. In the present study, we investigated the role of IL-17A in regulating C' in lung fibrosis. Microarray analyses of mRNA isolated from primary normal human small airway epithelial cells indicated that IL-17A (100 ng/ml; 24 h; n = 5 donor lungs) induces C' components (C' factor B, C3, and GPCR kinase isoform 5), cytokines (IL8, -6, and -1B), and cytokine ligands (CXCL1, -2, -3, -5, -6, and -16). IL-17A induces protein and mRNA regulation of C' components and the synthesis of active C' 3a (C3a) in normal primary human alveolar type II epithelial cells (AECs). Wild-type mice subjected to IL-17A neutralization and IL-17A knockout (il17a-/- ) mice were protected against bleomycin (BLEO)-induced fibrosis and collagen deposition. Further, BLEO-injured il17a-/- mice had diminished levels of circulating Krebs Von Den Lungen 6 (alveolar epithelial injury marker), local caspase-3/7, and local endoplasmic reticular stress-related genes. BLEO-induced local C' activation [C3a, C5a, and terminal C' complex (C5b-9)] was attenuated in il17a-/- mice, and IL-17A neutralization prevented the loss of epithelial C' inhibitors (C' receptor-1 related isoform Y and decay accelerating factor), and an increase in local TUNEL levels. RNAi-mediated gene silencing of il17a in fibrotic mice arrested the progression of lung fibrosis, attenuated cellular apoptosis (caspase-3/7) and lung deposition of collagen and C' (C5b-9). Compared to normals, plasma from IPF patients showed significantly higher hemolytic activity. Our findings demonstrate that limiting complement activation by neutralizing IL-17A is a potential mechanism in ameliorating lung fibrosis.-Cipolla, E., Fisher, A. J., Gu, H., Mickler, E. A., Agarwal, M., Wilke, C. A., Kim, K. K., Moore, B. B., Vittal, R. IL-17A deficiency mitigates bleomycin-induced complement activation during lung fibrosis.


Assuntos
Bleomicina/farmacologia , Ativação do Complemento/efeitos dos fármacos , Fibrose/metabolismo , Interleucina-17/deficiência , Interleucina-17/metabolismo , Pneumopatias/metabolismo , Idoso , Animais , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrose/genética , Imunofluorescência , Hemólise/genética , Hemólise/fisiologia , Humanos , Interleucina-17/genética , Pneumopatias/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real
17.
Eur J Pharmacol ; 809: 141-150, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28526339

RESUMO

Magnesium isoglycyrrhizinate as a hepatoprotective agent possesses immune modulation and anti-inflammation, and treats liver diseases. But its effects on immunological-inflammatory and metabolic profiles for metabolic syndrome with liver injury and underlying potential mechanisms are not fully understood. In this study, magnesium isoglycyrrhizinate alleviated liver inflammation and lipid accumulation in fructose-fed rats with metabolic syndrome. It also suppressed hepatic inflammatory signaling activation by reducing protein levels of phosphorylation of nuclear factor-kappa B p65 (p-NF-κB p65), inhibitor of nuclear factor kappa-B kinase α/ß (p-IKKα/ß) and inhibitor of NF-κB α (p-IκBα) as well as nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC) and Caspase-1 in rats, being consistent with its reduction of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and IL-6 levels. Furthermore, magnesium isoglycyrrhizinate modulated lipid metabolism-related genes characterized by up-regulating peroxisome proliferator-activated receptor-α (PPAR-α) and carnitine palmitoyl transferase-1 (CPT-1), and down-regulating sensor for fatty acids to control-1 (SREBP-1) and stearoyl-CoA desaturase 1 (SCD-1) in the liver of fructose-fed rats, resulting in the reduction of triglyceride and total cholesterol levels. These effective actions were further confirmed in fructose-exposed BRL-3A and HepG2 cells. The molecular mechanisms underpinning these observations suggest that magnesium isoglycyrrhizinate may inhibit NF-κB/NLRP3 inflammasome activation to reduce immunological-inflammatory response, which in turn may prevent liver lipid metabolic disorder and accumulation under high fructose condition. Thus, blockade of NF-κB/NLRP3 inflammasome activation and lipid metabolism disorder by magnesium isoglycyrrhizinate may be the potential therapeutic approach for improving fructose-induced liver injury with metabolic syndrome in clinic.


Assuntos
Frutose/efeitos adversos , Inflamassomos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Animais , Regulação para Baixo/efeitos dos fármacos , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , PPAR alfa/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
18.
PLoS One ; 12(2): e0171940, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28222108

RESUMO

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.


Assuntos
Autofagia/fisiologia , Sobrevivência Celular/fisiologia , NF-kappa B/fisiologia , Estresse Oxidativo/fisiologia , Proteínas de Ligação a RNA/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Proteína Sequestossoma-1/fisiologia , Animais , Western Blotting , Linhagem Celular , Modelos Animais de Doenças , Imunofluorescência , Degeneração Macular/etiologia , Degeneração Macular/fisiopatologia , Camundongos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Superóxidos/metabolismo , Regulação para Cima
19.
Artigo em Inglês | MEDLINE | ID: mdl-29377033

RESUMO

While our previous studies suggest that limiting bleomycin-induced complement activation suppresses TGF-ß signaling, the specific hierarchical interactions between TGF-ß and complement in lung fibrosis are unclear. Herein, we investigated the mechanisms underlying TGF-ß-induced complement activation in the pathogenesis of lung fibrosis. C57-BL6 mice were given intratracheal instillations of adenoviral vectors overexpressing TGF-ß (Ad-TGFß) or the firefly gene-luciferase (Ad-Luc; control). Two weeks later, mice with fibrotic lungs were instilled RNAi specific to receptors for C3a or C5a-C3ar or C5ar, and sacrificed at day 28. Histopathological analyses revealed that genetic silencing of C3ar or C5ar arrested the progression of TGF-ß-induced lung fibrosis, collagen deposition and content (hydroxyproline, col1a1/2); and significantly suppressed local complement activation. With genetic silencing of either C3ar or C5ar, in Ad-TGFß-injured lungs: we detected the recovery of Smad7 (TGF-ß inhibitor) and diminished local release of DAF (membrane-bound complement inhibitor); in vitro: TGF-ß-mediated loss of DAF was prevented. Conversely, blockade of the TGF-ß receptor prevented C3a-mediated loss of DAF in both normal primary human alveolar and small airway epithelial cells. Of the 52 miRNAs analyzed as part of the Affymetrix array, normal primary human SAECs exposed to C3a, C5a or TGF-ß caused discrete and overlapping miRNA regulation related to epithelial proliferation or apoptosis (miR-891A, miR-4442, miR-548, miR-4633), cellular contractility (miR-1197) and lung fibrosis (miR-21, miR-200C, miR-31HG, miR-503). Our studies present potential mechanisms by which TGF-ß activates complement and promotes lung fibrosis.

20.
Breastfeed Med ; 11: 461-468, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27643921

RESUMO

BACKGROUND: Suction pressure has been reported to be a key driving force of lactation. An infant's sucking at its mother's breasts is the major stimulus to post-natal prolactin (PRL) secretion, and PRL is the essential hormone for lactation and milk production. It is unknown what role suction pressure has in PRL secretion and milk supply postnatally. OBJECTIVES: To explore the relationship between the suction pressure, PRL level, lactation, and milk supply in breastfeeding mother-infant dyads. METHODS: Healthy women with normal full-term infants were enrolled (n = 122). Data collection included suction pressure, PRL level, and the mother's perception of both the onset of her lactation and her milk supply at 1 month. Suction pressure was measured with a pressure sensor connected to a tube placed alongside the nipple. The chemiluminescence method was used to quantify maternal serum PRL level both before and after a breastfeed to explore the effect of suckling on PRL increment. The mother's perception of the onset of her lactation was evaluated by the mother's sense of fullness in her breast. The mother's perception of milk supply was evaluated by using the H&H Lactation Scale. RESULTS: Data from 117 participants were included in this analysis. Low suction pressure, a shorter gestational age, a high pre-gestational body mass index (BMI), and high infant birth weight were associated with a smaller than average PRL increment. High suction pressure, longer sucking duration, and a low gestational weight gain were related to a mother's perception of an earlier onset of her lactation. However, low suction pressure, a high frequency of giving formula, a small PRL increment, and the mother's perception of a later onset of her lactation were correlated with her perception of an insufficient milk supply. CONCLUSIONS: Suction pressure varied directly with the post-natal PRL increment. Suction pressure was associated with the timing of the mother's perception of her lactation. Strong suction pressure by the infant is likely to enhance the mother's confidence in her lactation. Additionally, sucking duration, frequency of giving formula, maternal BMI, and infant birth weight played a role in early breastfeeding success.


Assuntos
Aleitamento Materno , Lactação/fisiologia , Período Pós-Parto/fisiologia , Prolactina/metabolismo , Comportamento de Sucção/fisiologia , Adulto , China/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Relações Mãe-Filho/psicologia , Mães/psicologia , Período Pós-Parto/psicologia , Prolactina/fisiologia , Sucção
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